INTERBACTERIAL COMPETITION in the BURKHOLDERIA CEPACIA COMPLEX Andrew Irving Perault a Dissertation Submitted to the Faculty At

Home , Burkholderia dolosa, Burkholderia multivorans, Enterobacter cloacae, Haemophilus, Proteus mirabilis, Salmonella enterica

INTERBACTERIAL COMPETITION IN THE BURKHOLDERIA CEPACIA COMPLEX

Andrew Irving Perault

A dissertation submitted to the faculty at the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor in Philosophy in the Department of Microbiology and Immunology in the School of Medicine.

Chapel Hill 2020

Approved by:

Peggy A. Cotter

Matthew C. Wolfgang

Brian P. Conlon

Elizabeth A. Shank

Alecia N. Septer

Scott H. Donaldson

ABSTRACT

Andrew Irving Perault: Interbacterial Competition in the Burkholderia cepacia Complex (Under the direction of Peggy A. Cotter)

Bacteria often live in multicellular communities composed of multiple species. Bacteria interact with one another during intimate cell-cell contact within such environments, and these interactions influence the ecology and evolution of multicellular communities. Polymicrobial communities reside within the respiratory tracts of individuals with the genetic disorder cystic fibrosis (CF). Polymicrobial respiratory infections are characteristic of CF disease, and canonical pathogens can dominate the airway communities during infections. Intriguingly, pathogenic species of the Burkholderia cepacia complex (Bcc) can establish infections within polymicrobial

CF respiratory tracts, even those dominated by the prevalent pathogen Pseudomonas aeruginosa, though these infections do not occur in young individuals and are limited to teenage and adult CF patients. Here we describe two mechanisms of cell contact-dependent interbacterial competition that provide Bcc pathogens ecological advantages: contact-dependent growth inhibition (CDI) and the type VI secretion system (T6SS).

We show the Bcc CF pathogen Burkholderia dolosa produces three CDI systems that facilitate killing of competitor cells during co-culture experiments. These systems deliver unique polymorphic toxins to target cells, and production of immunity proteins cognate to these toxins rescues target cells from killing. We show the genes encoding one B. dolosa CDI system are present in multiple sequenced Bcc strains, suggesting this mechanism of interbacterial competition may be prevalent among Bcc pathogens. Using the Bcc pathogen Burkholderia cenocepacia, we show that the T6SS of Bcc species mediates strong competition against a

iii variety of target bacteria. We describe the striking ability for this pathogen to use its T6SS to outcompete P. aeruginosa isolates from teenage and adult CF patients, but not P. aeruginosa isolates from young patients. Whole genome sequencing identified mutations in the susceptible

P. aeruginosa isolates that abrogate activity by their own T6SSs, thus inhibiting their retaliation to attack from B. cenocepacia and promoting their elimination from co-cultures. These competitive dynamics may explain why Bcc infections are limited to teenage and adult CF patients. Altogether CDI and T6S likely provide Bcc pathogens competitive advantages within the polymicrobial communities of the CF respiratory tract, promoting their colonization and infection potential, and thus may be attractive targets for therapies treating these infections.

ACKNOWLEDGEMENTS

I am fortunate to have wonderful support circles, both professionally and non- professionally, and therefore a plethora of individuals deserve my gratitude. For the sake of brevity, I will try to keep this brief…

First, I would like to thank my mentor Peggy A. Cotter. Academia can be a welcoming and supportive community, and graduate student trainees can be blessed by having terrific mentors; however, no one is luckier than me and the fellow students to be trained by Peggy.

Forget her passion for science, her excitement to discuss data, her love for helping trainees become great scientists, her ability to let loose; Peggy is just a great person. To get that and all the professional benefits with a mentor, what else could you ask for? I was lucky to have a similarly great mentor, Victor J. DiRita, before starting my graduate studies at UNC, and I thank him as well. I also would like to thank my thesis committee members. Although we only have formal meetings once a year, your mentorship and assistance in progressing my thesis research certainly extends beyond that. I also want to thank my unofficial mentors within the UNC

Microbiology & Immunology community and all the amazing individuals and support staff we have in our department.

I, of course, also want to thank my family and friends. At the very least, my family has been proud of me during my studies. This may seem minimal, but it is quite the driving force to continue. My family has also been curious about my research, excited for my next steps, and supportive in many other ways. I also need to thank Uncle Bill for putting a roof over my head during my graduate studies and regularly asking how my “sea monkeys” are doing. I find

v great fun in scientific research, but I have also been lucky to have great fun outside of lab, thanks to absolutely wonderful friends I have made over the years. My friends in North Carolina, including my graduate school colleagues, non-scientist friends, and all those who were members of the Cotter Lab, have made it so I will always cherish my time in graduate school, and I am excited to continue our friendships. My friends from before starting graduate school have brought me tremendous joy for many years, and I am lucky they will continue to do so long into my future. Also, I believe they still have not called me “nerd”!

TABLE OF CONTENTS

LIST OF FIGURES ...... ix

LIST OF TABLES ...... xi

LIST OF ABBREVIATIONS ...... xii

CHAPTER 1 – Introduction ...... 1

1.1 Microbial Communities ...... 1 1.2 Microbial Cooperation within Communities ...... 2 1.3 Microbial Competition within Communities ...... 2 1.4 Contact-Dependent Growth Inhibition ...... 3 1.5 Type VI Secretion Systems ...... 7 1.6 Polymicrobial Communities within the Cystic Fibrosis Respiratory Tract ...... 10 1.7 Pseudomonas aeruginosa ...... 12 1.8 The Burkholderia cepacia Complex ...... 15 1.9 Objectives of this Dissertation ...... 18 1.10 References ...... 21

CHAPTER 2 – Three Distinct Contact-Dependent Growth Inhibition Systems Mediate Interbacterial Competition by the Cystic Fibrosis Pathogen Burkholderia dolosa ...... 40

2.1 Summary ...... 40 2.2 Importance ...... 41 2.3 Introduction ...... 41 2.4 Materials and Methods ...... 43 2.5 Results ...... 48 2.6 Discussion ...... 56 2.7 References ...... 73

CHAPTER 3 – Host Adaptation Predisposes Pseudomonas aeruginosa to Type VI Secretion System-Mediated Predation by the Burkholderia cepacia Complex ...... 78

vii 3.1 Summary ...... 78 3.2 Introduction ...... 78 3.3 Results ...... 80 3.4 Discussion ...... 88 3.5 Method Details ...... 94 3.6 References ...... 114

CHAPTER 4 – Conclusion ...... 122

4.1 Interbacterial Competition in the Context of Cystic Fibrosis Infections ...... 122 4.2 My Contributions to the Burkholderia cepacia Complex Sociomicrobiology Field ...... 123 4.3 CDI System-Mediated Competition and Cooperation by the Burkholderia cepacia Complex ...... 124 4.4 T6SS-Mediated Burkholderia cepacia Complex Colonization and Infection of the Cystic Fibrosis Respiratory Tract ...... 127 4.5 Future Investigations for the Burkholderia cepacia Complex Sociomicrobiology Field .. 132 4.6 References ...... 135

APPENDIX ...... 142

viii LIST OF FIGURES

Figure 1.1. CDI Model ...... 19 Figure 1.2. T6SS model ...... 20 Figure 2.1. bcpAIOB loci in BdAU0158 ...... 62

Figure 2.2. The Bcp-1 and Bcp-2 CDI systems provide BdAU0158 a competitive advantage during in vitro growth ...... 63

Figure 2.3. The BdAU0158 bcp-3 locus encodes a functional CDI system that is not expressed under in vitro competition conditions ...... 64

Figure 2.4. BdAU0158 BcpO proteins are not required for CDI-mediated competition or resistance to CDI ...... 65

Figure 2.5. The Bcp-dependent community behaviors of autoaggregation and pigment production are not evident in BdAU0158 ...... 66

Figure 2.6. E. coli autotoxicity due to inducible production of the BdAU0158 Bcp-2 and Bcp-3 CT toxins, but not the Bcp-1 CT toxin ...... 67

Figure 2.7. The BdAU0158 Bcp-1 allele contains the same CT toxin- immunity pair as the BtE264 Bcp allele ...... 68

Figure 2.8. Orthologs of the BdAU0158 BcpA-CT and BcpI across several Burkholderia strains ...... 69 Figure 2.9. Production of BdAU0158 BcpA-1-CT and BcpI-1 by E. coli ...... 70 Figure 3.1. The BcAU1054 T6SS mediates interbacterial competition ...... 100

Figure 3.2. The BcAU1054 T6SS core cluster is homologous to the BcJ2315 T6SS core cluster, though contains insertions ...... 101 Figure 3.3. BcAU1054 ∆hcp does not have a growth defect ...... 102 Figure 3.4. Screening approach to identify probable BcAU1054 T6SS E-I pairs ...... 103 Figure 3.5. At least five BcAU1054 T6SS effectors mediate interbacterial competition ...... 104

Figure 3.6. Susceptibility of P. aeruginosa CF isolates to the BcAU1054 T6SS correlates with patient age ...... 105 Figure 3.7. BcAU1054 T6SS effectors exhibit target strain-specific variability in toxicity ...... 106

Figure 3.8. Restoration of H1-T6SS protein production can rescue host- adapted P. aeruginosa from T6SS-mediated elimination by BcAU1054 ...... 107

Figure 3.9. Additional Bcc pathogens kill host-adapted P. aeruginosa in a T6SS-dependent manner ...... 108

ix Figure 3.10. B. cenocepacia isolates from CF patients with concurrent P. aeruginosa infections outcompete their paired P. aeruginosa isolates under T6SS-permissive conditions ...... 109

x LIST OF TABLES

Table 2.1. Bacterial strains used in this study ...... 71 Table 2.2 Plasmids used in this study ...... 72

Table 3.1. Bioinformatically-predicted BcAU1054 T6SS E-I pairs and predicted effector enzymatic activities ...... 110

Table 3.2. Competition sensitivity, Hcp1 production, and putative T6SS- abrogating mutations of P. aeruginosa CF isolates used in this study ...... 111 Table 3.3. Bacterial strains used in this study ...... 112

xi LIST OF ABBREVIATIONS

ATPase adenosine triphosphatase

BcAU1054 Burkholderia cenocepacia strain AU1054

Bcc Burkholderia cepacia complex

BdAU0158 Burkholderia dolosa strain AU0158

BmCGD2M Burkholderia multivorans strain CGD2M bp base pair

BtE264 Burkholderia thailandensis strain E264

C.I. competitive index

CDI contact-dependent growth inhibition

CDS contact-dependent signaling

CF cystic fibrosis

CFTR cystic fibrosis transmembrane conductance regulator

CGD chronic granulomatous disease

Cm chloramphenicol

CT C terminus

DNA deoxyribonucleic acid

E-I effector-immunity

GTPase guanosine triphosphatase

IM inner membrane kDa kilodalton

Km kanamycin

LB lysogeny broth

LPS lipopolysaccharide

LSLB low salt lysogeny broth

xii mRNA messenger ribonucleic acid

NT N terminus

OD600 optical density, 600 nm

OM outer membrane

ORF open reading frame

PBS phosphate-buffered saline

PCR polymerase chain reaction

RNA ribonucleic acid

SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis sRNA small regulatory RNA

Tet tetracycline

TPS two-partner secretion tRNA transfer ribonucleic acid

T3SS type III secretion system

T5SSb type Vb secretion system

T6S type VI secretion

T6SS type VI secretion system

WT wild-type

xiii

CHAPTER 1 – INTRODUCTION

1.1 Microbial Communities

Bacteria are social organisms often residing within multicellular communities made up of one or many species (1, 2). These communities, typically referred to as biofilms, can be free- floating in aqueous environments or surface-attached, and are usually encased in an extracellular matrix composed of secreted macromolecules including polysaccharides, DNA, proteins, and lipids (3, 4). Due to the matrix surrounding biofilms, bacterial cells aggregate and can be in intimate cell-cell contact, promoting direct interactions between cells. Bacteria can also interact from afar within biofilms via secreted molecules (3, 5). These interactions have dramatic effects on the ecology and evolution of biofilm residents (6, 7).

Microbial communities are instrumental to our natural world. From their impact on biogeochemical cycling (8, 9) to their essentiality for plant growth (10), microbial communities influence everyday life. Humans are colonized by microbial communities at several body sites, including the skin, oral cavity, intestinal tract, and the female reproductive tract, where these bacteria are required for proper health of their host (11-14). Alternatively, human-associated microbial communities can have deleterious effects on their hosts and promote disease.

Polymicrobial infections form in the periodontal space (15), in the vagina (16), in chronic wounds

(17), and on medical-device implants (18), and these infections are typically difficult to treat due to the antibiotic-tolerant nature of biofilms (19).

1.2 Microbial Cooperation within Communities

Bacteria form beneficial interactions with kin cells and cells of different microbial strains and species within biofilms (20). Secreted molecules in the form of “public goods” improve nutrient acquisition by community members (21, 22), and bacteria digest complex macromolecules that feed into central metabolism more efficiently within biofilms compared to cells in isolation (23, 24). Human gut Bacteroides spp. are adept at degrading complex polysaccharides and releasing smaller molecules that serve as energy sources for other members of the intestinal microbiota (25). Additional secreted “public goods” enhance surface attachment and retention within the community (26, 27), as well as mediate communication and cooperative behaviors among cells (28). Cells unable to produce biofilm matrix components can be incorporated into a growing biofilm as long as matrix-producers are present (29). Due to the extracellular matrix and gradients of nutrients and oxygen, the physical nature of biofilms provides a protective shield against cellular stressors including antibiotics (19), and thus biofilm formation can be a cooperative behavior benefiting all community members.

1.3 Microbial Competition within Communities

Converse to the potential collective benefits of cooperative behaviors within biofilms is the high degree of intercellular competition that occurs in these communities (30-32).

Competitive behaviors are hypothesized to have a greater influence than cooperative behaviors on the ecology and evolution of microbial communities (33), and bacteria have evolved to gain multiple strategies to mediate interbacterial antagonism (32). Nutrients and physical space are often limited in polymicrobial communities (34), and thus members that are the most fit to compete for these resources can dominate. Bacteria can compete with one another across distances via the secretion and diffusion of toxic molecules, or can compete with close neighboring cells in cell contact-dependent manners (35). Antibiotics are specialized

2 metabolites produced by microorganisms that can diffuse throughout the environment and kill competitor cells (36). Degradative enzymes also act as diffusible antimicrobial weapons and provide competitive advantages to several bacterial species, including Myxococcus xanthus (37) and Staphylococcus spp. (38). Certain bacteria bleb off portions of their cellular membranes to form extracellular vesicles, which can deliver toxic cargo to competitor bacteria (39).

Pseudomonas aeruginosa (40), Streptomyces spp. (41, 42), M. xanthus (43), and Lysobacter

(44) have been shown to kill competitor bacteria by delivering toxins within extracellular vesicles.

Interbacterial competition dependent on intimate cell-cell contact can be mediated by complex secretory systems or by direct transfer of membrane components (32, 35). In addition to the battery of secreted antimicrobials produced by M. xanthus, this species delivers polymorphic toxins to Myxococcus target cells during an outer membrane (OM) fusion process termed “outer membrane exchange” (45, 46). Specialized secretion systems, including the type

IV, type Vb, type VI, and type VII secretion systems (T4SS, T5SSb, T6SS, and T7SS, respectively) also act as potent antibacterial weapons. The T4SS, T5SSb, and T6SS are widespread throughout Gram-negative bacteria (32), though T4SS-mediated interbacterial competition has only been described in the species Xanthomonas citri (47). The T7SS is a newly-characterized mechanism of interbacterial competition found in Gram-positive bacteria

(48, 49). Of the specialized secretion systems mediating cell contact-dependent interbacterial competition, the T5SSb (also referred to as contact-dependent growth inhibition, or CDI) and the

T6SS are the most characterized.

1.4 Contact-Dependent Growth Inhibition

CDI systems are composed of proteins belonging to the two-partner secretion (TPS) pathway proteins (also called T5SSb proteins) (50). CDI was first discovered in rat fecal

Escherichia coli isolate EC93, as EC93 could outcompete another E. coli strain (K12) upon cell-

3 cell contact (51). Further investigations revealed genes predicted to encode CDI systems are widespread in Proteobacteria and that a key component of these systems are immunity proteins, which protect against CDI toxins (52, 53). CDI systems fall into two broad classes: the

E. coli-type systems and the Burkholderia-type systems. E. coli-type systems are found in a-, b-, and g-proteobacteria, whereas Burkholderia-type systems are only found in members of the

Burkholderia genus and other closely-related species (52, 53). The major discerning characteristic between the two CDI classes is their operon layout. E. coli-type systems are encoded by cdiBAI loci (51), and Burkholderia-type systems are encoded by bcpAIOB loci (53). bcpO genes are present only in some Burkholderia-type CDI system-encoding loci, and the amino acid sequences of their encoded proteins are conserved across alleles (except for the signal sequences) (53, 54). No clear role exists for BcpO proteins, though B. thailandensis strain E264 (BtE264) and B. dolosa strain AU0158 (BdAU0158) mutants lacking the bcpO genes show a diminished ability to kill target cells via CDI (53, 54).

Despite their differences in genetic organization, E. coli-type and Burkholderia-type CDI systems function similarly. The CdiB/BcpB proteins are the transporter proteins of the TPS system, forming b barrels in the OM and allowing for translocation of the CdiA/BcpA passenger proteins (50). The CdiA/BcpA proteins are large exoproteins (typically >3000 amino acids) containing filamentous hemagglutinin repeats forming rigid b-helical rods on the surfaces of cells (Figure 1.1) (50). The C-terminal portions of CdiA/BcpA comprise the antibacterial toxins of CDI systems (52). Conserved amino acid motifs (VENN in E. coli-type systems, Nx(E/Q)LYN in Burkholderia-type systems) separate the N-terminal conserved domains encompassing the b- helical rods (~2800 amino acids), which exhibit high degrees of amino acid sequence identity among alleles within CDI system types, from the C-terminal toxin domains (~300 amino acids), which exhibit a striking diversity in sequence (50, 51, 53). CdiI/BcpI proteins are immunity proteins that function to block autotoxicity in CDI protein-producing cells, as well as protect cells

4 from CDI-mediated killing by the same kind (i.e., cells that have the same CDI system-encoding allele) (52, 53, 55). The amino acid sequences of C-terminal toxins (CdiA-CTs and BcpA-CTs) and their cognate CdiI/BcpI proteins co-vary, resulting in polymorphic toxin-antitoxin protein pairs (52, 53). Of the CdiA-CTs and BcpA-CTs characterized to date, most have either been shown to be or are predicted to function as nucleases, degrading DNA or tRNA within target cells (54, 56-61).

Both E. coli-type and Burkholderia-type CDI systems can function modularly. The DNA sequence encoding the CT of one allele can be replaced with DNA sequence encoding another allele’s CT, and these chimeric CdiA/BcpA proteins still function in CDI and toxicity is only prevented by production of the CdiI/BcpI protein cognate to the chimeric CdiA-CT/BcpA-CT (52,

55). Given the modular capabilities and the polymorphic nature of CDI systems, homologous recombination may promote diversification of these systems by swapping in alternative toxin- immunity-encoding sequences (62). Certain Burkholderia-type CDI system-encoding genes are predicted to be located in mobile genetic islands (63, 64), including the BtE264 bcpAIOB genes, which are mobile via transposon-mediated insertion (65). Therefore, homologous recombination and transposition may be common mechanisms to diversify and disseminate CDI system- encoding genes throughout microbial communities.

Recent findings have updated the predicted model of CDI toxin delivery into target cells.

Using EC93 as a model organism, the CdiA-CT was shown to remain in the periplasm of the cell, whereas the majority of the N-terminal conserved region, including the predicted receptor binding domain, was extracellular, forming a loop to thread the C terminus into the periplasm. It was proposed that upon CdiA binding to an OM receptor on the target cell, the C terminus would be secreted out of the periplasm and the CdiA-CT toxin would be delivered to the target cell (Figure 1.1) (66). While the OM receptor allowing for CDI toxin translocation into target cells has not been identified for Burkholderia-type CDI systems, the OM proteins BamA and

OmpC/OmpF serve as receptors for CdiA during CDI mediated by certain E. coli-type systems

5 (67, 68). Additionally, inner membrane (IM) translocator proteins, often ABC transporter membrane permeases, facilitate CdiA-CT delivery into the target cell cytoplasm where the toxin can elicit its effects (for nuclease toxins) (69). “Permissive factors” within target cells have also been identified that are required for toxicity of the delivered E. coli-type CDI toxins (57, 70, 71).

Further investigations into the OM receptors for CdiA proteins revealed that the surface exposed loops 6 and 7 of BamA and the surface-exposed loops 4 and 5 of OmpC are the domains that interact with an inhibitor cell’s CdiA, and that the amino acid sequences of these loops covary with the sequences of the predicted receptor-binding domains on CdiA proteins (68, 72-74).

These results suggested that a cell producing an E. coli-type CDI system can likely only deliver

CdiA-CT toxins to phylogenetically related target cells, possibly cells so closely related that they encode the same cdiBAI allele, and thus would be immune to toxicity. Moreover, the IM translocator proteins and “permissive factors” may add additional levels of target cell specificity for CdiA proteins and restrict potential CDI-mediated toxicity to closely related target cells, bringing into question the true ecological role of CDI systems in microbial communities.

Intercellular interactions besides competition have been demonstrated for the BtE264

CDI system. Biofilm formation by BtE264 is dependent on the BcpAOIB proteins, and catalytic residues required for the toxicity of this strain’s BcpA-CT are also required for promoting this cooperative behavior (75). BcpAIOB-mediated biofilm formation by BtE264 does not require intercellular killing, and thus the mechanism of promoting biofilm formation is not the release of biofilm matrix components from lysed cells (75). RNA-sequencing experiments comparing the transcriptomic responses in BtE264 cultures where wild-type (WT) BcpA-CT toxin was delivered between cells to the transcriptomic responses in BtE264 cultures where the non-toxic, non- biofilm-promoting BcpA-CT variant was delivered between cells revealed global gene expression changes elicited by the WT BcpA-CT (76). WT BcpA-CT included expression of genes encoding extracellular polysaccharide synthesis machinery and pilus proteins, both of which are structures likely important for biofilm formation. This CDI system-mediated

6 intercellular communication was termed contact-dependent signaling (CDS) (76). BdAU0158, a human pathogenic member of the Burkholderia cepacia complex (Bcc), shares a BcpAIOB system almost identical to that of BtE264, and this BdAU0158 BcpA protein promoted similar gene expression changes in BtE264 within BdAU0158-BtE264 co-cultures (76); moreover,

BdAU0158 and another Bcc pathogen Burkholderia multivorans produce functional CDI systems that kill target cells (54, 77). CDI system-mediated interbacterial competition and communication/cooperation may be widespread in Burkholderia spp.

1.5 Type VI Secretion Systems

Like CDI systems, the T6SS is a protein secretory apparatus in Gram-negative bacteria that mediates interbacterial competition upon cell-cell contact (78). From an evolutionary standpoint, T6SSs appear to be repurposed bacteriophages, as many of the structural components are analogous to T4 phage proteins (79-82). A membrane/baseplate complex made up of the proteins TssJ, TssM, TssL, TssK, and TssE anchors the T6S apparatus to the cell envelope and allows translocation of the apparatus across the cell wall and membranes

(82). VgrG and PAAR domain-containing proteins form the tip of the T6SS and puncture the target cell envelope upon secretion; these proteins associate with the membrane/baseplate complex first, followed by the assembly of additional apparatus proteins (82, 83). An inner tube made up of stacked hexameric rings of Hcp, surrounded by an outer tube of TssB and TssC proteins, next assemble at the baseplate (Figure 1.2A) (78, 84). Upon cell-cell contact, the

TssB/TssC sheath contracts to power propulsion of the Hcp tube and VgrG/PAAR tip into neighboring cells (Figure 1.2B) (85-87), and the ATPase ClpV disassembles the sheath proteins for their incorporation into subsequent T6S apparatuses (88). The TssA and TagA proteins associate at the distal end of the T6SS tube, and TagA anchors the Hcp tube and

TssB/TssC sheath to the inner membrane at the opposite side of the cell, stabilizing the apparatus until the secretion event (89, 90).

7 T6SSs mediate interbacterial competition by delivering proteinaceous effectors to target cells during secretion events. Effectors associate with either Hcp or VgrG proteins of the T6S apparatus (91, 92), and “evolved” VgrGs contain their own effector domains (79, 80, 92, 93).

The T6SS was first described as an anti-eukaryotic weapon (79), and many bacterial pathogens deliver effectors into host cells using T6S (94); however, T6SSs are predicted to be produced by

~25% of all Gram-negative bacteria (95), many of which are not pathogens, and thus interbacterial competition is believed to be the true ecological and evolutionary role of these systems (96). Antibacterial T6SS effectors have diverse molecular targets within prey cells, including membrane phospholipids, cell wall peptidoglycan, and nucleic acids (97). Recently characterized effectors also inhibit cell division by targeting FtsZ ring formation (98) and induce the stringent response by synthesizing (p)ppApp (99). Similar to CDI toxins, T6SS effectors have cognate antitoxins called immunity proteins, which are encoded by genes in immediate proximity to their cognate effector-encoding genes, composing effector-immunity (E-I)-encoding gene pairs. Immunity proteins protect against autotoxicity in T6SS-producing cells, as well as protect against kind-cell killing (97).

In contrast to CDI systems, target cell specificity is not known to restrict T6SS-mediated competition to closely related cells. Interspecies competition via T6S, including between members of different proteobacterial classes, has been documented (78, 100, 101), though

T6SS targeting of Gram-positive bacteria is not known to occur (102), perhaps due to the thick

Gram-positive cell wall acting as a shield against these antibacterial weapons. Studies using the

Pseudomonas aeruginosa reference strain PAO1, which produces three unique T6SSs, have shown that PAO1 is more T6SS-active in co-cultures with antagonistic bacteria than it is in co- cultures with non-aggressive bacteria (101, 103). Two hypotheses have been proposed for this behavior: T6SS-dueling, where PAO1 retaliates against T6SS-active neighbors using its own

T6SS (103), and the P. aeruginosa response to antagonism (PARA), where PAO1 activates aggressive behaviors like T6S upon sensing kin cell lysates (101). Therefore, T6S appears to

8 favorably target antagonistic competitor bacteria, at least for PAO1; however, it remains unknown whether other bacterial species exhibit similar specific T6SS activity.

Several studies have described T6SS-mediated interbacterial competition within higher host organisms. The plant pathogen Agrobacterium tumefaciens outcompetes bacterial targets via T6SS-activity during plant infection (104), and the plant commensal Pseudomonas putida uses its T6SS to outcompete multiple phytopathogens during plant colonization experiments

(105). Several human pathogens have been shown to outcompete bacterial targets in a T6SS- dependent manner during murine model infection, including Salmonella enterica serovar

Typhimurium (106), Shigella sonnei (107), and Vibrio cholerae (108), and T6SS-mediated competition improves the colonization of these pathogens. Human gut commensal Bacteroides spp. also use T6SSs for interbacterial competition and to gain a competitive advantage during intestinal colonization (109, 110). Moreover, metagenomic analyses of the human intestinal microbiota suggest T6S provides Bacteroides spp. and additional Bacteroidales spp. competitive advantages in the human gut (111, 112).

Although interbacterial competition is proposed to be the primary role of T6SSs, cooperative behaviors of the human pathogen Proteus mirabilis are influenced by T6SS activity.

P. mirabilis multicellular swarming is influenced by T6S, though contradictory models propose this is an indirect effect of T6SS activity due to intercellular killing (113), or that the T6SS delivers non-lethal proteins involved in self-identity that promote cooperation in the multicellular swarm (114, 115). Human pathogenic enteroaggregative E. coli and avian pathogenic E. coli exhibit T6SS-dependent biofilm formation (116). Our knowledge of the impact of T6S on biofilm formation and additional cooperative behaviors is likely limited, and like CDI, these systems may have diverse roles in addition to interbacterial competition. Given the promiscuous nature of

T6SSs and their prevalence among Gram-negative bacteria, T6S may play a critical role in determining the ecology and evolution within polymicrobial communities.

9 1.6 Polymicrobial Communities within the Cystic Fibrosis Respiratory Tract

Cystic fibrosis (CF) is an autosomal recessive genetic disorder affecting individuals of northern European descent (117). CF is the most prevalent life-threatening inheritable disease in such individuals, with approximately one in 3,000 live births affected (118), and approximately

1,000 new diagnoses occurring annually in the United States of America (119). Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the disease.

Hundreds of unique mutations in CFTR are known to lead to CF, though the vast majority of cases (~85% in the United States) are due to the F508del mutation resulting in the loss of the phenylalanine residue at position 508 from CFTR (117, 119). While life expectancy for CF patients used to be in the teens, improved therapies have increased life expectancy to approximately age 40, and modeling predicts a child born with CF today will likely live to over age 50 (118-120).

Disease-causing mutations in CFTR can impair production of, function of, or trafficking to the cytoplasmic membrane by CFTR, an apical anion channel transporting chloride and bicarbonate across the membrane (117, 121, 122). Mucosal epithelial cells are most affected by

CFTR mutations, with impaired ion conductance leading to dehydration of the mucus layer, ATP channel disruption, organelle acidification, and dysregulation of intracellular vesicle transport

(118, 123, 124). CF pathology is pronounced in several body sites: the intestines, where it causes the obstructive condition meconium ileus (125); the liver, where it blocks bile ducts and leads to focal biliary cirrhosis (126); the pancreas, promoting pancreatic insufficiency (127); and the respiratory tract, where disease manifestations cause the greatest number of deaths (119).

In the CF respiratory tract, dehydration of the mucus layer collapses cilia of respiratory epithelial cells, preventing their normal function in mucociliary transport, a necessary physical barrier to invading respiratory pathogens (118). This immobile mucus layer enhances mucus secretions by goblet cells and submucosal glands, as well as promotes submucosal gland hyperplasia, exacerbating mucus secretion. The abnormal mucus layer becomes dehydrated

10 due to impaired CFTR function (or lack thereof), leading to the accumulation of dense, dehydrated mucus in the airways, which serves as a hospitable environment for invading opportunistic pathogens (117). Moreover, impaired innate immune defenses in the CF airways may contribute to infections (128), including dysfunctional defensins and antimicrobial peptides

(possibly due to acidification of the mucus layer) (129, 130), as well as abrogated antimicrobial responses by neutrophils and biasing towards proinflammatory macrophages (131, 132).

Canonical bacterial CF respiratory pathogens include Haemophilus influenzae, Staphylococcus aureus, P. aeruginosa, and members of the Bcc, though Stenotrophomonas maltophilia,

Achromobacter spp., and nontuberculous mycobacteria (NTM) are emerging CF pathogens

(119, 133). Fungal pathogens can also infect CF patients, with Aspergillus fumigatus being the most concerning as it can cause invasive infections and allergic bronchopulmonary aspergillosis

(134). Chronic infections caused by these pathogens promote perpetual inflammatory responses, which damage the airways and lead to lung function decline and ultimately patient death (117, 118, 135).

Improvements in molecular techniques to identify microbial species have increased our understanding of polymicrobial communities in the CF airways. Although canonical pathogens can be the predominant organisms within these tissues (136-138), it is now clear that the CF respiratory tract harbors complex, dynamic microbial communities including nonpathogenic species (139). These techniques can also provide insight into the CF respiratory tract environment and physiology; for example, the detection of obligate anaerobic bacteria within CF respiratory samples suggests there are anaerobic, or at least microaerobic, pockets within the airways (140). Oral cavity microbiota species commonly colonize the CF respiratory tract and can predominate in the airways in the absence of canonical pathogens (141, 142). Species associated with nasal, skin, and intestinal microbiota are also detected in the CF respiratory tract (136, 143, 144). Given the rich microbial communities in CF patients, intercellular interactions likely occur within the airways and may influence disease progression (139).

11 Studies investigating such interactions are typically conducted ex vivo, as robust animal models for chronic CF infections have not been developed (145-147). P. aeruginosa and S. aureus are common focuses of sociomicrobiological investigations relevant to CF (148), and interactions between the two pathogens include competition (149, 150), alteration of antibiotic susceptibility

(151, 152), and virulence potentiation (153-155). Continued development of CF animal models will make interbacterial interaction studies in the context of disease more accessible; nevertheless such interactions have the potential to influence disease outcome in these patients

(139, 148).

1.7 Pseudomonas aeruginosa

P. aeruginosa is a Gram-negative bacterium typically residing in aqueous environments that notoriously causes a range of opportunistic infections in immunocompromised individuals

(156). Given its metabolic versatility and regulatory networks coordinating adaptation to varying environments, P. aeruginosa causes infections in multiple body sites, including the eyes, ears, heart, urinary tract, skin, and respiratory tract (157). P. aeruginosa can cause chronic infections in the lungs and in burns and other wounds, and increasing rates of multidrug resistance limit antibiotic treatment options (158). As it is one of the most common pathogens causing healthcare-associated infections (159), there is a great need for research improving our knowledge of how P. aeruginosa causes infections and persists within human patients.

P. aeruginosa respiratory infections often occur in hospitalized patients intubated by ventilators, on which the bacterium can reside in biofilms, or in CF patients (156). Flagella and type IV pili mediate attachment to the respiratory epithelium and biofilm formation; both surface structures are required in infection models (160-162), though isolates from chronic infections often down-regulate flagellar gene expression or are non-flagellated (163). During acute infections, the type III secretion system (T3SS) promotes virulence by injecting effectors directly into host cells that disrupt the cytoskeleton and induce necrosis (162, 164, 165). It is

12 hypothesized the T3SS promotes P. aeruginosa wound infection by antagonizing wound healing and enhances neutrophil influx to infected lungs during pneumonia (165). Additional secreted virulence factors include the translation inhibitor exotoxin A (166, 167), several tissue-degrading proteases (156), lipases and phospholipases that degrade respiratory surfactant (162), oxidative stress-inducing pyocyanin (168), and iron-scavenging siderophores (169, 170). During the proinflammatory and tissue-destructive acute phase of infection, P. aeruginosa can breach the epithelial barrier and case fatal septicemia (157, 171). Quorum sensing, a density-dependent mechanism of intercellular communication and behavioral coordination, regulates virulence and biofilm formation by P. aeruginosa, and this communication is required during infection of animal models (172, 173).

If not cleared by the immune response or antibiotic treatment during acute infection, P. aeruginosa can transition to long term chronic infections of the respiratory tract where the bacteria typically reside within biofilms (156, 174). Despite the rich microbial communities in the

CF airways, P. aeruginosa can be the predominant organism during chronic infections, making up over 90% of all bacterial cells (136-138). During the evolutionary process of host adaptation, mutations are selected for that promote biofilm formation and also reduce cytotoxicity and inflammatory potential (163, 175-178). Such adaptations include the loss of flagella and type IV pili, lack of T3SS activity, outer membrane alterations that decrease immune stimulation (179-

181), conversion to a mucoidy state that may protect against immune responses (182), and inactivation of the lasR quorum sensing system (183). The Gac/Rsm global regulatory network controls transition from acute infection to chronic infection (184). GacS and GacA form a two- component system (GacS being the sensor kinase, and GacA being the response regulator), activating production of the small regulatory RNAs (sRNAs) RsmY and RsmZ when phosphorelay through GacSA occurs. RsmY/Z inhibit the post-transcriptional regulator RsmA and alter the translation of many gene products (184, 185). GacSA activity and subsequent

RsmY/Z production represses acute virulence factors including flagella, type IV pili, and the

13 T3SS, but induces expression of chronic infection-associated genes important for biofilm matrix production (pel and psl), siderophores, and the T6SS (157). GacSA phosphorelay is controlled by additional sensor kinases: RetS acts as a GacSA inhibitor (185), whereas LadS promotes

GacSA phosphorelay (186).

Mutations in retS and gacS/gacA often determine the virulent lifestyle of P. aeruginosa within the CF respiratory tract. During early, acute phases of infection, RetS inhibits GacSA phosphorelay (184, 185); however, mutations can be selected for in retS that abrogate the activity of its protein product and allow for GacSA phosphorelay, and thus biofilm formation and other chronic infection behaviors (184, 187, 188). Given GacSA phosphorelay and RsmY/Z are required for production of T6SS proteins by P. aeruginosa, certain isolates from CF chronic infections can be T6SS-active (189), which may benefit this pathogen in the polymicrobial CF respiratory tract. After transitioning to a chronic infection lifestyle, often mediated by retS mutagenesis, subsequent mutations can be selected for in gacS/gacA (190-192), potentially abrogating GacSA phosphorelay and T6SS activity. In agreement with this, certain CF chronic infection isolates of P. aeruginosa are not T6SS-active (189). T6SS proteins of P. aeruginosa are immunogenic (189), so there may be a selective pressure to lose T6SS activity in later stages of infection.

P. aeruginosa is a model organism used to study T6S, as it has three separate T6SSs

(the H1-, H2-, and H3-T6SS) (193). Both the H1- and H2-T6SSs mediate interbacterial competition, though the H1-T6SS is a much stronger antibacterial weapon (194-196). GacSA regulates production of proteins associated with all three T6SSs, via repression of RsmA, and the transcriptional regulator AmrZ also acts as a negative regulator of the H2-T6SS (196).

Studies using P. aeruginosa strain PAO1 have described intricate posttranslational regulation of

T6SS activity. Assembly of the H1-T6SS requires the TagQRST regulatory system associated with the cell envelope. TagQRST controls phosphorylation of the kinase PpkA (197), which in turn phosphorylates the scaffolding protein Fha1 to initiate T6SS apparatus assembly (103, 198,

14 199). The phosphatase PppA dephosphorylates Fha1, a necessary step in the disassembly of the T6SS, promoting the formation of subsequent T6S apparatuses (199). TagQRST, PpkA,

PppA, and Fha1 are all required for efficient T6SS activity and interbacterial competition (103,

199). It is hypothesized that the TagQRST system senses membrane perturbations and responds by activating PAO1 T6SS assembly, and membrane disruption may be due to T6SS attack from a neighboring competitor cell, thus promoting the T6SS-dueling behavior of PAO1

(103).

1.8 The Burkholderia cepacia Complex

The Bcc includes at least 17 species belonging to the Burkholderia genus that exhibit genetic diversity, but are phenotypically similar (200, 201). Bcc bacteria are found ubiquitously in the environment, often in soil and aqueous environments (201). In the soil, Bcc bacteria typically form beneficial symbioses with plants and prevent fungal infections (202), but can also be significant phytopathogens of onions and other commercial crops (203). The first identified

Bcc bacterium originated from an onion infection, and was initially named Pseudomonas cepacia (204). 16S rRNA gene sequencing and DNA-DNA hybridization techniques have since determined Bcc species belong to the Burkholderia genus, not the Pseudomonas genus (205).

Bcc bacteria have tremendous metabolic potential, including the ability to metabolize pollutants such as trichloroethylene, and thus have been used in bioremediation efforts (202, 206).

Despite their beneficial capabilities in promoting plant growth and reversing pollution, certain members of the Bcc cause serious opportunistic infections in immunocompromised patients (201). Humans most susceptible to Bcc infections are those with CF, but Bcc species are also problematic pathogens for individuals suffering from chronic granulomatous disease

(CGD). CGD is a primary immune disorder that impairs the oxidative killing response of neutrophils, and thus Bcc pathogens can survive within the respiratory tracts of these immunocompromised patients, where they are the second-most common cause of death (201,

15 207). Similar to P. aeruginosa, Bcc pathogens can establish long-term infections in CF patients due to dysfunctional mucociliary transport and immune responses in the airways. The first description of Bcc bacteria as CF pathogens resulted from an epidemic during the early 1980s at the Hospital for Sick Children in Toronto (208). These patients showed more severe symptoms and lung function decline than patients infected with P. aeruginosa (208), including necrotizing pneumonia, septicemia, and rapid death, a condition now referred to as “cepacia syndrome” (133, 201). Cepacia syndrome rarely occurs in patients infected by other canonical

CF pathogens, and thus Bcc infections are of great concern among this patient population

(209). Bcc pathogens can also transmit person-to-person and cause epidemics at CF treatment centers (210-213).

Compared to S. aureus and P. aeruginosa, Bcc infections are rare, occurring in 3-5% of

CF patients within the United States (S. aureus and P. aeruginosa infect ~70% and ~45% of CF patients in the United States, respectively) (119). For unknown reasons, Bcc pathogens only infect older CF patients, typically teenagers and adults, with a median age at first infection of 20 years; conversely, S. aureus and P. aeruginosa are common in young patients, with S. aureus infecting 60-80% of patients 10 years old or younger, and a median age at first infection of 5 years for P. aeruginosa (119). Although Bcc pathogens can infect CF patients not culturing positive for P. aeruginosa, Bcc-P. aeruginosa co-infections are common, and in such cases, P. aeruginosa respiratory burdens are typically lower than in patients solely infected by P. aeruginosa (208, 214). Like P. aeruginosa, Bcc pathogens can also be the predominant organisms in the CF respiratory tract during infections (137, 138). Notable Bcc pathogens are

Burkholderia cenocepacia, B. multivorans, and B. dolosa, with B. cenocepacia and B. multivorans causing the most infections. Although B. multivorans has replaced B. cenocepacia over recent years as the most prevalent Bcc pathogen in the CF patient population (133, 200),

B. cenocepacia (specifically the ET-12, PHDC, and Midwest lineages) has been the most virulent pathogen over the past few decades (211, 215, 216).

16 Bcc bacteria exhibit a striking degree of intrinsic antibiotic resistance, making infections difficult to treat clinically (201). Resistance is often mediated by drug efflux pumps, decreased

OM permeability to antibiotics, and degradative enzymes including chromosomally-encoded b- lactamases (217-219). A notable example of antibiotic resistance within the Bcc is the ability of these bacteria to use penicillin as a sole carbon source (220). In addition to their antibiotic resistance potential, Bcc pathogens produce an array of classical virulence factors, though roles in pathogenesis have not been demonstrated for all (201). Bcc bacteria produce a lipopolysaccharide (LPS) layer of the OM that is unique compared to other Gram-negative bacteria. Distinct chemical components of the LPS (specifically 4-amino-4-deoxyarabinose moieties attached to lipid A) inhibit the activity of antimicrobial peptides produced during the innate immune response (221, 222). Moreover, Bcc LPS is highly endotoxic and likely promotes inflammatory pathology during pneumonia and septicemia (223-225). Flagella are important for

Bcc pathogen invasion of host cells, where these bacteria typically reside during infection (214), and flagellar mutants cause no overt disease in animal models of infection (226, 227). The B. cepacia epidemic strain marker, a genetic locus present in the ET-12 B. cenocepacia lineage, encodes a quorum sensing system that is required for virulence in a rat model of infection (228).

Cable pili and a 22-kDa adhesin are produced by certain B. cenocepacia lineages and mediate attachment to the respiratory epithelium (229-231). Genes encoding a T3SS and one or more

T6SSs are present in Bcc pathogens; disruptions in the T3SS-encoding locus and one T6SS- encoding locus attenuate virulence in murine models of infection (232, 233).

T6S has been studied in B. cenocepacia, mostly from the standpoint of its role in targeting host cells and promoting pathogenesis. By delivering the effector TecA (234), the B. cenocepacia T6SS inactivates macrophage Rho GTPases, disrupting their actin cytoskeleton, impairing phagocytosis, and inhibiting NADPH oxidase assembly onto vacuoles containing B. cenocepacia (235-238). TecA-induced cytoskeletal collapse activates the caspase-1 inflammasome and pyroptosis, a mechanism of inflammatory cell death, in infected

17 macrophages (234, 239, 240), exacerbating inflammation during murine infection. Pyroptosis activation by TecA is required for survival of B. cenocepacia-infected WT mice, though the role of T6SS-mediated inflammation in CF models in unknown. Recent bioinformatic analysis suggests this T6SS is prevalent among Bcc species (referred to as T6SS-1), though some species harbor additional T6SSs of unknown function (241). The T6SS-1 mediates modest interbacterial killing by B. cenocepacia strain H111 (241), but it remains unclear whether other

Bcc pathogens, or additional T6SSs, have antibacterial functions. Given the ability of Bcc pathogens to establish infections in the polymicrobial CF respiratory tract, T6SS-mediated interbacterial competition may play an important role during initial colonization and/or prolonged infection.

1.9 Objectives of this Dissertation

CF is a polymicrobial disease characterized by complex, dynamic microbial communities in the airways that are often dominated by canonical pathogens. As interbacterial interactions likely occur in the CF respiratory tract and may influence pathogen colonization and overall disease, we hypothesize interbacterial competition provides pathogens advantages over other bacteria within the airways. Using Bcc bacteria as model organisms, we study CDI- and T6SS- mediated interbacterial competition through the following aims:

Aim 1: Characterize the potential CDI systems of B. dolosa and their ability to kill

competitor bacteria.

Aim 2: Assess T6SS-mediated competition by B. cenocepacia in the context of

colonization dynamics of the P. aeruginosa-infected CF respiratory tract.

Figure 1.1. CDI Model. The CDI system-producing cell (inhibitor cell) displays CdiA/BcpA on the cell surface by translocation across CdiB/BcpB. The toxic CT domain (CdiA-CT/BcpA-CT) remains within the periplasm of the inhibitor cell before delivery to the target cell. Upon receptor- binding domain interaction with the target cell OM receptor (indicated by black arrow), the CdiA- CT/BcpA-CT is delivered to the target cell. CdiI/BcpI immunity proteins remain in the cytoplasm of the inhibitor cell and protect against autotoxicity. Model not drawn to scale.

Figure 1.2. T6SS model. (A) T6SS apparatus assembled at the inhibitor cell envelope. The legend depicts T6SS proteins. (B) Before T6SS attack (left), the apparatus remains within the cytoplasm of the inhibitor cell (orange cell). Upon contacting the target cell (purple cell), the TssBC sheath contracts to deliver the Hcp tube, VgrG/PAAR tip, and associated effectors into the target cell (right). Model not drawn to scale.

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CHAPTER 2 – THREE DISTINCT CONTACT-DEPENDENT GROWTH INHIBITION SYSTEMS MEDIATE INTERBACTERIAL COMPETITION BY THE CYSTIC FIBROSIS PATHOGEN BURKHOLDERIA DOLOSA1

2.1 Summary

The respiratory tracts of individuals afflicted with cystic fibrosis (CF) harbor complex polymicrobial communities. By an unknown mechanism, species of the Gram-negative

Burkholderia cepacia complex, such as Burkholderia dolosa, can displace other bacteria in the

CF lung, causing cepacia syndrome, which has a poor prognosis. The genome of B. dolosa strain AU0158 (BdAU0158) contains three loci that are predicted to encode Contact-Dependent growth Inhibition (CDI) systems. CDI systems function by translocating the toxic C-terminus of a large exoprotein directly into target cells, resulting in growth inhibition or death unless the target cells produce a cognate immunity protein. We demonstrate here that each of the three bcpAIOB loci in BdAU0158 encodes a distinct CDI system that mediates interbacterial competition in an allele-specific manner. While only two of the three bcpAIOB loci are expressed under the in vitro conditions tested, the third conferred immunity under these conditions due to the presence of an internal promoter driving expression of the bcpI gene. One BdAU0158 bcpAIOB allele is highly similar to bcpAIOB in Burkholderia thailandensis strain E264 (BtE264), and we showed that their BcpI proteins are functionally interchangeable, but Contact-Dependent Signaling (CDS) phenotypes were not observed in BdAU0158. Our findings suggest that the CDI systems of

BdAU0158 may provide this pathogen an ecological advantage during polymicrobial infections of the CF respiratory tract.

1 First published in the Journal of Bacteriology, November 2018, Volume 200, e00428-18

40 2.2 Importance

Human-associated polymicrobial communities can promote health and disease, and interbacterial interactions influence the microbial ecology of such communities. Polymicrobial infections of the cystic fibrosis respiratory tract impair lung function and lead to death of individuals suffering from this disorder; therefore, a greater understanding of these microbial communities is necessary for improving treatment strategies. Bacteria utilize contact-dependent growth inhibition systems to kill neighboring competitors and maintain their niche within multicellular communities. Several cystic fibrosis pathogens have the potential to gain an ecological advantage during infection via contact-dependent growth inhibition systems, including

Burkholderia dolosa. Our research is significant as it has identified three functional contact- dependent growth inhibition systems in B. dolosa that may provide this pathogen a competitive advantage during polymicrobial infections.

2.3 Introduction

Bacteria often reside in complex polymicrobial communities in which intra- and interspecies interactions influence community structure (1-4), and interbacterial competition has been proposed to have a greater impact on microbial ecology and evolution within polymicrobial environments than interbacterial cooperation (5). While complex microbial communities, such as the microbiota of the intestinal and vaginal tracts, can promote the health of their hosts (6, 7), microbial communities can also arise in diseased tissues and exacerbate morbidity, such as in the respiratory tracts of cystic fibrosis (CF) patients. Several bacterial pathogens, including

Staphylococcus aureus, Pseudomonas aeruginosa, and members of the Burkholderia cepacia complex (Bcc), are notorious for dominating the CF airways, (8, 9). Infections by Bcc pathogens typically do not arise until teenage years or adulthood and can rapidly progress to “cepacia syndrome,” which is a fatal, necrotizing pneumonia accompanied by bacteremia (10, 11).

Strikingly, Bcc pathogens can replace other pathogenic species as the predominant organisms

41 during polymicrobial infections, though the mechanisms underlying this behavior remain unknown (12-14).

Contact-dependent growth inhibition (CDI) is a mechanism of interbacterial competition in which the toxic C terminus (CT) of a large exoprotein belonging to the two-partner secretion

(TPS) family is delivered directly from one bacterium to another, resulting in death or growth arrest of the recipient cell (15). Autotoxicity is prevented in CDI+ cells by the production of an immunity protein that binds to the CT, blocking its activity(15). Genes encoding CDI systems are widespread among Gram-negative bacteria and are polymorphic in nature, with different alleles encoding unique CT toxins and cognate immunity proteins, and thus protection against toxicity by immunity proteins occurs in an allele-specific manner (16).

Two classes of CDI systems have been described to date: the Burkholderia-type and the

Escherichia coli-type (15-17). E. coli-type systems are encoded by cdiBAI genes, with cdiA encoding the toxic exoprotein, cdiB encoding its TPS transporter partner protein, and cdiI encoding the immunity protein (15). Burkholderia-type systems are encoded by bcpAIOB genes

(17). bcpA and bcpB encode the exoprotein and transporter TPS proteins, respectively, bcpI encodes the immunity protein, and a fourth open reading frame (ORF), bcpO, encodes a small protein of unknown function (17). In both E. coli- and Burkholderia-type CDI systems, the N- terminal ~2800 amino acids of CdiA and BcpA proteins, respectively, are conserved among closely-related species, while the C-terminal ~300 amino acids vary greatly, as do the CdiI and

BcpI proteins, allowing for toxin-antitoxin heterogeneity across different CDI system-encoding alleles (16, 18). Burkholderia- and E. coli-type CDI systems also contain distinct motifs separating the conserved and variable domains of the toxic exoproteins; Nx(E/Q)LYN in BcpA proteins and VENN in CdiA proteins (16, 17). Burkholderia-type CDI systems are further classified into two different phylogenetic groups – Class I and Class II – based on the amino acid sequences of the BcpB and BcpO proteins, and the conserved region of BcpA (17, 19).

BcpO proteins across different Burkholderia Class I alleles are nearly identical, save for the N-

42 terminal ~20 aa-long signal sequence that co-varies with BcpA-CT and BcpI, whereas the bcpO genes of Class II are not similar across different alleles (17, 19).

Most information available for Burkholderia-type CDI systems is for the Class I allele in

B. thailandensis strain E264 (BtE264) (17, 19-21). We and others have shown that the BtE264

BcpAIOB proteins compose a functional CDI system (17), and that chimeric BtE264 strains producing the BcpA-CTs and BcpI proteins of pathogenic B. pseudomallei strains outcompete neighboring cells in a CDI-dependent manner (19, 22, 23). Our laboratory has also discovered that the BtE264 CDI system induces gene expression changes that promote cooperative behaviors between kin cells (i.e. cells that contain identical Bcp alleles) (24), a phenomenon we call “contact-dependent signaling” (CDS). We hypothesize that delivery of the BcpA-CT to a neighboring kin cell and subsequent BcpA-CT-BcpI binding forms a signaling complex that leads to expression of genes that confer community behaviors including autoaggregation, biofilm formation, and pigment production (18, 21, 24).

Burkholderia dolosa, a member of the Bcc that caused a deadly epidemic in CF patients in Boston during the 1990s (25, 26), can transmit from human-to-human and lead to significant decline in lung function compared to uninfected CF patients (27, 28). The genome of B. dolosa strain AU0158 (BdAU0158) contains three bcpAIOB loci, each potentially encoding a CDI system; one Class I allele and two Class II alleles. The goal of this study was to characterize the bcpAIOB loci and potential CDI systems of BdAU0158 using the native pathogenic strain.

2.4 Materials and Methods

Bacterial strains and growth conditions.

All strains used in this study are listed in Table 2.1 of the supplemental material. B. dolosa strains were maintained in either LB (NaCl concentration of 10 g/L) or low salt LB (LSLB,

NaCl concentration of 5 g/L), while B. thailandensis strains were exclusively maintained in

LSLB. Antibiotics were added to select for growth of various Burkholderia strains at the following

43 concentrations: 250 µg/mL kanamycin, 50 µg/mL tetracycline, and/or 20 µg/mL chloramphenicol. E. coli strains were maintained in LB, and antibiotics were added at the following concentrations when appropriate: 100 µg/mL ampicillin, 50 µg/mL kanamycin, 10

µg/mL tetracycline, and/or 35 µg/mL chloramphenicol. Diaminopimelic acid (DAP) was added at a concentration of 200 µg/mL to cultures maintaining E. coli strain RHO3. All strains were grown overnight with aeration at 37˚C (unless indicated otherwise).

Construction of plasmids and mutant strains.

All plasmids used in this study are listed in Table 2.2 of the supplemental material, and were delivered to BdAU0158 or BtE264 cells via conjugation with E. coli RHO3 strains harboring these plasmids. BdAU0158 ∆bcp-1, BdAU0158 ∆bcp-2, BtE264 ∆bcpAIOB, BtE264 attTn7::Cm, and BtE264 ∆bcpAIOB attTn7::Km were constructed previously (17, 24). Allelic exchange plasmids for generating BdAU0158 in-frame deletion mutants were constructed on the pEXKm5 backbone (29). Briefly, ~500 bp upstream and including the first three codons of the ORF(s) to be deleted were fused via overlap extension PCR to the last three codons of the ORF(s) and

~500 bp downstream sequence, and constructs were cloned into pEXKm5.

Plasmids to deliver cassettes to the attTn7 sites of the BdAU0158 and BtE264 chromosomes were constructed using the pUC18Tmini-Tn7T backbone (30). Cassettes for generating antibiotic-resistant BdAU0158 strains were delivered to the BdAU0158 chromosome via triparental mating with E. coli RHO3 strains harboring either pUC18T-miniTn7-Km (for kanamycin resistance) or pUC18T-miniTn7-Tet (for tetracycline resistance), as well as the

RHO3 strain harboring the transposase-encoding helper plasmid pTNS3 (17). To complement

BdAU0158 and BtE264 bcp locus deletion mutants with various bcpI genes, the genes of interest were cloned into pUCS12Km (pUC18T-miniTn7-Km plasmid with the constitutive

BtE264 ribosomal S12 subunit gene promoter cloned immediately 5’ to the multiple cloning site), and these PS12-driven constructs were delivered to a neutral site in the chromosome via

44 triparental mating with E. coli RHO3 harboring pTNS3. Plasmids containing lacZ reporter cassettes were generated on the pUClacZ backbone (pUC18T-miniTn7-Km with promoterless lacZ cloned into the multiple cloning site), with promoters of interest inserted immediately 5’ to the lacZ gene. lacZ reporter cassettes were delivered to the BdAU0158 chromosome via triparental mating with E. coli RHO3 harboring pTNS3.

Interbacterial competition experiments.

All competitions between BdAU0158 strains followed our previously developed protocol

(17), with minor adjustments. Cells from overnight cultures were washed in phosphate buffered saline (PBS) and diluted to an OD600 value of 0.2. Equal volumes of inhibitor and target cells were mixed, and 20 µL spots of cell suspensions were plated on LSLB agar and allowed to dry.

Once spots had dried, competitions were incubated at 37˚C for 48 hours (h). To determine the starting ratios for competitions, the cell suspensions containing inhibitor and target bacteria were serially diluted and 20 µL spots were plated onto selective media (LSLB/Km250 or

LSLB/Tet50) and incubated at 37˚C. Following 48 h co-culture, cells were sampled from the edge of the colony biofilms, resuspended in 1 mL PBS, serially diluted, and 20 µL spots of serial dilutions were plated onto selective media (LSLB/Km250 or LSLB/Tet50) and incubated at 37˚C.

Competitions involving BtE264 were conducted similarly, except that co-cultures were incubated at room temperature for 24 h. Colony counts for inhibitor and target bacteria at the starting and

24 h or 48 h time points were used to determine the competitive index (C.I.) for each competition experiment, following the calculation shown below,

��ℎ�� /�� = !" !" ��ℎ��!#/��!# with tx representing either the 24 h or 48 h time points, and t0 representing starting time point. A positive Log10 C.I. indicates the inhibitor strain outcompeted the target strain, a negative Log10

C.I. indicates the target strain outcompeted the inhibitor strain, and a Log10 C.I. » 0 indicates no

45 competition in favor of either strain.

b-galactosidase activity assays.

Cells from overnight cultures were washed in PBS and diluted to an OD600 value of 0.2.

20 µL spots of cell suspensions were plated on LSLB agar, and after spots had dried, cells were incubated at 37˚C for 48 h. For the co-culture with BdAU0158 bcp-3C and BdAU0158 attTn7::Pbcp-3-lacZ, strains were mixed at a 1:1 ratio, 20 µL spots were plated onto LSLB, and incubated at 37˚C for 48 h. Following incubation, entire colony biofilms were resuspended in 1 mL PBS, diluted 1:10 in Z-buffer + 0.27% b-mercaptoethanol (Fisher Scientific), and 250 µL were removed to measure OD600 values. To permeabilize cells, 50 µL chloroform and 10 µL

0.1% sodium dodecyl sulfate were added to the remaining 750 µL cell suspensions, samples were vortexed, and allowed to settle. 50 µL permeabilized cells and 50 µL 4 mg/mL ortho-

Nitrophenyl-b-galactoside were added to 150 µL Z-buffer, and OD420 values were measured every minute over a 20-minute time course. OD420 values for two time points within the linear range and the corresponding change in time were used to calculate b-galactosidase activity with the following formula:

Δ�� -�� = $%# × 1000 Δ� × 0.05 �� × ��&##

Z-buffer was prepared (per litre) as follows: 50 mM Na2HPO4 anhydrous, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4 (anhydrous).

Intracellular toxicity assays.

E. coli BL21(DE3) cells harboring IPTG-inducible plasmids were grown overnight in

LB+kanamycin. Cells were washed in PBS, subcultured in 50 mL LSLB+kanamycin at a starting

OD600 of 0.02 (with D-glucose added at a final concentration of 0.2% for non-induced cultures), and grown at 37˚C with aeration for 7 h. Samples were taken hourly to determine OD600 values

46 of the cultures, as well as to plate serial dilutions on LB agar+kanamycin supplemented with

0.2% D-glucose to determine cell viability. At the 2 h time point, IPTG was added at a final concentration of 0.5 mM to induce expression of bcp constructs. Production of BcpA-1-CT and

BcpI-1 was assessed via separation in a 12% SDS-PAGE gel and Coomassie staining.

Aggregation assays.

Cells from overnight cultures were washed in PBS and inoculated into 2 mL M63 minimal medium (3 g/L KH2PO4, 7 g/L K2HPO4, 2 g/L (NH4)2SO4, 0.5 g/L FeSO4, 0.2% glucose,

1 mM MgSO4, 0.4% glycerol, 0.01% casamino acids) at a starting OD600 of 0.2. Aggregation cultures were grown on a rotator drum at 37˚C for 24 h. After 24 h, culture tubes were taken off the rotator drum, incubated statically at 25˚C for ~30 minutes (min) to allow cells to settle, and the OD600 values of settled cultures were measured. The tubes were then vigorously vortexed to homogenize the cultures, and the OD600 values of vortexed cultures were measured.

Pigment production assays.

Cells from overnight cultures were washed twice in PBS, diluted to an OD600 value of

0.2, and 20 µL spots were plated onto LSLB agar. After spots had dried, plates were wrapped in paraffin film and incubated at 25˚C for 14 days (d).

Bioinformatic analyses.

BtE264 BcpB homologs were identified using the National Center for Biotechnology

Information Blastp suite. Burkholderia strains harboring the same BcpA-CT as the allele shared by BtE264 and BdAU0158 were identified using the Burkholderia Genome Database (31). ORF predictions were conducted with the Geneious 8 software package (32), and genome comparisons using the Mauve plugin (33) were conducted on Geneious 8. Protein alignments were conducted using the Clustal Omega online server (34). Protein structure predictions were

47 conducted using the Phyre2 online server (35). Signal sequence identification was performed using the SignalP 4.1 online server (36).

2.5 Results

The Burkholderia dolosa AU0158 (BdAU0158) genome contains three bcpAIOB alleles.

By searching for homologs of the BtE264 bcpB gene, we previously detected two bcpAIOB alleles in BdAU0158 (17). Further investigation revealed a third bcpAIOB locus in

BdAU0158. All three loci resemble Burkholderia-type CDI system-encoding genes, with the gene order being bcpAIOB and the presence of a fourth ORF, bcpO, between bcpI and bcpB.

We will refer to these bcpAIOB loci as bcp-1 (locus tags AK34_RS22045-AK34_RS22035, chromosome 1), bcp-2 (locus tags AK34_RS06120-AK34_RS06110, chromosome 2), and bcp-

3 (locus tags AK34_RS04315-AK34_RS04310, chromosome 2) (Figure 2.1A). The BdAU0158 bcp-1 allele belongs to the Class I family of bcpAIOB alleles, whereas the BdAU0158 bcp-2 and

BdAU0158 bcp-3 alleles belong to the Class II family. Accordingly, the BcpA proteins encoded by the bcp-2 allele (BcpA-2) and the bcp-3 allele (BcpA-3) are more similar to one another than they are to the BcpA protein encoded by the bcp-1 allele (BcpA-1) (Figure 2.1A). Strikingly, the

BdAU0158 bcp-1 allele is highly similar to the bcp allele of BtE264 (Figure 2.1B), which we previously determined encodes a CDI system in BtE264 (17). The BdAU0158 BcpA-1 and the

BtE264 BcpA proteins share 83.0% amino acid identity overall, with 86.4% amino acid identity at the C termini (CTs). By contrast, the BdAU0158 BcpA-2 and BcpA-3 proteins share 79.7% identity overall, with only 19.9% similarity between their CTs (Figure 2.1B). Phyre2 analysis (35) predicts all three BdAU0158 BcpA-CTs to be nucleases. All three BdAU0158 bcp loci contain potential bcpO genes, with the gene of the bcp-1 locus (bcpO-1) being nearly identical to

BtE264 bcpO (BdAU0158 BcpO-1 and BtE264 BcpO share 94.4% identity at the amino acid level after removal of the signal sequence). ORF prediction software (32) detects two potential

ORFs in the intergenic region between bcpI-2 and bcpB-2, and three potential ORFs in the

48 intergenic region between bcpI-3 and bcpB-3 (Figure 2.1A).

The BdAU0158 bcp-1 and bcp-2 loci encode functional CDI systems.

To determine if the bcp loci of BdAU0158 encode functional CDI systems, mutant strains containing unmarked, in-frame deletions lacking all but the first three codons of bcpA and the last three codons of bcpB in each of the bcp loci were generated (∆bcp-1, ∆bcp-2, and ∆bcp-3), and these mutants were competed against WT BdAU0158. All competitions were conducted on

LSLB agar for 48 h at 37˚C with 1:1 initial inhibitor:target ratios. WT BdAU0158 outcompeted the

∆bcp-1 mutant by approximately 4 logs (Figure 2.2A) and outcompeted the ∆bcp-2 mutant by approximately 2.5 logs (Figure 2.2B). To determine if the competitive exclusion in favor of WT

BdAU0158 was CDI-dependent, the ∆bcp-1 and ∆bcp-2 mutants were complemented at the attTn7 site with either cognate bcpI genes (bcpI-1 for the ∆bcp-1 mutant, bcpI-2 for the ∆bcp-2 mutant) or heterologous bcpI genes (bcpI-2 for the ∆bcp-1 mutant, bcpI-1 for the ∆bcp-2 mutant), with all bcpI genes present in trans under the control of the constitutive promoter of the

BtE264 ribosomal S12 subunit gene (PS12). The ∆bcp-1 mutant was only rescued from killing by

WT BdAU0158 (Log10 C.I.»0) when provided the cognate bcpI-1 gene in trans (Figure 2.2A), and the ∆bcp-2 mutant was only rescued from killing by WT BdAU0158 when provided its cognate bcpI-2 gene in trans (Figure 2.2B). We do not observe growth rate differences between

WT BdAU0158 and the ∆bcp mutants during in vitro growth, and the lack of competition between WT inhibitor strains and target mutant strains complemented with cognate bcpI genes suggest the ability of WT BdAU0158 to outcompete the ∆bcp-1 and ∆bcp-2 strains is solely due to CDI. These data indicate that the BdAU0158 bcp-1 and bcp-2 loci encode functional CDI systems that can kill or inhibit the growth of neighboring cells lacking cognate BcpI proteins.

Unlike the BdAU0158 ∆bcp-1 and ∆bcp-2 mutant strains, the ∆bcp-3 mutant was not outcompeted by WT during 48 h co-culture on solid medium (Figure 2.2C). Two possible explanations for this finding are that the bcp-3 locus does not encode a functional CDI system,

49 or that the bcp-3 locus is not expressed under the in vitro competition conditions used.

Unlike the BdAU0158 bcp-1 and bcp-2 loci, the bcp-3 locus is not expressed under in vitro competition conditions.

To investigate expression of the BdAU0158 bcp-1, bcp-2, and bcp-3 loci, promoter-lacZ fusions (Pbcp-1-lacZ, Pbcp-2-lacZ, and Pbcp-3-lacZ) were constructed and delivered to the attTn7 site of BdAU0158. Reporter strains were grown in monoculture under the same conditions used for competition experiments (LSLB agar at 37˚C for 48 h), and b-galactosidase activity was measured. Consistent with results from the competition experiments, the bcp-1 and bcp-2 promoters were active under these conditions (Figure 2.3A). Pbcp-1 was more active than Pbcp-2, which suggests the bcp-1 locus is expressed to a higher degree than the bcp-2 locus, which may explain why Bcp-1-mediated CDI is more potent than Bcp-2-mediated CDI (Figures 2.2A and 2.2B). b-galactosidase activity assays showed that Pbcp-3 is not active under the conditions used for competitions (Figure 2.3A), providing an explanation for why WT BdAU0158 did not outcompete the ∆bcp-3 mutant.

The BdAU0158 bcp-3 locus encodes a functional CDI system.

To determine if the BdAU0158 bcp-3 locus encodes a functional CDI system, a strain constitutively expressing the locus (bcp-3C) was generated by replacing the native bcp-3

C promoter region with the PS12 constitutive promoter. BdAU0158 bcp-3 outcompeted the ∆bcp-3 mutant by 5 logs (Figure 2.3B), with one competition resulting in no ∆bcp-3 cells being recovered from the co-culture (red-filled circle on Figure 2.3B). The BdAU0158 ∆bcp-3 mutant was protected from killing by BdAU0158 bcp-3C when bcpI-3 was supplied in trans. Surprisingly,

C WT BdAU0158 cells were not outcompeted by BdAU0158 bcp-3 , even though the native Pbcp-3 appears to be inactive under these conditions (Figures 2.3A and 2.3B). We hypothesized three

50 scenarios to explain this result: (1) low-level expression of the bcp-3 locus occurs that cannot be detected by promoter activity assays but leads to sufficient BcpI-3 production to resist Bcp-3- mediated CDI, (2) CDI attack induces bcp-3 expression in target cells, or (3) an internal promoter in the bcp-3 locus exists that separately drives expression of bcpI-3.

An internal promoter in the BdAU0158 bcp-3 locus separately drives expression of bcpI-

To investigate the possibility that CDI induces bcp-3 expression in target cells, the

C BdAU0158 Pbcp-3-lacZ reporter strain was mixed at a 1:1 ratio with the BdAU0158 bcp-3 inhibitor strain (which produces all three CDI systems), and this co-culture was incubated at

37˚C for 48 h on LSLB agar. The Pbcp-3-lacZ reporter strain is not susceptible to killing via CDI as it contains the bcpI genes of all three bcp loci. b-galactosidase activity in the BdAU0158 Pbcp-3- lacZ reporter strain was no greater after co-culture with the BdAU0158 bcp-3C inhibitor strain than after mono-culture (Figures 2.3A and 2.3C). These results indicate that CDI attack (Bcp-1-,

Bcp-2-, or Bcp-3-mediated) does not induce bcp-3 expression in a target cell.

To determine if an internal promoter resides in the BdAU0158 bcp-3 locus that drives expression of bcpI-3, the last 500 bp of bcpA-3 (the sequence immediately upstream and including the bcpI-3 start codon) were cloned into the lacZ expression cassette and the cassette was delivered to the BdAU0158 chromosome, generating the reporter strain BdAU0158 attTn7::PbcpI-3-lacZ. This strain produced ~1500 units of b-galactosidase activity after 48 h growth at 37˚C on LSLB agar (Figure 2.3C), indicating that a promoter (PbcpI-3) resides at the 3’ end of the BdAU0158 bcpA-3 gene that appears to drive expression of bcpI-3, allowing for production of the BcpI-3 antitoxin even when the remainder of the bcp-3 locus is not expressed.

51 CDI attack and resistance to CDI do not require the BdAU0158 BcpO proteins.

A characteristic of Burkholderia-type CDI system-encoding loci is the presence of a fourth ORF, bcpO. The function(s) of BcpO proteins have not been determined, although a

BtE264 ∆bcpO mutant is partially defective at outcompeting BtE264 ∆bcpAIOB via CDI (17), indicating that the BtE264 BcpO protein is important for CDI but is not required for this activity.

The BcpO protein predicted to be encoded by the BdAU0158 bcp-1 locus (BcpO-1) shares

90.4% identity at the amino acid level with BtE264 BcpO. There are two and three predicted

ORFs between bcpI and bcpB in the BdAU0158 bcp-2 and bcp-3 loci, respectively (Figure

2.1A).

Strains containing unmarked, in-frame deletion mutations in each of the BcpO-encoding regions of BdAU0158 were generated, resulting in ∆bcpO-1, ∆bcpO-2, and ∆bcpO-3 mutants

(deletion schematics shown in Figure 2.4). These mutants were competed on LSLB agar for 48 h at 37˚C against their parental, bcp-intact strains or their respective locus deletion mutants to determine if the BdAU0158 BcpO proteins are required for resistance to CDI or for the ability to kill target cells via CDI, respectively. In accordance with the effect of BcpO on BtE264 CDI (17), the BdAU0158 ∆bcpO-1 mutant had approximately a 1-log defect in CDI-mediated killing of the

BdAU0158 ∆bcp-1 target strain compared to WT. However, BcpO-1 was not required for resistance to Bcp-1-mediated killing, as the ∆bcpO-1 mutant was not outcompeted by WT

BdAU0158 (Figure 2.4A). BcpO-2 was not important for CDI in BdAU0158, as the ∆bcpO-2 mutant outcompeted the ∆bcp-2 mutant as well as WT outcompeted ∆bcp-2, and the inability of

WT to outcompete the ∆bcpO-2 mutant shows that BcpO-2 was not required for resistance to

Bcp-2-mediated CDI (Figure 2.4B). Competitions investigating the role of BcpO-3 had to be conducted in the bcp-3C background to ensure these genes were expressed. As shown in

Figure 2.4C, BdAU0158 bcp-3C and the bcp-3C strain lacking bcpO-3 (bcp-3C∆bcpO-3) were equally able to outcompete ∆bcp-3 targets, indicating that BcpO-3 was not required for Bcp-3- mediated CDI. Given that target cells with the native bcp-3 locus promoter are not outcompeted

52 by the constitutively-expressing strain (Figure 2.3B), a ∆bcpO-3 mutant in the WT background was used to determine if BcpO-3 is required for resistance to CDI. In agreement with results from competitions investigating BcpO-1 and BcpO-2, the BdAU0158 ∆bcpO-3 mutant was not susceptible to CDI by a BdAU0158 bcp-3C inhibitor (Figure 2.4C). In fact, the ∆bcpO-3 mutant had a slight growth advantage compared to the inhibitor (similar to the ∆bcp-3 attTn7::bcpI-3 and WT targets in Figure 2.3B), possibly due to the energetic cost of constitutively producing the

Bcp-3 proteins in the bcp-3C strain. Together, these data suggest that the BcpO proteins of

BdAU0158 do not play a crucial role in CDI.

BdAU0158 does not exhibit Bcp-dependent community behaviors.

Given that BdAU0158 produces three CDI systems, one of which is identical to the

BtE264 CDI system, we hypothesized that BdAU0158 could exhibit similar Bcp-dependent cooperative behaviors (i.e., contact-dependent signaling [CDS]) as those seen in BtE264, and that these behaviors may promote infection of the CF respiratory tract. We investigated autoaggregation and pigment production by WT BdAU0158, a panel of mutants that lack one

CDI system (∆bcp-1, ∆bcp-2, and ∆bcp-3) or all three CDI systems (∆∆∆), and the strain constitutively expressing the bcp-3 locus (bcp-3C). Autoaggregation of liquid cultures that were grown rotating for 24 h at 37˚C in minimal medium was assessed by measuring the OD600 value of a culture after sitting stationary at 25˚C for ~30 minutes and the OD600 value of the same culture following vigorous vortexing. A vortexed:settled OD600 ratio greater than 1 indicates that autoaggregation of cells occurred during liquid growth (as seen with WT BtE264 in Figure 2.5A), whereas a vortexed:settled OD600 ratio of ~1 indicates that cells did not autoaggregate (as seen with BtE264 ∆bcpAIOB in Figure 2.5A). All BdAU0158 strains had vortexed:settled OD600 ratios of ~1, suggesting that BdAU0158 does not autoaggregate under these conditions and that the production of CDI systems, or lack thereof, does not influence this phenotype. Pigment production was assessed by growth of BdAU0158 cells on LSLB agar for 48 h at 37˚C, and

53 subsequent incubation at 25˚C for up to 14 d. Under these conditions, WT BtE264 produces a dark beige pigment, whereas the BtE264 ∆bcpAIOB mutant remains white (Figure 2.5B). WT

BdAU0158 colony biofilms appear darker than those of the BtE264 ∆bcpAIOB mutant, though this coloration is not dependent on the Bcp proteins, as mutants lacking bcp loci and the bcp-3C strain appear similar in color to WT BdAU0158 (Figure 2.5B). These results indicate that

BdAU0158 does not perform Bcp-dependent cooperative behaviors similar to those seen in

BtE264; however, it is possible that the BdAU0158 Bcp proteins do mediate community-based phenotypes, and that the assays used to detect BtE264 CDS and its associated phenotypes cannot detect such behaviors in BdAU0158.

Toxicity of BdAU0158 CDI toxins in E. coli.

To determine if the BdAU0158 BcpA-1-CT, BcpA-2-CT, and BcpA-3-CT are sufficient for toxicity, inducible expression plasmids were generated as derivatives of pET-28(a). Each plasmid contained nucleotide sequences encoding one BcpA-CT (Nx(E/Q)LYN motif through

BcpA stop codon), a CT plus its cognate BcpI (Nx(E/Q)LYN motif through BcpI stop codon), or one BcpI alone. E. coli BL21(DE3) cells harboring these plasmids were grown in LSLB broth and expression was either induced with isopropyl b-D-1-thiogalactopyranoside (IPTG) or repressed by D-glucose. Over the time course, OD600 was measured and aliquots were plated on solid medium to assess cell viability. To our surprise, production of the BdAU0158 BcpA-1-

CT was not toxic in E. coli, but production of BcpI-1 was slightly toxic (Figure 2.6B). We detected production of both the BcpA-1-CT and BcpI-1 by E. coli using sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) (Figure 2.9). Although induction of bcp-1 did not cause a decrease in OD600 (Figure 2.6A), indicating cells were not lysing, the viability of

BcpI-1-producing cells over time decreased compared to cells producing either BcpA-1-CT or

BcpA-1-CT and BcpI-1 concurrently (Figure 2.6B). Cells producing BcpA-1-CT and BcpI-1 concurrently were protected from the toxic effects of BcpI-1, suggesting that binding of the CT to

54 BcpI-1 inhibits toxicity of the immunity protein. We hypothesize that the portion of BdAU0158

BcpA-1-CT being produced in E. coli is either larger or smaller than the true toxic protein, and that improper folding or processing of the CT inhibits its toxic effects. The fact that BcpI-1 is toxic in E. coli is more perplexing, as it is not toxic when produced in BdAU0158 (Figure 2.2A).

Phyre2 analysis (35) predicts that BcpI-1 contains a DNA-binding domain. One possibility is that when BcpI-1 is produced in excess without its cognate BcpA-1-CT, it may have deleterious effects in E. coli via interactions with genomic DNA.

Unlike BdAU0158 BcpA-1-CT, BcpA-2-CT and BcpA-3-CT were toxic when produced in

E. coli (Figures 2.6D and 2.6F). Production of BcpA-2-CT caused the OD600 to plateau (Figure

2.6C) and resulted in a 4-log decrease in cell viability (Figure 2.6D). BcpA-2-CT-producing cells that concurrently produced BcpI-2 were partially rescued from the toxic effects of the BcpA-2-

CT, and production of BcpI-2 alone had no deleterious effects on E. coli other than the presumed energetic cost of protein production (Figure 2.6D). Similar results were found for bcp-

3; induction did not cause a decrease in OD600 (Figure 2.6E), but BcpA-3-CT production caused a drastic decrease in cell viability that was partially rescued by concurrent production of BcpI-3

(Figure 2.6F). BcpI-3 appears to protect against the toxic effects of the BcpA-3-CT less well than BcpI-2 protects against the effects of BcpA-2-CT (Figure 2.6D vs Figure 2.6F), though at least partial protection was detected in each case. In these co-producing strains, the BcpA-CTs and BcpI proteins are presumably produced at 1:1 ratios, which may be the reason complete protection against CT-induced toxicity was not detected. It is possible that stoichiometry needs to favor the immunity proteins in order to fully inhibit autotoxicity – a situation that may occur under normal conditions as BcpA proteins are exported out of the cell, or may occur if bcpI- specific promoters are widespread throughout bcpAIOB loci.

The BtE264 Bcp and BdAU0158 Bcp-1 alleles are functionally redundant.

The similarity of the predicted amino acid sequences of BtE264 BcpA and BdAU0158

55 BcpA-1 (Figure 2.1B) suggests they are functionally redundant. Previous work in our laboratory identified residues required for the catalytic activity of BtE264 BcpA-CT (E3064 and K3066) and substituting alanines for these residues abrogated CDI activity (20). Glutamate and lysine residues are found at the same positions in BdAU0158 BcpA-1-CT (Figure 2.7A). In addition to the similarities of BtE264 BcpA-CT and BdAU0158 BcpA-1-CT, BtE264 BcpI and BdAU0158

BcpI-1 are nearly identical (Figure 2.7B). To investigate functional interchangeability, BtE264

∆bcpAIOB and BdAU0158 ∆bcp-1 mutants were provided with the heterologous bcpI gene delivered to the attTn7 site, generating BdAU0158 ∆bcp-1 attTn7::bcpIE264 and BtE264

∆bcpAIOB attTn7::bcpIAU0158-1. During 48 h co-culture on LSLB agar at 37˚C, the BdAU0158

∆bcp-1 mutant was rescued from Bcp-1-mediated CDI by the BdAU0158 WT inhibitor when it constitutively expressed the BtE264 bcpI gene (Log10 C.I.»0) (Figure 2.7C). Similarly, during 24 h co-culture on LSLB agar at 25˚C, the BtE264 ∆bcpAIOB mutant was rescued from Bcp- mediated CDI by the BtE264 WT inhibitor when it constitutively expressed the BdAU0158 bcpI-1 gene (Log10 C.I.»0) (Figure 2.7D). The ability of each BcpI protein to protect against CDI in the heterologous species provides experimental evidence that BdAU0158 and BtE264 share the same CDI system-encoding allele.

2.6 Discussion

CDI systems are present in a broad range of Gram-negative bacteria, including many that are pathogenic for animals or plants. Most studies of CDI function have used Escherichia coli strain EC93 as a model for E. coli-type systems or Burkholderia thailandensis strain E264 as a model for Burkholderia-type systems (15-17, 19, 22, 23, 37-45). Some experiments have used E. coli or B. thailandensis strains producing chimeric CdiA or BcpA proteins with toxin domains (and cognate immunity proteins) from pathogens (16, 19, 22, 23, 46), with a smaller number, limited to E. coli-type systems, investigating CDI in the pathogen itself (41, 47, 48). In this work, we used the epidemic Bcc isolate BdAU0158 and showed that its three distinct CDI

56 systems, including two Class II Burkholderia-type systems, are capable of killing and/or arresting the growth of neighboring bacteria. Whole genome sequence exists for two additional

B. dolosa strains, PC543 and LO6 (also referred to as B. cepacia strain LO6). The genomes of these strains contain three bcpAIOB loci, and the potential proteins encoded by these loci are

100% identical at the amino acid level to the Bcp-1, Bcp-2, and Bcp-3 proteins of BdAU0158, suggesting that CDI by B. dolosa is not limited to strain BdAU0158.

While the BdAU0158 Bcp-1 and Bcp-2 CDI systems mediated interbacterial competition under laboratory conditions, Bcp-3-mediated CDI was only detected when the region 5’ to the start of bcpA-3 was replaced with the constitutively active S12 promoter. These results are consistent with our lacZ reporter fusion analyses. The 500 bp and 300 bp DNA fragments corresponding to the regions immediately 5’ to bcpA-1 and bcpA-2, respectively, resulted in substantial b-galactosidase activity when present upstream of lacZ, while the corresponding fragment from bcp-3 resulted in no detectable b-galactosidase activity under the same laboratory conditions. Together with the fact that the gene 5’ to bcpA-3 is oriented in the opposite direction, the most likely explanation for the results we obtained for bcp-3 is that the

500 bp fragment does contain the promoter for bcp-3, but that this promoter is regulated such that it is not activated under standard laboratory conditions. Little is known about the regulation of any CDI system-encoding genes. In E. coli-type systems, the cdiBAI genes are expressed under laboratory conditions only in strain EC93 (15, 16, 47), and in B. thailandensis E264, the bcpAIOB genes appear to be tightly regulated such that only about one in 1,000 bacteria express the genes at a high level under laboratory conditions (17). Future experiments will be aimed at identifying transcription start sites and investigating how and why the bcp loci are differentially regulated in BdAU0158.

Because the bcp-3 locus appears to be transcriptionally silent under standard laboratory growth conditions, we were surprised to find that WT BdAU0158 was not outcompeted by the

57 strain expressing bcp-3 constitutively (BdAU0158 bcp-3C). This result led us to search for a promoter for bcpI-3 within the 3’ end of bcpA-3. Although the transcription start site has yet to be determined, the region immediately 5’ to bcpI-3 was sufficient to drive lacZ expression, suggesting that transcription initiation in this region in the native locus results in sufficient BcpI protein production to confer protection from BcpA-3-mediated toxicity. While it would seem to be advantageous for bacteria to produce all immunity proteins at a low level constitutively, our study is the first demonstration, to our knowledge, of a promoter for bcpI (or cdiI) within a bcpAIOB (or cdiBAI) operon that is independent of that driving transcription of the rest of the operon. For E. coli-type CDI systems, “orphan” cdiA-CT/cdiI modules have been identified that encode functional CdiA-CT toxins and CdiI immunity proteins but are located outside cdiBAI loci

(37). These orphan modules, which exist in the genomes of several pathogens containing CDI system-encoding genes (37, 49), share similarities with recombination hotspot (rhs) loci and can contribute to diversification of the CDI systems in these species via recombination with the cdiBAI genes. There is no evidence that promoters exist for these orphan modules or the corresponding orphan cdiI genes (37), and orphan bcpA-CT/bcpI modules have not been detected in Burkholderia spp.

The role of the additional ORF between bcpI and bcpB in Burkholderia-type CDI system- encoding loci (which we named bcpO) remains enigmatic. In Class I Burkholderia-type loci, the bcpO genes are highly homologous. They are predicted to encode small lipoproteins that lack localization of lipoproteins (Lol) avoidance signals, suggesting they localize to the inner leaflet of the outer membrane. The N-terminal halves of the 53 aa mature polypeptides are rich in prolines and the C-terminal halves are rich in tryptophans. As with BtE264 (17), deletion of bcpO-1 in BdAU0158 resulted in a modest decrease in CDI activity, but the mechanism underlying this phenotype is unknown. Our investigations into whether BcpO contributes to CDS or cooperative behaviors have yielded inconclusive results so far. The “bcpO” genes in Class II

Burkholderia-type loci bear little similarity to each other or to bcpO genes in Class I alleles and

58 hence should probably be renamed. Deletion of these ORFs in BdAU0158 bcp-2 and bcp-3 had no effect on CDI activity under the conditions tested.

Although we did not detect CDS in BdAU0158, the assays used for these experiments were developed to describe CDS in BtE264 (24) and thus may not be specific for Bcp-mediated community behaviors in other strains. Future investigation into CDS by BdAU0158 and other

Burkholderia spp. containing bcpAIOB loci is warranted. Given the growing appreciation for the impact of bacterial cooperation on pathogenesis (50-54), the virulence of diverse Gram-negative bacterial pathogens may be influenced by CDS.

Production of BdAU0158 BcpA-2-CT and BcpA-3-CT by E. coli resulted in autotoxicity that was partially ablated by concurrent production of BcpI-2 and BcpI-3, respectively.

Conversely, E. coli producing BcpA-1-CT did not exhibit reduced viability, despite the likelihood that this polypeptide contains the toxic domain of BcpA-1. For some E. coli-type CDI systems, cytoplasmic “permissive factors” are required for activity of a delivered toxin within a target cell

(38, 42, 43). A requirement for a toxicity-promoting factor specific to Burkholderia, or to

BdAU0158, may explain the lack of toxicity of BdAU0158 BcpA-1-CT in E. coli. Alternatively, it is possible that the BcpA-1-CT polypeptide that we selected to produce in E. coli BL21(DE3) is not the toxic molecule delivered by the BdAU0158 Bcp-1 CDI system. Indeed, the precise BcpA or

CdiA polypeptide that is delivered to the cytoplasm of target cells, and whether it is modified in any way, is not known for any CDI system. We are currently conducting experiments to identify the BcpA polypeptides that are delivered during CDI and CDS in BtE264 and other Burkholderia strains.

Also unexpected was the finding that production of BdAU0158 BcpI-1 was toxic in E. coli. BcpI-1 is not toxic when produced in BdAU0158 and the nearly identical BtE264 BcpI protein is not toxic when produced in BtE264 (17). Phyre2 analysis predicts a DNA-binding domain in BcpI-1. A possible explanation for its toxicity when massively over-produced in E. coli is that it interacts with genomic DNA in a way that blocks an essential function, such as

59 replication or transcription of an essential gene(s). Whether BcpI from either BdAU0158 or

BtE264 actually binds DNA is unknown, but would be consistent with a role for BcpI, in complex with BcpA-CT, in controlling changes in gene expression during CDS (18, 24).

The high degree of similarity between bcp-1 of BdAU0158 and bcpAIOB of BtE264, and the functional redundancy of the BcpA (24) and BcpI proteins (this work), suggests that these genes represent the same allele. Genomic analyses suggest that both Burkholderia- and E. coli- type CDI system-encoding genes reside on genomic islands that were mobile at some time in the past, and perhaps still are (55-59). We identified 11 Burkholderia strains potentially harboring the same CDI system as BdAU0158 and BtE264 by searching for orthologs of

BdAU0158 BcpA-CT and BcpI (Figure 2.8). The Mauve software package (33) detected evidence of synteny surrounding the bcpAIOB loci between the three B. dolosa strains

(BdAU0158, BdPC543, and BdLO6). Close examination of these orthologous BcpA-CT and

BcpI proteins revealed that although they are highly similar across the different strains, variation in the amino acid sequence exists in the C-terminal portion of BcpA-CT (extreme CT, BcpA-

ECT) and the N-terminal portion of BcpI (BcpI-NT) (Figure 2.8). We separated these strains into three groups – the BdAU0158/BtE264 group, the BgluBGR1 group, and the BtE444 group – based on the degree of variation of the BcpA-ECTs and BcpI-NTs compared to the BdAU0158

BcpA-ECT and BcpI-NT, respectively. Variation in BcpA-ECT and BcpI-NT sequence, along with the lack of synteny between the regions surrounding bcpAIOB in these strains, suggests that if these genes were acquired horizontally, there has been substantial evolution since that time. Though we demonstrated that the BcpA and BcpI proteins of BdAU0158 and BtE264 function in the heterologous species ((24) and this work), it is unknown whether that holds true for all strains on this list. We hypothesize that the C-terminus of BcpI is required for protection against toxicity of the BcpA-CT, given that the BcpI proteins of BdAU0158 and BtE264 block

BcpA-CT toxicity in the heterologous species despite exhibiting BcpI-NT sequence variation.

Though purely speculative, perhaps these variable regions of BcpA-CT and BcpI are important

60 for CDS, and that delivery of an “identical” BcpA-CT, from a CDI standpoint, will not elicit CDS in a target cell producing a variable BcpI. Future comparative analyses of these variable alleles will be informative to the mechanisms of both CDI and CDS.

Aside from the investigation of P. aeruginosa CDI by Melvin et al. (48), our study is the only demonstration of multiple functional CDI systems in a single pathogenic species. Given the polymicrobial nature of the CF respiratory tract, CDI may provide BdAU0158, as well as P. aeruginosa, a competitive advantage during host infection. The genomes of strains of several

Bcc species, including B. cenocepacia, B. multivorans, and B. vietnamiensis, contain possible

CDI system-encoding genes and thus these pathogens may benefit from CDI activity during infection. Evolutionary genomic analysis of B. dolosa isolates from CF patients over a 16-year period (all originating from the Boston epidemic) revealed that identical nonsynonymous mutations arose in bcpA-2 across multiple patients, suggesting there is strong selective pressure promoting parallel adaptive evolution of this CDI-encoding gene within the human host

(26). Additionally, Bcc species are found ubiquitously in the environment, especially in soil (60), and thus BdAU0158 could employ its CDI systems to outcompete potential competitors in diverse settings. It is hypothesized that competitive behaviors are the strongest force shaping microbial ecology (5); therefore BdAU0158 and other Bcp-producing Bcc pathogens may utilize

CDI to gain a foothold in the complex polymicrobial environments of the CF respiratory tract and establish long-term, devastating infections in these patients.

Figure 2.1. bcpAIOB loci in BdAU0158. (A) Schematic of the BcpAIOB proteins encoded by the BdAU0158 bcp-1 (BdAU0158-1), bcp-2 (BdAU0158 bcp-2), and bcp-3 (BdAU0158-3) loci, as well as the BcpAIOB proteins encoded by the BtE264 bcp locus. Grey coloration corresponds to conserved regions of the proteins, whereas variable regions spanning BcpA-CT, BcpI, and BcpO are denoted in yellow (for the BtE264 and BdAU0158-1 alleles), purple (for the BdAU0158-2 allele), and blue (for the BdAU0158-3 allele). Potential BcpO proteins encoded by the BdAU0158 bcp-2 and bcp-3 loci are indicated by slashed boxes. (B) Amino acid alignments of BtE264 BcpA and BdAU0158 BcpA-1, and of BdAU0158 BcpA-2 and BdAU0158 BcpA-3. Residue similarity is denoted by greyscale, with black indicating identical residues and white indicating disparate residues. Black triangles above and below alignments represent Nx(E/Q)LYN motifs.

Figure 2.2. The Bcp-1 and Bcp-2 CDI systems provide BdAU0158 a competitive advantage during in vitro growth. (A) Competition assays between BdAU0158 WT inhibitor cells and ∆bcp- 1, ∆bcp-1 attTn7::bcpI-1, and ∆bcp-1 attTn7::bcpI-2 target cells. (B) Competition assays between BdAU0158 WT inhibitor cells and ∆bcp-2, ∆bcp-2 attTn7::bcpI-2, and ∆bcp-2 attTn7::bcpI-1 target cells. (C) Competition assays between BdAU0158 WT inhibitor cells and ∆bcp-3 and ∆bcp-3 attTn7::bcpI-3 target cells. For each competition, results from three separate biological replicates, each with three technical replicates (except for the WT vs ∆bcp-3 attTn7::bcpI-3 competitions in (C), which show two biological replicates, each with three technical replicates). Solid horizontal lines represent mean Log10C.I. values. Dotted line (Log10C.I. = 0) indicates no competitive advantage for inhibitor or target strain. ****P<0.0001, Mann-Whitney test.

Figure 2.3. The BdAU0158 bcp-3 locus encodes a functional CDI system that is not expressed under in vitro competition conditions. (A) b-galactosidase activity assays for promoters of the BdAU0158 bcp-1, bcp-2, and bcp-3 loci. A BdAU0158 strain harboring a constitutively expressed lacZ reporter (PS12-lacZ) served as a positive control, whereas a BdAU0158 strain harboring a lacZ reporter without a promoter (promoterless) served as a negative control. (B) Competition assays between BdAU0158 bcp-3C inhibitor cells and ∆bcp-3, ∆bcp-3 attTn7::bcpI- 3, and WT target cells. Dotted line (Log10C.I. = 0) indicates no competitive advantage for inhibitor or target strain. Red-filled circle indicates a competition from which no target cells were recovered following 48 h co-culture. (C) b-galactosidase activity assays for the BdAU0158 Pbcp-3 C C reporter strain co-cultured with BdAU0158 bcp-3 (bcp-3 vs Pbcp-3-lacZ), as well as the BdAU0158 PbcpI-3 reporter strain (PbcpI-3-lacZ), with PS12-lacZ and promoterless positive and negative controls, respectively, as in (A). b-galactosidase activity assays in (A) and (C) show results from two biological replicates, each with three technical replicates. ***P<0.001, ****P<0.0001, Unpaired t test. Competition assays in (B) show results from three biological replicates, each with three technical replicates. Solid horizontal lines represent mean Log10C.I. values. ****P<0.0001, Mann-Whitney test.

Figure 2.4. BdAU0158 BcpO proteins are not required for CDI-mediated competition or resistance to CDI. (A) Competition assays between BdAU0158 WT inhibitor cells and ∆bcp-1 target cells, BdAU0158 ∆bcpO-1 inhibitor cells and ∆bcp-1 target cells, and BdAU0158 WT inhibitor cells and ∆bcpO-1 target cells (right of dashed vertical line). (B) Competition assays between BdAU0158 WT inhibitor cells and ∆bcp-2 target cells, BdAU0158 ∆bcpO-2 inhibitor cells and ∆bcp-2 target cells, and BdAU0158 WT inhibitor cells and ∆bcpO-2 target cells (right of dashed vertical line). (C) Competition assays between BdAU0158 bcp-3C inhibitor cells and ∆bcp-3 target cells, BdAU0158 bcp-3C∆bcpO-3 inhibitor cells and ∆bcp-3 target cells, and BdAU0158 bcp-3C inhibitor cells and ∆bcpO-3 target cells (right of dashed vertical line). bcpO gene deletion schematics are shown above each graph. For each competition, results from two separate biological replicates, each with three technical replicates. Solid horizontal lines represent mean Log10C.I. values. Dotted horizontal line (Log10C.I. = 0) indicates no competitive advantage for inhibitor or target strain. *P<0.05, Mann-Whitney test.

Figure 2.5. The Bcp-dependent community behaviors of autoaggregation and pigment production are not evident in BdAU0158. (A) Autoaggregation assays of BdAU0158 and BtE264 cultures grown in minimal medium, measured by determining the ratio of OD600 values of vortexed and settled cultures. Only BtE264 WT cells exhibit autoaggregation. Results from three separate biological replicates, with mean ratios plotted. Mean ratios compared to non- autoaggregating BtE264 ∆bcpAIOB to determine if cells autoaggregated. ****P<0.0001, Student’s t-test. n.s., not significant. (B) Pigment production assays of BdAU0158 and BtE264 colony biofilms grown on LSLB agar. Only BtE264 exhibits Bcp-dependent pigment production. Images are representative of at least three biological replicates.

Figure 2.6. E. coli autotoxicity due to inducible production of the BdAU0158 Bcp-2 and Bcp-3 CT toxins, but not the Bcp-1 CT toxin. (A, C, E) OD600 values of cultures of E. coli BL21(DE3) strains producing BdAU0158 Bcp CT toxins (red dotted lines), co-producing CT toxins and BcpI proteins (green dotted lines), and producing BcpI proteins (blue dotted lines) of the Bcp-1 (A), Bcp-2 (C), and Bcp-3 (E) CDI systems. (B, D, F) Cell viability, as measured by CFU/mL, of the Bcp-1 protein-producing (B), Bcp-2 protein-producing (D), and Bcp-3 protein-producing (F) E. coli BL21(DE3) cultures. Solid lines represent cultures in which BdAU0158 Bcp protein production was repressed by the addition of 0.2% glucose. For each intracellular toxicity assay, means from three biological replicates plotted.

Figure 2.7. The BdAU0158 Bcp-1 allele contains the same CT toxin-immunity pair as the BtE264 Bcp allele. (A) Amino acid sequence alignment of the BdAU0158 BcpA-1-CT and BtE264 BcpA-CT. The glutamate and lysine residues required for toxicity are boxed in orange. (B) Amino acid sequence alignment of the BdAU0158 BcpI-1 and BtE264 BcpI proteins. For (A) and (B), identical residues highlighted in black, similar residues highlighted in grey, and disparate residues not highlighted. (C) Competition assays between BdAU0158 WT inhibitor cells and ∆bcp-1 and ∆bcp-1 attTn7::bcpIE264 target cells. (D) Competition assays between BtE264 WT inhibitor cells and ∆bcpAIOB and ∆bcpAIOB attTn7::bcpIAU0158-1 target cells. For each competition, results from three separate biological replicates, each with three technical replicates. Solid horizontal lines represent mean Log10C.I. values. Dotted line (Log10C.I. = 0) indicates no competitive advantage for inhibitor or target strain. ****P<0.0001, Mann-Whitney test.

Figure 2.8. Orthologs of the BdAU0158 BcpA-CT and BcpI across several Burkholderia strains. Yellow indicates regions of amino acid sequence identity. Regions of amino acid sequence variation are indicated by shades of pink (BdAU0158/BtE264 group), purple (BgluBGR1 group), and green (BtE444 group), with percent identity to the corresponding BdAU0158 sequence shown below. Members of the BdAU0158/BtE264 group have variable sequences that are more similar to each other than they are to members of the BgluBGR1 and BtE444 groups. BdPC543, B. dolosa strain PC543; BdLO6, B. dolosa strain LO6 (also called B. cepacia LO6); BcATCC 25416, B. cepacia strain ATCC 25416; BtE254, B. thailandensis strain E254; Bt2002721723, B. thailandensis strain 2002721723; BgluBGR1, B. glumea strain BGR1; BglaATCC 25417, B. gladioli strain ATCC 25417; BtE444, B. thailandensis strain E444; BtPhuket 4W-1, B. thailandensis strain Phuket W-1; BtUSAMRU Malaysia #20, B. thailandensis strain USAMRU Malaysia #20; B. sp. MSMB1835, species unknown.

Figure 2.9. Production of BdAU0158 BcpA-1-CT and BcpI-1 by E. coli BL21(DE3) as visualized by Coomassie staining of whole cell lysates separated on a 12% SDS-PAGE gel. Cells were harvested after 6 h growth (4 h post IPTG-induction). The accumulation of BcpA-1-CT by the induced strains harboring the plasmid containing bcpA-1-CT (left two lanes) and the plasmid containing bcpA-1-CT and bcpI-1 (right two lanes) is indicated by the arrowhead. The wedge indicates production of BcpI-1, which only occurs in the induced strain harboring the plasmid containing bcpA-1-CT and bcpI-1. The sizes of the two indicated proteins match the predicted sizes of BcpA-1-CT and BcpI-1.

70 Table 2.1. Bacterial strains used in this study. Strain Genotype Source or reference B. dolosa BdAU0158 BcRLR* ∆bcp-1 Garcia et al. 2016 ∆bcp-2 Garcia et al. 2016 ∆bcp-3 This study ∆∆∆ BdAU0158 ∆bcp-1∆bcp-2∆bcp-3 This study BdAU0158 BdAU0158 attTn7::Km This study BdAU0158 BdAU0158 attTn7::Tet This study ∆bcp-1 BdAU0158 ∆bcp-1 attTn7::Km This study ∆bcp-1 BdAU0158 ∆bcp-1 attTn7::Tet This study ∆bcp-1 attTn7::bcpI-1 BdAU0158 ∆bcp-1 attTn7::bcpI-1-Km This study ∆bcp-1 attTn7::bcpI-2 BdAU0158 ∆bcp-1 attTn7::bcpI-2-Km This study ∆bcp-2 BdAU0158 ∆bcp-2 attTn7::Km This study ∆bcp-2 attTn7::bcpI-2 BdAU0158 ∆bcp-2 attTn7::bcpI-2-Km This study ∆bcp-2 attTn7::bcpI-1 BdAU0158 ∆bcp-2 attTn7::bcpI-1-Km This study ∆bcp-3 BdAU0158 ∆bcp-3 attTn7::Km This study ∆bcp-3 attTn7::bcpI-3 BdAU0158 ∆bcp-3 attTn7::bcpI-3-Km This study bcp-3C BdAU0158WpAP29 This study ∆bcp-3 BdAU0158 ∆bcp-3 attTn7::Tet This study ∆bcp-3 attTn7::bcpI-3-Tet BdAU0158 ∆bcp-3 attTn7::bcpI-3-Tet This study Pbcp-1-lacZ BdAU0158 attTn7::Pbcp-1-lacZ This study Pbcp-2-lacZ BdAU0158 attTn7::Pbcp-2-lacZ This study

Pbcp-3-lacZ BdAU0158 attTn7::Pbcp-3-lacZ This study

PS12-lacZ BdAU0158 attTn7::PS12-lacZ This study lacZ BdAU0158 attTn7::lacZ This study

PbcpI-3-lacZ BdAU0158 attTn7::PbcpI-3-lacZ This study

∆bcp-1 attTn7::bcpIE264 BdAU0158 ∆bcp-1 attTn7::bcpIE264-Tet This study ∆bcpO-1 BdAU0158 ∆bcpO-1 attTn7::Km This study ∆bcpO-2 BdAU0158 ∆bcpO-2 attTn7::Tet This study bcp-3C∆bcpO-3 BdAU0158WpAP29 ∆bcpO-3 This study ∆bcpO-3 BdAU0158 ∆bcpO-3 attTn7::Tet This study B. thailandensis BtE264 Brett et al. 1998 ∆bcpAIOB Anderson et al. 2012 BtE264 BtE264 attTn7::Cm Anderson et al. 2012 ∆bcpAIOB BtE264 ∆bcpAIOB attTn7::Km Anderson et al. 2012

∆bcpAIOB attTn7::bcpIAU0158-1 BtE264 ∆bcpAIOB attTn7::bcpIAU0158-1-Km This study E. coli BL21(DE3) pAP30 pET-28(a)::bcpA-1-CT This study BL21(DE3) pAP31 pET-28(a)::bcpA-1-CT—bcpI-1 This study BL21(DE3) pAP46 pET-28(a)::bcpI-1 This study BL21(DE3) pAP32 pET-28(a)::bcpA-2-CT This study BL21(DE3) pAP33 pET-28(a)::bcpA-2-CT—bcpI-2 This study BL21(DE3) pAP47 pET-28(a)::bcpI-2 This study BL21(DE3) pAP34 pET-28(a)::bcpA-3-CT This study BL21(DE3) pAP35 pET-28(a)::bcpA-3-CT—bcpI-3 This study BL21(DE3) pAP48 pET-28(a)::bcpI-3 This study *Burkholderia cepacia Research Laboratory and Repository, University of Michigan, Ann Arbor, MI USA.

71 Table 2.2 Plasmids used in this study. Plasmid Backbone Description Antibiotic Source or Resistance Reference pEXKm5 -- Allelic exchange vector Km López et al. 2009 pAP10 pEXKm5 To delete BdAU0158 bcp-3 Km This study pAP49 pEXKm5 To delete BdAU0158 bcpO-1 Km This study pAP50 pEXKm5 To delete BdAU0158 bcpO-2 Km This study pAP51 pEXKm5 To delete BdAU0158 bcpO-3 Km This study pUC18Tmini- -- To deliver KmR cassette to Amp, Km Choi et al. 2008 Tn7T-Km attTn7 site pUC18Tmini- pUC18Tmini-Tn7T- To deliver TetR cassette to Amp, Tet Anderson et al. Tn7T-Tet Km attTn7 site 2012 pTNS3 -- Helper plasmid to deliver Amp Choi et al. 2005 cassettes to attTn7 site pUCS12Km pUC18Tmini-Tn7T- To deliver PS12-driving Amp, Km Anderson et al. Km cassettes to the attTn7 site 2012 pAP3 pUCS12Km To deliver BdAU0158 bcpI-1 Amp, Km This study to attTn7 site pAP5 pUCS12Km To deliver BdAU0158 bcpI-2 Amp, Km This study to attTn7 site pAP42 pUC18Tmini-Tn7T- To deliver BdAU0158 bcpI-3 Amp, Tet This study Tet (constitutively expressed) to attTn7 site pAP29 pUCS12Km 1st 501 bp of BdAU0158 Amp, Km This study bcpA-3, used to generate BdAU0158 bcp-3C pMA43 pUC18Tmini-Tn7T- To deliver BtE264 bcpI Amp, Tet Anderson et al. Tet (constitutively expressed) to 2012 attTn7 site pUClacZ pUC18Tmini-Tn7T- To deliver promoterless lacZ Amp, Km Anderson et al. Km to attTn7 site 2012 pECG10 pUClacZ To deliver PS12-lacZ to attTn7 Amp, Km Anderson et al. site 2012 pAP23 pUClacZ 500 bp upstream of Amp, Km This study BdAU1058 bcp-1, to generate BdAU0158 Pbcp-1-lacZ reporter pAP22 pUClacZ 300 bp upstream of Amp, Km This study BdAU1058 bcp-2, to generate BdAU0158 Pbcp-2-lacZ reporter pAP24 pUClacZ 500 bp upstream of Amp, Km This study BdAU1058 bcp-3, to generate BdAU0158 Pbcp-3-lacZ reporter pAP61 pUClacZ Last 500 bp of BdAU1058 Amp, Km This study bcpA-3, to generate BdAU0158 PbcpI-3-lacZ reporter pET-28(a) -- IPTG-inducible expression Km Novagen vector pAP30 pET-28(a) BdAU0158 bcpA-1-CT Km This study pAP31 pET-28(a) BdAU0158 bcpA-1-CT—bcp- Km This study I-1 pAP46 pET-28(a) BdAU0158 bcpI-1 Km This study pAP32 pET-28(a) BdAU0158 bcpA-2-CT Km This study pAP33 pET-28(a) BdAU0158 bcpA-2-CT—bcp- Km This study I-2 pAP47 pET-28(a) BdAU0158 bcpI-2 Km This study pAP34 pET-28(a) BdAU0158 bcpA-3-CT Km This study pAP35 pET-28(a) BdAU0158 bcpA-3-CT—bcp- Km This study I-3 pAP48 pET-28(a) BdAU0158 bcpI-3 Km This study

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CHAPTER 3 – HOST ADAPTATION PREDISPOSES PSEUDOMONAS AERUGINOSA TO TYPE VI SECRETION SYSTEM-MEDIATED PREDATION BY THE BURKHOLDERIA CEPACIA COMPLEX1

3.1 Summary

Pseudomonas aeruginosa (Pa) and Burkholderia cepacia complex (Bcc) species are opportunistic lung pathogens of individuals with cystic fibrosis (CF). While Pa can initiate long- term infections in younger CF patients, Bcc infections only arise in teenagers and adults. Both

Pa and Bcc use type VI secretion systems (T6SS) to mediate interbacterial competition. Here, we show that Pa isolates from teenage/adult CF patients, but not those from young CF patients, are outcompeted by the epidemic Bcc isolate Burkholderia cenocepacia strain AU1054

(BcAU1054) in a T6SS-dependent manner. The genomes of susceptible Pa isolates harbor

T6SS-abrogating mutations, the repair of which, in some cases, rendered the isolates resistant.

Moreover, seven of eight Bcc strains outcompeted Pa strains isolated from the same patients.

Our findings suggest that certain mutations that arise as Pa adapts to the CF lung abrogate

T6SS activity, making Pa and its human host susceptible to potentially fatal Bcc superinfection.

3.2 Introduction

The respiratory tracts of individuals suffering from the genetic disorder cystic fibrosis

(CF) are hospitable environments for microorganisms, and thus CF patients harbor complex, dynamic microbial communities in their airways that can include opportunistic pathogens (1-4).

Pseudomonas aeruginosa and certain members of the Burkholderia cepacia complex (Bcc), a

1 Co-authors: Courtney E Chandler, David A Rasko, Robert K Ernst, Matthew C Wolfgang, Peggy A Cotter

78 taxonomic group containing at least 17 Burkholderia spp. (5), cause devastating infections in CF patients (4, 6, 7). While P. aeruginosa infects young CF patients and is the most common opportunistic CF pathogen by early adulthood, Bcc infections are less common and, for unknown reasons, limited to older CF patients, typically teenagers and adults (8). Unlike other

CF pathogens, Bcc strains are more frequently associated with person-to-person spread (9-11) and can progress to a fatal necrotizing pneumonia and bacteremia termed “cepacia syndrome”

(4, 12). While P. aeruginosa-Bcc co-infections occur within CF patients, the two pathogens do not colocalize. P. aeruginosa is mostly found extracellular in the airway lumen and Bcc within phagocytes; however, the P. aeruginosa burden in co-infected patients tends to be lower than in patients infected by P. aeruginosa alone (13).

Given the polymicrobial nature of the CF respiratory tract, interbacterial interactions likely occur in these tissues and may influence disease progression (1, 14-16). Interbacterial competition is hypothesized to be one of the strongest determinants of ecology and evolution within polymicrobial communities (17). A prevalent and well-understood mechanism of interbacterial competition is the type VI secretion system (T6SS) (18, 19), which is predicted to be present in ~25% of Gram-negative bacteria (20). T6SSs use a bacteriophage-like mechanism to deliver effector proteins directly into target bacterial or eukaryotic cells (21-24).

Antibacterial T6SS effectors disrupt diverse biological processes within target cells, and cognate immunity proteins protect T6SS-producing cells from autotoxicity (18, 25, 26). Type VI secretion

(T6S) has been studied in the Bcc pathogen Burkholderia cenocepacia strain J2315 (BcJ2315), which produces a T6SS that is important for infection of macrophages and influences the immune response to this pathogen (27-30). Recent bioinformatic analysis has detected T6SS- encoding genes throughout the Bcc, with one system (referred to as T6SS-1) predominating; however, several species encode multiple T6SSs (31). The B. cenocepacia strain H111 T6SS was shown to have modest antibacterial activity (31), though the potential antibacterial role of

T6SSs in other Bcc pathogens remains unknown.

79 P. aeruginosa produces three separate T6SSs (the H1-, H2-, and H3-T6SSs), and while both the H1- and H2-T6SSs are antibacterial weapons, the H1-T6SS is the stronger mediator of interbacterial competition (23, 32, 33). The P. aeruginosa H1-T6SS is under intricate regulation at both the post-transcriptional and post-translational level. Phosphorelay through the GacSA two-component system activates T6SS protein production via the regulatory small RNAs

(sRNAs) RsmY and RsmZ, which relieve RsmA-mediated repression of T6SS transcript translation (34-38). Moreover, a threonine phosphorylation pathway regulates T6SS assembly and function via signal transduction through the membrane-associated TagQRST proteins,

Fha1, and the kinase and phosphatase PpkA and PppA, respectively (39-42).

P. aeruginosa undergoes dramatic evolution within the CF respiratory tract to transition to a chronic infection lifestyle (43, 44), and evolutionary analyses have detected mutations in gacS/gacA as well as T6SS structural genes (45-47). Given Bcc infections establish later in the lives of CF patients compared to P. aeruginosa infections, we hypothesized that host adaptation by P. aeruginosa may open the door to subsequent Bcc infections if the resident P. aeruginosa community loses T6SS activity. In this study, we conducted experiments to test this hypothesis.

3.3 Results

The BcAU1054 T6SS mediates interbacterial competition

We selected Burkholderia cenocepacia strain AU1054 (BcAU1054), which was isolated from the bloodstream of a CF patient who succumbed to the infection, for our studies (9, 48).

The BcAU1054 genome encodes a predicted T6SS on chromosome 1 (BCEN_RS13060, tssM, through BCEN_RS13170, tssL) (Figure 3.1A). Presumably due to errors during the whole genome sequencing of this strain (assembly GCA_000014085.1), multiple genes in this region were annotated as pseudogenes. We PCR amplified and sequenced these regions and found that each gene is intact, encoding a full-length protein. Compared to the T6SS-encoding cluster of B. cenocepacia strain J2315 (BcJ2315), the BcAU1054 T6SS gene cluster contains an

80 additional region (BCEN_RS13075 (tai1) through BCEN_RS13110 (vgrG1)) that includes two predicted effector-immunity (E-I)-encoding gene pairs (tae1-tai1 and tle1-tli1) (Figure 3.2).

Immediately 3’ to the BcAU1054 core cluster (with the 5’ to 3’ direction corresponding to the sequence numbering in Figure 3.1A) is another additional region containing vgrG2, the predicted E-I-encoding pair tne1-tni1, and several genes predicted to encode proteins involved in phage or other mobile genetic elements.

To identify additional genes with the potential to encode E-I pairs, we first analyzed genes located near the seven annotated vgrG genes (Figure 3.1A). VgrG proteins, along with

PAAR-repeat proteins, form the puncturing tip of the T6SS needle and typically associate with effectors encoded by nearby genes (18, 21, 49). Based on previous nomenclature (18), we named predicted cell membrane-degrading effectors Tle for T6SS lipase effector, nucleic acid- degrading effectors Tne for T6SS nuclease effector, cell wall-degrading effectors Tae for T6SS amidase effector, and used Tpe for the T6SS pore-forming effector. We named cognate immunity proteins Tli, Tni, Tai, and Tpi. Three of the predicted E-I-encoding gene pairs (tle1-tli1, tle3-tli3, and tpe1-tpi1) are found near ORFs encoding proteins with the domain of unknown function (DUF) 4123, which is a highly conserved chaperone domain for T6SS effectors (50)

(Figure 3.1A). Protein secondary structure analysis using Phyre2 (51) and HHpred (52) predicted antibacterial enzymatic activities for all potential effectors (Table 3.1). Tae1 and Tle4 are not encoded by genes closely associated with vgrG genes, but have predicted antibacterial activities. A duplication of the 3’ end of tagL flanking tae1-tai1 (Figure 3.1A) suggests these predicted E-I-encoding genes inserted into the BcAU1054 T6SS core cluster via a transposon, and secondary structure analysis predicts a glycosyl hydrolase domain in Tae1. We identified the tle4-tli4 gene pair by searching for DUFs shared among E-I-encoding genes, as DUF3304 is only present in the BcAU1054 genome within tli1, tli3, and tli4 (Table 3.1).

To determine whether the BcAU1054 T6SS mediates interbacterial competition, we generated an unmarked, in-frame deletion mutation in hcp, which encodes the inner tube

81 protein of the T6SS apparatus. Over 5 h co-culture, BcAU1054 outcompeted Escherichia coli

DH5a by ~2-logs, whereas BcAU1054 ∆hcp had no competitive advantage (Figure 3.1B).

BcAU1054 also had a T6SS-dependent competitive advantage over another Bcc pathogen,

Burkholderia dolosa strain AU0158 (BdAU0158), as the parental strain outcompeted BdAU0158 by ~4-logs over 5 h, whereas BcAU1054 ∆hcp was outcompeted by BdAU0158 (Figure 3.1C).

Ectopic expression of hcp from the attTn7 site of the genome partially restored the ability of

BcAU1054 ∆hcp to outcompete E. coli DH5a and BdAU0158 (Figures 3.1B and 3.1C).

Importantly, BcAU1054 ∆hcp did not have a growth defect compared to the parental strain, suggesting growth rate differences were not a factor determining competitive fitness (Figure

3.3). The BcAU1054 T6SS, therefore, is a potent antibacterial weapon capable of killing competitor bacteria.

At least five BcAU1054 T6SS effectors mediate interbacterial competition

To determine which predicted effectors are involved in T6SS-mediated interbacterial competition by BcAU1054, we generated nine mutants, each containing an unmarked, in-frame deletion mutation in one of the predicted E-I-encoding gene pairs. We screened these mutants by engineering them to produce green fluorescent protein (GFP) and co-culturing them, individually, with either wild-type (WT) or ∆hcp BcAU1054 strains for ~20 h, and then measuring

GFP fluorescence intensity and the OD600 of the co-cultures. For four of the mutants (∆tle1∆tli1,

∆tne1∆tni1, ∆tne2∆tni2, and ∆tpe1∆tpi1), the GFP/OD600 values for co-cultures with WT

BcAU1054 were about half of what they were for co-cultures with BcAU1054 ∆hcp, indicating these mutants were outcompeted in a T6SS-dependent manner, presumably because they lack the immunity protein that is specific for the cognate toxic effector (Figure 3.4). To measure competition quantitatively, we then competed each of these four mutants against WT and ∆hcp

BcAU1054 strains. In each case, the E-I deletion mutant was outcompeted by its parental strain

82 in a T6SS-dependent manner (Figure 3.5A). Ectopic expression of the cognate immunity gene in each mutant rescued it from T6SS-mediated killing by the parental BcAU1054 strain (Figure

3.5A), providing evidence that Tle1-Tli1, Tne1-Tni1, Tne2-Tni2, and Tpe1-Tpi1 are true antibacterial E-I pairs associated with the BcAU1054 T6SS.

To determine if there are additional E-I-encoding gene pairs in BcAU1054, we generated a mutant lacking all nine predicted E-I-encoding gene pairs (BcAU1054 ∆9E-I) and assessed its ability to outcompete target bacteria. This nonuple mutant outcompeted BdAU0158 by ~3.5-logs

(slightly less than WT BcAU1054) (Figure 3.5B), indicating that at least one more effector delivered by the BcAU1054 T6SS exists.

Susceptibility of P. aeruginosa CF isolates to the BcAU1054 T6SS correlates with patient age

Since Bcc pathogens are known to establish infections in the polymicrobial CF respiratory tract, we next sought to determine whether the BcAU1054 T6SS targets the prevalent CF pathogen P. aeruginosa. In competition experiments against P. aeruginosa reference strain PAO1, PAO1 outcompeted both WT and ∆hcp strains of BcAU1054, though showed a slightly (~0.5-log) greater ability to outcompete T6SS-active than T6SS-inactive

BcAU1054 (Figure 3.6A). These results are consistent with two theories on the regulation of

H1-T6SS activity by PAO1: T6SS-dueling (42), in which the PAO1 H1-T6SS only deploys following antagonism by a neighboring cell, and the P. aeruginosa response to antagonism

(PARA) (53), in which PAO1 activates aggressive behaviors, like T6SS activity, following detection of kin cell lysates. The ability of PAO1 to outcompete BcAU1054 was solely dependent on the H1-T6SS, as a transposon insertion in vipA1, which encodes a necessary structural protein of the H1-T6SS (42), rendered PAO1 susceptible to being outcompeted by

BcAU1054 (Figure 3.6A).

83 PAO1 was originally isolated from a wound infection and has undergone decades of laboratory passage and diversification (54-57). We therefore reasoned that PAO1 may not accurately represent potential T6SS-mediated interactions between Bcc pathogens and P. aeruginosa strains relevant to CF infection. To address this concern, we used collections of P. aeruginosa strains isolated from CF patients (58, 59). BcAU1054 did not outcompete any of the

P. aeruginosa strains isolated from infants or young children (three years old or younger) and was often slightly outcompeted by these P. aeruginosa strains (Figure 3.6B). By contrast,

BcAU1054 had the striking ability to outcompete nearly half of the P. aeruginosa strains isolated from teenagers and adults (11-31 years old) in a T6SS-dependent manner, oftentimes efficiently enough to prevent recovery of any P. aeruginosa from the co-cultures (Figure 3.6D). We also determined if the nonuple E-I deletion mutant of BcAU1054 could outcompete the susceptible P. aeruginosa strains. Although BcAU1054 ∆9E-I was strongly outcompeted by PAO1, it retained a competitive advantage against C078C, C120C, C123D, and CEC118 (Figure 3.7). Intriguingly,

C120C was less susceptible to killing by BcAU1054 ∆9E-I than were C078C, C123D, and

CEC118, (Figure 3.7) suggesting C120C is less sensitive to the unidentified effector(s) associated with the BcAU1054 T6SS, and that BcAU1054 T6SS effectors exhibit target strain- specific variability in toxicity. The ability of BcAU1054 to efficiently kill P. aeruginosa from older

CF patients correlates with the clinical presentation of Bcc infections, which do not occur in young CF patients and solely arise in teenagers and adults (8).

Host-adapted P. aeruginosa isolates that are sensitive to the BcAU1054 T6SS harbor

T6SS-abrogating mutations

We sequenced the genomes of all P. aeruginosa clinical isolates (not from co-infections) used in our study. Of the four T6SS-susceptible teenage/adult isolates (C078C, C120C, C123D, and CEC118), three contain mutations in the gacS or gacA genes, which encode a two- component system required for T6SS protein production (Table 3.2) (34, 38, 60). C078C

84 contains a missense mutation in gacS (gacSG1715A), resulting in the variant protein GacSG572D.

C123D contains a large genomic deletion spanning the gacS gene and the nearby pirRSA genes, which encode a siderophore iron-acquisition system (61). C120C contains a premature stop codon in gacA (gacAC349T). The C078C genome also contains a premature stop codon in pppA (pppAG111A). PppA is a post-translational regulator of H1-T6SS activity (39), and is required for efficient T6SS-mediated competition (42). CEC118 has a small deletion in fha1

(fha1∆404-424) resulting in the loss of seven amino acid residues from Fha1, another post- translational regulator of H1-T6SS activity (39).

To investigate if the ability of BcAU1054 to outcompete P. aeruginosa strains isolated from teenage/adult CF patients correlates with a loss of H1-T6SS activity in the P. aeruginosa strains, we assessed production of Hcp1, the major subunit protein of the H1-T6SS inner tube, during growth on agar. Every P. aeruginosa isolate that was outcompeted by the BcAU1054

T6SS showed either negligible or diminished Hcp1 production compared to PAO1 (Figures

3.6D and 3.6E). The isolates CEC121, CEC122, and CEC116, which were not outcompeted by the BcAU1054 T6SS, produced Hcp1 at levels similar to PAO1 (Figure 3.6E). Conversely, every P. aeruginosa strain isolated from an infant or young child except CEC32 produced Hcp1 at or near levels similar to PAO1 (Figure 3.6C), which correlates with their resistance to T6SS- dependent outcompetition by BcAU1054 (Figure 3.6B).

Restoration of H1-T6SS protein production can rescue host-adapted P. aeruginosa from

T6SS-mediated elimination by BcAU1054

Phosphorelay through the GacSA two-component system activates production of the sRNAs RsmY and RsmZ, which are required for T6SS protein production by P. aeruginosa (34-

38). To determine if lack of gacS/gacA function is responsible for susceptibility of the P. aeruginosa strains with mutations in these genes, we introduced a plasmid (pJN-rsmZ) into

C120C, C123D, and C078C to express rsmZ (induced by arabinose) independent of the GacSA

85 phosphorelay (62, 63). We also introduced the vector backbone (pJN105) into these strains as a negative control. In competitions against BcAU1054 on agar containing 0.1% arabinose, C120C pJN-rsmZ was rescued from T6SS-mediated elimination by BcAU1054, while C120C harboring the pJN105 was not (Figure 3.8A). Likewise, C120C pJN-rsmZ, but not C120C pJN105, produced Hcp1 when grown under inducing conditions (Figure 3.8D). C123D pJN-rsmZ (Figure

3.8B) and C078C pJN-rsmZ (Figure 3.8C) were still strongly outcompeted by the BcAU1054

T6SS, and pJN-rsmZ did not promote Hcp1 production by these isolates (Figure 3.8D).

Because C078C also contains a premature stop codon in pppA, we delivered the WT pppA gene under control of a constitutive promoter to the attTn7 site, and also introduced pJN-rsmZ into this strain, but expression of these genes failed to rescue this strain from T6SS-mediated elimination by BcAU1054 (Figures 3.8E and 3.8F). Lastly, we delivered the WT fha1 gene to the attTn7 site of CEC118, as this isolate has a truncated fha1 (fha1∆404-424), but constitutive expression of full-length fha1 did not rescue CEC118 from being outcompeted by BcAU1054

(Figure 3.8G). It is not surprising that constitutive expression of full-length pppA and full-length fha1 did not rescue C078C and CEC118, respectively, as both isolates were defective for Hcp1 production (Figure 3.6E); moreover, the natively-produced truncated PppA and Fha1 variants could act as dominant negatives in these strains.

Additional Bcc pathogens kill host-adapted P. aeruginosa in a T6SS-dependent manner

To investigate whether T6SS-mediated killing of host-adapted P. aeruginosa is a common feature of Bcc strains, we used Burkholderia multivorans strain CGD2M (BmCGD2M) and BdAU0158, which encode predicted T6SS-1 systems. BmCGD2M encodes one additional predicted T6SS, and BdAU0158 encodes two additional predicted T6SSs. We generated plasmid disruption mutations in the tssC1 genes of these strains’ T6SS-1 clusters (BmCGD2M tssC1::pAP82 and BdAU0158 tssC1::pAP83), and competed these mutants and the parental strains against P. aeruginosa strains PAO1 and C078C. BmCGD2M had a slight (~1-log) T6SS-

86 dependent competitive advantage against PAO1, and strongly outcompeted the P. aeruginosa host-adapted isolate C078C using its T6SS (Figure 3.9A). Although BdAU0158 was outcompeted by PAO1, possibly via T6SS-dueling or PARA on the part of PAO1, BdAU0158 outcompeted C078C by ~3-logs in a T6SS-dependent manner (Figure 3.9B). These data suggest T6S may provide many Bcc pathogens a competitive advantage against host-adapted

P. aeruginosa.

B. cenocepacia isolates from CF patients with concurrent P. aeruginosa infections outcompete their paired P. aeruginosa isolates under T6SS-permissive conditions

Our data, together with data from other groups (45-47), suggest T6SS-abrogating mutations can accumulate as P. aeruginosa evolves within the CF respiratory tract to transition to a chronic infection lifestyle, and that patients infected by T6SS-null P. aeruginosa may be susceptible to Bcc superinfections. To explore this hypothesis further, we acquired eight B. cenocepacia-P. aeruginosa coinfection pairs, each isolated from a separate CF patient with concurrent B. cenocepacia and P. aeruginosa infections. During co-culture on agar, seven of the eight B. cenocepacia isolates outcompeted their paired P. aeruginosa isolate by as little as

~1-log (BcAU7523 vs. PaAU7618) or as great as ~4-logs (BcAU29704 vs. PaAU29744) (Figure

3.10A). Since genetic manipulation of recent human isolates is often not possible, we took advantage of the fact that growth in shaking liquid cultures is non-permissive for T6SS-mediated competition (23, 32, 64, 65), likely because cells are not in contact long enough to allow T6SS effector delivery to target cells. The competitive advantages of B. cenocepacia isolates over their P. aeruginosa paired isolates dropped dramatically during shaking liquid growth compared to growth on agar (Figure 3.10B); while some isolates (BcAU7523, BcAU22760) only experienced an ~1-log decrease in competitive index, others (BcAU4392, BcAU19695,

BcAU29704) experienced an ~3-log decrease in competitive index. In fact, liquid growth

87 provided two P. aeruginosa isolates (PaAU7618 and PaAU19694) a competitive advantage over their paired B. cenocepacia isolates (Figure 3.10B).

To identify potential genetic explanations for the competitive disadvantages of the P. aeruginosa co-infection isolates, we PCR-amplified the genes involved in H1-T6SS production/activity that are mutated in the P. aeruginosa strains isolated from adults for which we have whole-genome sequence information (Table 3.2) and sequenced these PCR products.

Three isolates contain mutations in gacS or gacA (PaAU7618 contains a gacAG175A mutation resulting in GacAG59S, PaAU19694 contains a gacAC162A mutation resulting in GacAD54E, and

PaAU23781 contains a premature stop codon in gacS (gacSG1568A)) (Table 3.2). PaAU4391 contains a small deletion in fha1 (fha1∆405-425) that is nearly identical to the mutation in CEC118

(fha1∆404-424) (Table 3.2). Western blotting showed negligible or diminished Hcp1 production by five P. aeruginosa co-infection isolates (PaAU7618, PaAU10617, PaAU19694, PaAU22775, and PaAU23781) compared to PAO1 (Figure 3.10C). PaAU4391, PaAU5159, and PaAU29744 produced Hcp1 at levels similar to PAO1 (Figure 3.10C), though PaAU4391 has an fha1∆405-425 mutation that may abrogate T6SS activity without affecting protein production. PaAU5159 and

PaAU29744 may harbor mutations in other genes important for post-translational regulation of

T6SS activity. These results suggest that Bcc pathogens may only be able to invade a P. aeruginosa-colonized CF respiratory tract if the P. aeruginosa population, or at least a subpopulation, has evolved to lose T6SS activity.

3.4 Discussion

Although the propensity of Bcc pathogens to infect only older CF patients, and to cause superinfections in those colonized with P. aeruginosa, has been appreciated for many years

(66-68), the underlying reasons for this apparent selectivity are unknown. During our investigation of T6S in the Bcc, we found that none of the P. aeruginosa strains in our study that were isolated from infant or child CF patients were susceptible to T6SS-mediated killing by

88 BcAU1054. Conversely, almost half of the strains isolated from teenage and adult CF patients were outcompeted by BcAU1054 in a T6SS-dependent manner, with susceptible P. aeruginosa strains often being completely eliminated from the co-cultures. Additional Bcc pathogens

(BmCGD2M and BdAU0158) also efficiently outcompeted susceptible P. aeruginosa strains via

T6SS activity, and seven of eight B. cenocepacia strains from patients with concurrent P. aeruginosa infections outcompeted their paired P. aeruginosa strains under conditions promoting T6SS-mediated interactions. These data suggest that one reason Bcc pathogens are restricted to infecting older CF patients is because only in these patients are resident P. aeruginosa susceptible to T6SS-mediated competition by Bcc bacteria.

We found that differential susceptibility of P. aeruginosa strains to T6SS-mediated competition by Bcc pathogens depends on T6SS functionality in P. aeruginosa. Disruption of vipA1 to inactivate the H1-T6SS in PAO1 converted it from being resistant to T6SS-mediated competition by BcAU1054 to being outcompeted by four logs. For the susceptible P. aeruginosa strains isolated from teenagers or adults, we found that all harbor mutations predicted to abrogate production and/or function of their T6SSs, all failed to produce substantial amounts of

Hcp1, and in one strain, elimination by BcAU1054 was prevented by activating production of its

T6SS proteins. Consistent with these observations, B. cenocepacia isolates strongly outcompeted their co-isolated P. aeruginosa strains only when the bacteria were co-cultured on a solid surface (conducive to contact-dependent interactions) and not when co-cultured in liquid medium. Furthermore, the P. aeruginosa co-infection isolates were typically deficient in Hcp1 production. These data indicate that, at least for the P. aeruginosa strains studied here, the main factor in determining susceptibility to T6SS-mediated competition by Bcc bacteria is whether P. aeruginosa produces a functional T6SS.

The T6S-abrogating mutations we identified in P. aeruginosa CF isolates in this study fell into two classes: those in genes encoding post-translational regulators of T6SS activity (pppA and fha1), and those in genes encoding the GacSA two-component regulatory system. Fha1 is

89 required for the initial assembly of the T6S apparatus, whereas PppA is required for disassembly of apparatuses and recycling of T6SS proteins into new apparatuses. Mutations in pppA or fha1 are expected to prevent efficient T6SS activity without affecting production of individual T6SS components (39, 42). Consistent with this expectation, Hcp1 was detectable in

PaAU4391, which contains a small deletion in fha1, but this co-infection isolate was outcompeted by its paired B. cenocepacia isolate. By contrast, mutations in gacA or gacS are expected to prevent production of the entire T6S apparatus. GacS is one of four hybrid sensor kinases that controls phosphorylation, and hence activation, of the GacA response regulator.

LadS functions with GacS to activate GacA, while RetS blocks GacS activity, thereby inhibiting

GacA activation (37, 69). The PA1611-encoded sensor kinase promotes GacA activation by relieving RetS inhibition of GacS (70). When active, GacA induces production of two sRNAs,

RsmY and RsmZ, which bind to, and prevent activity of, RsmA, a pleiotropic global regulator that impedes translation of many target genes (71, 72). When not inhibited by RsmY or RsmZ,

RsmA activity results in production of factors associated with acute infection (e.g., flagella, type

III secretion, type IV pili) and lack of production of factors and phenotypes associated with chronic infection (e.g., exopolysaccharide production, biofilm, T6S). The

RetS/PA1611/LadS/GacSA signaling pathway is therefore considered to function as a switch between acute and chronic infection modes (34, 37, 73).

While there is evidence that the genes encoding the RetS/PA1611/LadS/GacSA signaling pathway are intact when P. aeruginosa establishes infection initially in the CF lung, mutations arise in retS within some strains over time (e.g., 11/36 clone types in the 2015 Marvig et al. study), and, at least for those studied, all retS-mutated strains acquire subsequent mutations in gacS/gacA or rsmA (45, 46). Our data are consistent with these reports, as three out of nine teenage/adult P. aeruginosa isolates used in our study were gacS/gacA mutants and also contained nonsynonymous retS mutations (these isolates were also defective in Hcp1 production). Three out of eight of the P. aeruginosa co-infection isolates contained gacS/gacA

90 mutations and did not produce Hcp1; their retS statuses are unknown. Thus, there appears to be a selection for lack of GacSA activity following mutation of retS within the CF respiratory tract, and we envisage that this selection could be either T6S-independent or T6S-dependent; a

Gac-regulated target other than T6S may drive this selection, with loss of T6S being simply a consequence of Gac inactivation, or T6SS activity itself could be what is selected against. We and others (45, 47) have detected mutations in genes encoding proteins specific for T6SS assembly and function in P. aeruginosa strains isolated from older CF patients, supporting the hypothesis that T6S may be disadvantageous to P. aeruginosa during chronic infection in the

CF lung.

Why might it be beneficial for P. aeruginosa to lose T6SS activity in later stages of host colonization? Given the polymicrobial nature of the CF respiratory tract, it is reasonable to hypothesize that maintaining a potent antibacterial weapon like the T6SS would be beneficial.

However, P. aeruginosa T6SS proteins are immunogenic (74), and avoiding the host immune response could be equally, or more, beneficial. Additionally, production of T6SSs is energetically costly, and while T6SS-mediated competition may be worth the cost during early stages of infection, it may be beneficial to stop producing these structures once P. aeruginosa has established its niche. As indicated by the proportion of reads in metagenomic samples, P. aeruginosa can constitute over 90% of all bacterial cells within the airways of certain CF patients (2, 3, 75). Under these conditions, T6S-mediated inter-species competition should not be required. Loss of T6S by predominant bacterial strains colonizing humans has been shown with gut resident Bacteroides spp., as T6SS-proficient Bacteroides are more prevalent in the unstable infant gut microbiota than they are in adult gut microbiota where individual Bacteroides spp. or strains predominate (76). One might expect that similar selective pressures would also act on Bcc during CF infection. However, the B. cenocepacia T6SS is required for murine infection (27), and at least some Bcc strains produce a T6S-dependent effector (TecA) that promotes intracellular survival within macrophages (28-30), suggesting there is a strong

91 selective advantage for Bcc pathogens to remain T6SS-active during infection of the CF respiratory tract.

Although P. aeruginosa and Bcc bacteria ultimately colonize different sites in the CF airways (13), Bcc pathogens must traverse the lumen, where P. aeruginosa can exist in large populations, before invading host cells. Therefore, transient Bcc-P. aeruginosa interactions likely occur, and our data support the hypothesis that the outcome of these interactions depends on the T6S proficiency of the resident P. aeruginosa. P. aeruginosa populations within individual CF patients exhibit genotypic and phenotypic diversity across different regions of the respiratory tract (77), and thus Bcc bacteria may only need to interact with a subpopulation of P. aeruginosa that has lost T6SS activity in order to initiate an infection and invade host cells.

Experiments using animal models and human microbiome analyses have shown that T6SS- mediated competition occurs within mammalian intestines (76, 78-81), though it is not known whether such interactions occur in the CF respiratory tract. These questions would be better addressed with animal models of CF disease. Unfortunately, a dearth of robust, efficient animal models for chronic bacterial infections has inhibited progress in the understanding of these infections (82-84).

While our data are consistent with T6SS-mediated competition between Bcc pathogens and P. aeruginosa playing a role in susceptibility of older CF patients to the Bcc, we hypothesize that additional factors play important roles in preventing Bcc infections in infants and young children. Staphylococcus aureus is the most prevalent pathogen of young CF patients (8), and

S. aureus colonization could preclude infection by Bcc pathogens. Additionally, changes in the immune response, physiology, and/or nutritional environment of the CF respiratory tract over time could cause these tissues to be more hospitable to Bcc pathogens later in the lives of CF patients compared to those in infants and children. CF patients are also often on antibiotic regimens to treat opportunistic infections, and regular use of antibiotics may promote Bcc pathogen colonization of older patients. Other unknown factors could also be at play.

92 In our studies, competition mediated by the T6SS-1 provided strong competitive advantages to three Bcc pathogens (BcAU1054, BmCGD2M, and BdAU0158) against host- adapted P. aeruginosa. Gene clusters encoding the T6SS-1 are prevalent throughout the Bcc

(31), suggesting that T6SS-mediated killing of host-adapted P. aeruginosa may be a common asset of Bcc pathogens. The role of additional T6SSs in Bcc pathogens remains unknown, but it appears that interbacterial antagonism is mostly mediated by the T6SS-1, at least under the conditions used in this study. Our investigation of the BcAU1054 T6SS revealed four bona fide antibacterial E-I pairs; however, our bioinformatic prediction of E-I pair-encoding genes missed at least one gene pair, as BcAU1054 ∆9E-I maintained a strong competitive advantage against

BdAU0158. The unidentified effector(s) is/are not encoded by gene(s) near vgrG genes, nor are there shared domains between the effectors we identified and the unidentified effector(s), suggesting the unidentified effector(s) may be members of an uncharacterized class of T6SS toxins. Future studies will identify the full repertoire of BcAU1054 T6SS E-I pairs. Our screening for antibacterial effectors and follow-up competition experiments were specific to intrastrain antagonism (BcAU1054 vs. BcAU1054) under one condition (LSLB agar at 37˚C). The predicted

E-I pairs that our screen suggested were not important for intrastrain competition may be important for interstrain/interspecies competition or competition under different conditions (e.g., temperature, salt, pH); similar conditional efficiency has been demonstrated for P. aeruginosa

T6SS effectors (85). Supporting this hypothesis, BcAU1054 ∆9E-I outcompeted T6SS-null P. aeruginosa teenage/adult isolates to varying degrees, suggesting the additional, unidentified effector(s) have prey cell-specific activity.

There is growing appreciation for the genotypic and phenotypic diversity of P. aeruginosa within the CF respiratory tract (43, 44, 77). Although reference strains are powerful tools for studying bacterial pathogens, they do not always perfectly represent the strains currently infecting humans. Our investigations illuminate differences between PAO1 and recently collected P. aeruginosa CF isolates specific to T6SS-mediated competition against Bcc pathogens, as well

93 as demonstrate varying abilities of P. aeruginosa CF isolates to compete against BcAU1054.

Our data support a model in which resident P. aeruginosa populations must evolve to lose T6SS activity in order for Bcc pathogens to colonize the CF respiratory tract. If true, not only is the Bcc

T6SS an important colonization factor, but assessing the T6S potential of resident P. aeruginosa could predict susceptibility of CF patients to deadly Bcc superinfections.

3.5 Method Details

Bacterial strains and growth conditions

All bacterial strains in this study were cultured in low salt lysogeny broth (LSLB: 10 g/L tryptone, 5 g/L yeast extract, 5 g/L sodium chloride) or on LSLB agar (1.5% agar). Antibiotics to select for Burkholderia strains were used at the following concentrations, when applicable: 30

µg/mL gentamicin, 250 µg/mL kanamycin, 50 µg/mL trimethoprim, 40 µg/mL tetracycline.

Antibiotics to select for P. aeruginosa strains were used at the following concentrations, when applicable: 20 or 35 µg/mL chloramphenicol, 20 µg/mL nalidixic acid, 50µg/mL trimethoprim, 75

µg/mL gentamicin, 40 µg/mL tetracycline. 20 µg/mL nalidixic acid was used to select for E. coli

DH5a, when applicable.

Genetic manipulations

E. coli strain RHO3 was used to conjugate plasmids into Burkholderia spp. and P. aeruginosa. The pEXKm5 allelic exchange vector (86) was used to generate unmarked, in- frame deletion mutations in BcAU1054. Briefly, ~500 nucleotides 5’ to and including the first three codons of the gene to be deleted were fused to ~500 nucleotides 3’ to and including the last three codons of the gene by overlap extension PCR and cloned into pEXKm5. Following selection of BcAU1054 merodiploids with the plasmids integrated into the chromosome, cells were grown for 4 h in YT broth (10 g/L yeast extract, 10 g/L tryptone) at 37˚C with aeration, subcultured 1:1000 in fresh YT broth, and grown overnight at 37˚C with aeration. After overnight

94 growth, cells that lost the cointegrated plasmid following the second homologous recombination step were selected on YT agar (1.5% agar) containing 25% sucrose and 100 µg/mL 5-bromo-4- chloro-3-indoxyl-b-D-glucuronide (X-Gluc). Deletion mutants were screened for by PCR and verified by sequencing regions spanning the deletions.

The pUC18T-mini-Tn7T suite of plasmids (87) was used to deliver antibiotic resistance gene cassettes to the attTn7 sites of BcAU1054 and P. aeruginosa. The trimethoprim resistance-conferring plasmid pUC18T-mini-Tn7T-Tp was generated in this study by restriction digesting out dhfRII from pUC18T-mini-Tn7-Tp-PS12-mCherry (88) using MscI and NcoI and ligating into digested pUC18T-mini-Tn7T-Km (87) lacking nptII (the kanamycin resistance- conferring gene). pUC18-miniTn7-kan-gfp (89) was used to generate GFP-producing BcAU1054

E-I deletion mutants. Complemented BcAU1054 mutant strains (with either hcp or T6SS immunity-encoding genes) were generated by PCR amplifying the genes of interest and cloning the sequences into pUCS12Km (90). BcAU1054 strains constitutively expressing lacZ were generated using pECG10 (90). P. aeruginosa isolates C078C and CEC118 were complemented with pppA and fha1, respectively, by cloning these sequences into pUCS12Km, digesting out the genes and upstream constitutive promoter PS12, and cloning these fragments into pUC18T- mini-Tn7T-Tet (90). For all pUC18T-mini-Tn7T-based cassette delivery to the attTn7 sites of

BcAU1054 and P. aeruginosa, the transposase-encoding pTNS3 helper plasmid was used in triparental conjugation. BmCGD2M tssC1::pAP82 and BdAU0158 tssC1::pAP83 were generated by cloning ~500 internal nucleotides of the tssC1 genes into pUC18T-mini-Tn7T-Km, conjugating the plasmids into BmCGD2M and BdAU1058, and selecting for plasmid cointegrants on kanamycin.

Interbacterial competition experiments

All competition experiments were conducted for 5 h on LSLB agar at 37˚C, with an ~1:1 starting cell ratio of inhibitor and target strains, unless stated otherwise. Cells were collected

95 from overnight liquid cultures, centrifuged for 2 min at 15,000 rpm, washed in 1X phosphate buffered saline (PBS), diluted to an OD600 of 1.0, and equal volumes of inhibitor and target cells were mixed. For BcAU1054 vs. E. coli DH5a competitions, BcAU1054 1.0 OD600 cell suspensions were diluted 1:3 in 1X PBS before mixing with DH5a 1.0 OD600 cell suspensions to attain an ~1:1 starting cell ratio. 20 µL spots of mixtures were plated on LSLB agar in 24-well plates, allowed to dry, and incubated at 37˚C for 5 h. Starting mixtures were also serially diluted and plated on antibiotic-containing selective media to enumerate inhibitor and target strains at the initial time point. Following 5 h, competition spots were resuspended in 1 mL 1X PBS within wells, serially diluted, and plated on antibiotic-containing selective media to enumerate inhibitor and target strains. Colony counts at the initial and 5 h time points allowed for competitive index

(C.I.) calculations as follows: C.I. = (inhibitort5/targett5)/(inhibitort0/targett0). A positive log10 C.I. indicates the inhibitor strain outcompeted the target strain, a negative log10 C.I. indicates the target strain outcompeted the inhibitor strain, and a log10 C.I. of ~0 indicates neither strain had a competitive advantage.

Liquid competitions between B. cenocepacia-P. aeruginosa co-infection isolates were set up following the above protocol, except 20 µL of cell mixtures were inoculated into 1 mL

LSLB and grown for 5 h at 37˚C shaking at 220 rpm. For competitions between BcAU1054 and

P. aeruginosa clinical isolates, BcAU1054 WT and ∆hcp strains constitutively expressing lacZ were used and inocula/competitions were plated onto antibiotic-containing LSLB agar with 40

µg/mL 5-bromo-4-chloro-3-indolyl b-D-galactopyranoside (X-Gal) to help differentiate between

P. aeruginosa and BcAU1054 colonies. Competitions between BcAU1054 and pJN- rsmZ/pJN105-harboring P. aeruginosa teenage/adult isolates were conducted on LSLB agar containing 0.1% L-arabinose.

96 BcAU1054 T6SS E/I screen

Co-cultures were set up following the same protocol as in the interbacterial competition experiments. For monocultures, cell suspensions (at an OD600 of 1.0) were mixed 1:1 with 1X

PBS before plating. For co-cultures and mono-cultures, 20 µL spots were plated on LSLB agar within 24-well plates, spots were allowed to dry, and plates were incubated at 37˚C for ~20 h.

Following incubation, the cultures were resuspended in 1 mL 1X PBS within wells, 100 µL were added to 96-well plates, and OD600 values and GFP fluorescence intensities (485 nm excitation,

3 530 nm emission) were measured on a PerkinElmer Wallac VICTOR TM plate reader.

Hcp1 immunoblotting

P. aeruginosa strains were swabbed onto LSLB agar and grown overnight at 37˚C. For pJN-rsmZ/pJN105-harboring P. aeruginosa teenage/adult isolates, strains were swabbed onto

LSLB agar containing 75 µg/mL gentamicin and 0.1% L-arabinose and grown overnight at 37˚C.

Following overnight incubation, cells were scraped off plate, resuspended in 1 mL cold 1X PBS, centrifuged for 2 min at 15,000 rpm, washed in 1 mL cold 1X PBS, and diluted to an OD600 of

5.0. Cells were then centrifuged for 2 min at 15,000 rpm and resuspended in 200 µL 2X SDS-

PAGE sample loading buffer (6X SDS-PAGE sample loading buffer: 375 mM Tris-HCl, 9% sodium dodecyl sulfate (SDS), 50% glycerol, 0.03% bromophenol blue, 1.3 M b- mercaptoethanol), boiled at 99˚C for 15 min, and samples were sheared 10 times through a

26G needle. Samples were resolved on 12% SDS-PAGE gels (5 µL loaded), transferred to nitrocellulose membranes, and membranes were blocked with 5% (w/v) non-fat dry milk in 1X

PBS for 1 h with rotation at room temperature (RT). Membranes were then washed three times in 1X PBS and incubated with a-Hcp1 peptide antibody (diluted 1:1000 in 5% (w/v) non-fat dry milk in 1X PBS+0.1% Tween®20 (PBS-T)) for 1 h with rotation at RT. Membranes were then washed three times in 1X PBS-T, incubated with IRDye® 800CW-conjugated a-rabbit IgG

97 antibody (diluted 1:25,000 in 5% (w/v) non-fat dry milk in 1X PBS-T) for 30 min with rotation at

RT, washed three times in 1X PBS, and imaged on a LI-COR Odyssey® fluorescence imager.

Sequencing

Genomic DNA was purified from P. aeruginosa isolates C078C, C120C, and C123D using the Promega Wizard Genomic DNA Purification Kit. Paired-end TruSeq (Illumina) libraries were generated and sequenced on the Illumina MiSeq 2X150 platform at the High-Throughput

Sequencing Facility at the University of North Carolina at Chapel Hill. Demultiplexed FASTQ files were mapped to the PAO1 reference genome (assembly GCA_000006765.1) using the

Geneious Prime standard assembler. Sequencing reads can be accessed in BioProject

PRJNA609958.

To sequence P. aeruginosa CEC isolate genomes, genomic DNA was isolated using a

GenElute Bacterial Genomic DNA Kit (Sigma Aldrich, NA2110; St. Louis, MO) following kit instructions with the following exception: all DNA was eluted in 400uL of ultra-pure DEPC- treated water (ThermoFisher Scientific, Waltham, MA). Concentration of DNA preps was determined using a NanoDrop 1000 (ThermoFisher Scientific, Waltham, MA). All preps were stored at -20C. The 150bp sequencing reads from the Illumina platform were assembled using spades v.3.7.1 with careful mismatch correction and the assemblies were filtered to contain only contigs ≥500bp with ≥5X k-mer coverage. The assemblies were further examined for characteristics that would suggest the genome was of high quality (<400 contigs) and potentially

P. aeruginosa. All reads and assemblies are deposited at NCBI under BioProject

PRJNA607994.

Specific P. aeruginosa co-infection isolate genes were sequenced by PCR-amplifying genes of interest and submitting the PCR products for Sanger sequencing.

98 Bioinformatic analysis of BcAU1054 T6SS-encoding genes and effector proteins

The BcAU1054 and BcJ2315 (genome assembly GCA_000009485.1) T6SS-encoding core clusters were aligned in Geneious Prime using the Mauve plugin (91). Phyre2 (51) and

HHpred (52) were used to predict the secondary structures and catalytic activities of potential

BcAU1054 T6SS effector proteins.

Figure 3.1. The BcAU1054 T6SS mediates interbacterial competition. (A) Core cluster (top two lines) and accessory genes encoding the BcAU1054 T6SS and bioinformatically-predicted effector-immunity (E-I) pairs (see Table 3.1). The legend indicates function of protein products. White ORFs encode DUF4123 T6SS adapter proteins, or proteins not known to be associated with the T6SS. (B and C) Interbacterial competition experiments between inhibitor BcAU1054 and target E. coli (B) and BdAU0158 (C). WT, ∆hcp, and ∆hcp attTn7::hcp BcAU1054 inhibitor strains used in each. Circles represent individual co-cultures from two biological replicates, each with four technical replicates. Solid horizontal lines represent mean log10 C.I. values. Dotted horizontal lines (log10 C.I. = 0) indicate no competitive advantage for either inhibitor or target. *P<0.05, **P<0.0005, Mann-Whitney test.

100

Figure 3.2. The BcAU1054 T6SS core cluster is homologous to the BcJ2315 T6SS core cluster, though contains insertions. The genomic regions encoding the T6SSs of BcAU1054 and BcJ2315 were aligned to each other using Mauve. Orange shading represents regions of homology. All the structural protein- encoding genes are homologous between the two strains, though the BcAU1054 core cluster has multiple insertions – regions spanning tae1-tai1, vgrG1-tle1, vgrG2-tni2, and genes downstream of the core cluster predicted to encode proteins involved in phage and mobile genetic elements (see Figure 3.1).

101

Figure 3.3. BcAU1054 ∆hcp does not have a growth defect. 5 h co-cultures between BcAU1054 WT and ∆hcp strains. Circles represent individual co- cultures from two biological replicates, each with four technical replicates. Solid horizontal line represents mean log10 C.I. Dotted horizontal line (log10 C.I. = 0) indicates no competitive advantage for either inhibitor or target.

102

Figure 3.4. Screening approach to identify probable BcAU1054 T6SS E-I pairs. (A) GFP/OD600 values for ~20 h co-cultures between WT and ∆hcp BcAU1054 inhibitor strains and BcAU1054 E-I deletion mutants constitutively producing GFP, as well as GFP/OD600 values for ~20 h monocultures of each strain. GFP measured at 485 nm excitation and 530 nm emission. Data resulting from at least two biological replicates, with height of bar representing mean GFP/OD600 value and error bars representing one standard deviation. Dotted horizontal line represents baseline GFP/OD600 value, taken from non-GFP-producing WT and ∆hcp BcAU1054 monocultures. See Figure 3.1 and Table 3.1 for location of E-I-encoding gene pairs and predicted effector activity, respectively. (B) 5 h interbacterial competition experiment between WT and ∆hcp BcAU1054 inhibitor strains and BcAU1054 ∆tae2∆tai2 target strain. Circles represent individual co-cultures (four technical replicates for each competition). Solid horizontal lines represent mean log10 C.I. values. Dotted horizontal line (log10 C.I. = 0) indicates no competitive advantage for either inhibitor or target.

103

Figure 3.5. At least five BcAU1054 T6SS effectors mediate interbacterial competition. (A) Interbacterial competition experiments between WT and ∆hcp BcAU1054 inhibitor strains and BcAU1054 mutants lacking E-I-encoding genes, including mutants complemented with cognate immunity genes. Probable E-I pairs identified by screen shown in Figure 3.4. (B) Interbacterial competition experiments between WT, ∆hcp, and ∆9E-I BcAU1054 inhibitor strains and BdAU0158 target. For (A and B), circles/diamonds represent individual co-cultures from two biological replicates, each with four technical replicates. Grey-filled diamonds represent competitions from which no target cells were recovered. Solid horizontal lines represent mean log10 C.I. values. Dotted horizontal lines (log10 C.I. = 0) indicate no competitive advantage for either inhibitor or target. ***P<0.0005, Mann-Whitney test.

104

Figure 3.6. Susceptibility of P. aeruginosa CF isolates to the BcAU1054 T6SS correlates with patient age. (A) Interbacterial competition experiments between WT and ∆hcp BcAU1054 inhibitor strains and WT and vipA1::Tn PAO1 target strains. (B) Interbacterial competition experiments between WT and ∆hcp BcAU1054 inhibitor strains and P. aeruginosa infant/child CF isolate targets. (C) Immunoblotting for Hcp1 production by PAO1 and P. aeruginosa infant/child CF isolates. (D) Interbacterial competition experiments between WT and ∆hcp BcAU1054 inhibitor strains and P. aeruginosa teenage/adult CF isolate targets. (E) Immunoblotting for Hcp1 production by PAO1 and P. aeruginosa teenage/adult CF isolates. For (A, B, and D), circles/diamonds represent individual co-cultures from two biological replicates, each with four technical replicates. Grey-filled diamonds represent competitions from which no target cells were recovered. Solid horizontal lines represent mean log10 C.I. values. Dotted horizontal lines (log10 C.I. = 0) indicate no competitive advantage for either inhibitor or target. *P<0.05, **P<0.005, ***P<0.0005, Mann-Whitney test. For (C and E), non-specific band above Hcp1 serves as loading control. Blots are representative of at least two experiments.

105

Figure 3.7. BcAU1054 T6SS effectors exhibit target strain-specific variability in toxicity. Interbacterial competition experiments between BcAU1054 ∆9E-I inhibitor strain and PAO1, C078C, C120C, C123D, and CEC118 P. aeruginosa target strains. Circles/diamonds represent individual co-cultures from two biological replicates, each with four technical replicates. Grey- filled diamonds represent competitions from which no target cells were recovered. Solid horizontal lines represent mean log10 C.I. values. Dotted horizontal line (log10 C.I. = 0) indicates no competitive advantage for either inhibitor or target. ***P<0.0005, Mann-Whitney test.

106

Figure 3.8. Restoration of H1-T6SS protein production can rescue host-adapted P. aeruginosa from T6SS- mediated elimination by BcAU1054. (A, B, and C) Interbacterial competition experiments between WT and ∆hcp BcAU1054 inhibitor strains and P. aeruginosa teenage/adult CF isolates C120C (A), C123D (B), and C078C (C) harboring the pJN-rsmZ and pJN105 (EV) plasmids. Competitions conducted on agar containing 0.1% arabinose. (D) Immunoblotting for Hcp1 production by C120C, C078C, and C123D isolates harboring pJN-rsmZ and pJN105 (EV) during growth on agar containing 0.1% arabinose, as well as Hcp1 production by PAO1 for comparison. Non-specific band above Hcp1 serves as loading control. The blot is representative of at least two experiments. (E) Interbacterial competition experiments between WT and ∆hcp BcAU1054 inhibitor strains and C078C attTn7::pppA target strain. (F) Interbacterial competition experiments between WT and ∆hcp BcAU1054 inhibitor strains and C078C attTn7::pppA target strains harboring pJN-rsmZ and pJN105 (EV). Competitions conducted on agar containing 0.1% arabinose. (G) Interbacterial competition experiments between WT and ∆hcp BcAU1054 inhibitor strains and CEC118 attTn7::fha1 target strain. For (A, B, C, E, F, and G), circles/diamonds represent individual co-cultures from two biological replicates, each with four technical replicates. Grey-filled diamonds represent competitions from which no target cells were recovered. Solid horizontal lines represent mean log10 C.I. values. Dotted horizontal lines (log10 C.I. = 0) indicate no competitive advantage for either inhibitor or target. n.s.=not significant, ***P<0.0005, Mann-Whitney test.

107

Figure 3.9. Additional Bcc pathogens kill host-adapted P. aeruginosa in a T6SS- dependent manner. (A) Interbacterial competition experiments between WT and tssC1::pAP82 BmCGD2M inhibitor strains and PAO1 and C078C target strains. (B) Interbacterial competition experiments between WT and tssC1::pAP83 BdAU0158 inhibitor strains and PAO1 and C078C target strains. For (A and B), circles/diamonds represent individual co-cultures from two biological replicates, each with four technical replicates. Grey-filled diamonds represent competitions from which no target cells were recovered. Solid horizontal lines represent mean log10 C.I. values. Dotted horizontal lines (log10 C.I. = 0) indicate no competitive advantage for either inhibitor or target. *P<0.05, ***P<0.0005, Mann-Whitney test.

108

Figure 3.10. B. cenocepacia isolates from CF patients with concurrent P. aeruginosa infections outcompete their paired P. aeruginosa isolates under T6SS-permissive conditions. (A and B) Interbacterial competition experiments between B. cenocepacia-P. aeruginosa co- infection isolate pairs on agar (A) and in shaking liquid culture (B). For agar competitions in (A), circles represent individual co-cultures from two biological replicates, each with four technical replicates. For shaking liquid competitions in (B), circles represent individual co-cultures from three biological replicates. Solid horizontal lines represent mean log10 C.I. values. Dotted horizontal lines (log10 C.I. = 0) indicate no competitive advantage for either inhibitor or target. (C) Immunoblotting for Hcp1 production by P. aeruginosa co-infection isolates, as well as Hcp1 production by PAO1 for comparison. Non-specific band above Hcp1 serves as loading control. The blot is representative of at least two experiments.

109 Table 3.1. Bioinformatically-predicted BcAU1054 T6SS E-I pairs and predicted effector enzymatic activities.

E-I Pair Locus Tag Associated Predicted Notes vgrG Effector Activity tle1-til1 BCEN_RS13090- vgrG1 Phospholipase BCEN_RS13095 tle2-tli2 BCEN_RS06980- vgrG3 Phospholipase Referred to as tle5-tli5 in BCEN_RS06975 (92) tle3-tli3 BCEN_RS25350- vgrG7 Phospholipase BCEN_RS25355 tle4-tli4 BCEN_RS23700- None Phospholipase tle4 has an RHS domain; BCEN_RS23705 tli4 has DUF3304, which otherwise is present in the BcAU1054 genome only in tli1 and tli3 tne1-tni1 BCEN_RS13180- vgrG2 Nuclease (not annotated) tne2-tni2 BCEN_RS16675- vgrG5 Nuclease tni2 not annotated with new Bcen_3345 (old locus tags locus tag) tae1-tai1 BCEN_RS13080- None Amidase May have inserted into BCEN_RS13075 T6SS core cluster via transposon (see duplication of 3’ end of tagL in Figure 3.1) tae2-tai2 BCEN_RS10100- vgrG4 Amidase Two annotated ORFs BCEN_RS10105- (10100 and 10105) have BCEN-RS10110 glycosylhydrolase domains and may encode an amidase effector, sequencing errors may have split into two ORFs tpe1-tpi1 BCEN_RS22905- vgrG6 Pore-forming toxin Tpe1 has homology to V. BCEN_RS22900 cholerae VasX (93) and B. thailandensis BTH_I2691 (94); tpi1 is misannotated – full-length gene is 969 bps (126 bps missing from 5’ end in annotation)

110 Table 3.2. Competition sensitivity, Hcp1 production, and putative T6SS-abrogating mutations of P. aeruginosa CF isolates used in this study.

P. Patient Susceptibility Hcp-1 Putative T6SS- RetS aeruginosa age (in to BcAU1054 production abrogating substitutions CF Isolate years) at T6SS (or to B. mutation(s) in gacS/gacA isolation cenocepacia mutants paired isolate) Infant/Child Isolates CEC32 1 - - N/A N/A CEC36 <1 - + N/A N/A CEC42 2 - + N/A N/A CEC44 2 - + N/A N/A CEC66 1 - + N/A N/A CEC73 3 - + N/A N/A CEC83 1 - + N/A N/A CEC87 <1 - + N/A N/A CEC112 3 - + N/A N/A Teenage/Adult Isolates C078C 31 +++ - gacSG1715A (GacSG572D), RetSA46V, pppAG111A (PppATRUNC) RetSR144H C120C 12 +++ - gacAC349T (GacATRUNC) RetSA46V, RetSL856Q C123D 19 +++ - ∆gacS RetSA46V CEC118 17 +++ - fha1∆404-424(Fha1∆134-140) N/A CEC119 25 + - N/D N/A CEC120 11 + - N/D N/A CEC121 19 - + N/A N/A CEC122 18 +/- + N/A N/A CEC116 11 - + N/A N/A Co-Infection Isolates PaAU4391 39 +++ + fha1∆405-425(Fha1∆135-141) N/A PaAU5159 26 ++ + N/D N/A PaAU7618 33 + - gacAG175A (GacAG59S) N/D PaAU10617 17 - - N/D N/A PaAU19694 36 +++ - gacAC162A (GacAD54E) N/D PaAU22775 38 + - N/D N/A PaAU23781 39 + - gacSG1568A (GacSTRUNC) N/D PaAU29744 33 +++ + N/D N/A

Isolates grouped into infant/child isolates, teenage/adult isolates, and co-infection isolates, with patient age at isolation provided. For susceptibility to competition against B. cenocepacia and Hcp1 production status, see Figures 3.6 and 3.10. N/A, not applicable. N/D, not determined.

111 Table 3.3. Bacterial strains used in this study.

Strain Name Source WGS Identifier Burkholderia cepacia complex strains BcAU1054 BcRLR* GCA_000014085.1 BcAU1054 ∆hcp This paper BcAU1054 ∆hcp attTn7::hcp This paper BcAU1054 ∆tle1∆tli1 This paper BcAU1054 ∆tle1∆tli1 attTn7::tli1 This paper BcAU1054 ∆tle2∆tli2 This paper BcAU1054 ∆tle3∆tli3 This paper BcAU1054 ∆tle4∆tli4 This paper BcAU1054 ∆tne1∆tni1 This paper BcAU1054 ∆tne1∆tni1 attTn7::tni1 This paper BcAU1054 ∆tne2∆tni2 This paper BcAU1054 ∆tne2∆tni2 attTn7::tni2 This paper BcAU1054 ∆tae1∆tai1 This paper BcAU1054 ∆tae2∆tai2 This paper BcAU1054 ∆tpe1∆tpi1 This paper BcAU1054 ∆tpe1∆tpi1 attTn7::tpi1 This paper BcAU1054 ∆9E-I This paper BcAU1054 attTn7::lacZ This paper BcAU1054 ∆hcp attTn7::lacZ This paper BmCGD2M BcRLR* GCA_000182295.1 BmCGD2M tssC1::pAP82 This paper BdAU0158 BcRLR* GCA_000959505.1 BdAU0158 tssC1::pAP83 This paper BcAU4392 BcRLR* BcAU5161 BcRLR* BcAU7623 BcRLR* BcAU10618 BcRLR* BcAU19695 BcRLR* BcAU22760 BcRLR* BcAU23782 BcRLR* BcAU29704 BcRLR* Pseudomonas aeruginosa strains PAO1 (54) Wolfgang Lab collection GCA_000006765.1 PAO1 vipA1::Tn (95) CEC32 BioProject PRJNA607994 CEC36 BioProject PRJNA607994 CEC42 BioProject PRJNA607994 CEC44 BioProject PRJNA607994 CEC66 BioProject PRJNA607994 CEC73 BioProject PRJNA607994 CEC83 BioProject PRJNA607994 CEC87 BioProject PRJNA607994 CEC112 BioProject PRJNA607994 C078C (96) (referred to as BC236) BioProject PRJNA609958 C078C attTn7::pppA This paper C120C (96) (referred to as BC238) BioProject PRJNA609958 C123D (96) (referred to as BC239) BioProject PRJNA609958 CEC118 BioProject PRJNA607994 CEC118 attTn7::fha1 This paper CEC119 BioProject PRJNA607994 CEC120 BioProject PRJNA607994 CEC121 BioProject PRJNA607994 CEC122 BioProject PRJNA607994 CEC116 BioProject PRJNA607994 PaAU4391 BcRLR*

112 PaAU5159 BcRLR* PaAU7618 BcRLR* PaAU10617 BcRLR* PaAU19694 BcRLR* PaAU22775 BcRLR* PaAU23781 BcRLR* PaAU29744 BcRLR* Eschericia coli strains DH5a Cotter Lab collection *Burkholderia cepacia Research Laboratory and Repository, University of Michigan, Ann Arbor, MI USA.

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121

CHAPTER 4 – CONCLUSION

4.1 Interbacterial Competition in the Context of Cystic Fibrosis Infections

Complex polymicrobial communities are characteristic of CF respiratory disease (1), and shifts in overall community diversity can occur on a daily basis and sometimes at the onset of pulmonary symptom exacerbation (i.e., at points of dramatic lung function decline) (2, 3).

Microbiome analyses suggest anaerobic species increase in abundance at the onset of pulmonary exacerbation (4), though it remains unknown whether these anaerobic blooms promote or are a result of disease progression. Despite the complex, dynamic microbial communities in the CF airways, canonical pathogens including S. aureus, P. aeruginosa, and

Bcc species can dominate these environments and cause long-lasting infections (2, 5, 6). There are likely a multitude of reasons why these pathogens are adept at predominating the CF respiratory tract. Perhaps CF pathogens are able to withstand the host immune response and persist in this inflammatory environment, whereas non-pathogenic members originating from the nasal, oral, skin, and intestinal microbiota are less able to do so. Although non-pathogens may not elicit strong inflammation, pathogen-induced inflammation may shift the airway environment to a state that is more hospitable to pathogens than non-pathogens. Inflammation may increase the ecological competitiveness of pathogens by directly damaging non-pathogens, or by providing nutrients that only pathogens can exploit. A similar phenomenon promotes S. enterica serovar Typhimurium upon infection of the intestines (7). Additionally, the nutritional environment of the CF airways may promote the growth of canonical pathogens over the growth of non-pathogenic species independent of inflammation. The degradation of CF respiratory

122 mucins by oral microbiota species provides nutrients for P. aeruginosa (8), and similar resource liberation may enhance colonization by other CF pathogens.

Efficient use of resources by CF pathogens to the detriment of non-pathogenic species would be an example of exploitative competition, which provides advantages to community members without direct cellular damage to their competitors (9). Conversely, interference competition involves direct mechanisms of interbacterial antagonism that provide competitive advantages to community members by killing or inhibiting the growth of competitors (9). In addition to the mechanisms of contact-mediated competition by Bcc pathogens that I have investigated in this thesis, several examples of interference competition benefiting CF pathogens have been described (10, 11). P. aeruginosa interferes with cellular respiration by S. aureus and Bcc species via the production of diffusible quinolones and hydrogen cyanide, respectively (12, 13); however, in the case of hydrogen cyanide toxicity towards Bcc species, some P. aeruginosa CF isolates have reduced killing potential compared to reference strain counterparts (P. aeruginosa strains PAO1 and PA14) (13). These differences are analogous to the T6SS-mediated competition dynamics between Bcc pathogens and P. aeruginosa I have detailed in Chapter 3 of this dissertation. The ability of Bcc pathogens to cause superinfections in P. aeruginosa-infected CF patients (14-17), and oftentimes predominate the CF airways over

P. aeruginosa and other bacteria (2, 3, 18), suggest competitive mechanisms promoting Bcc pathogens are important during human colonization and infection. In this dissertation, I describe two specialized secretion system-mediated mechanisms of interbacterial competition that may provide ecological advantages to Bcc pathogens in the CF respiratory tract.

4.2 My Contributions to the Burkholderia cepacia Complex Sociomicrobiology Field

Although Bcc infections are rare compared to those caused by P. aeruginosa and S. aureus (19), they can be devastating and cause rapid lung function decline and death in CF patients (14, 20). Research enhancing our understanding of Bcc biology and pathogenesis will

123 promote the development of prophylactic and therapeutic options to reduce Bcc infection burden, and improved treatments are needed given the high degree of antibiotic resistance in these pathogens (21). My research investigating cell contact-mediated interbacterial competition among Bcc pathogens has revealed two mechanisms by which these bacteria compete with neighboring bacteria, and such mechanisms may promote Bcc colonization and infection of the

CF respiratory tract.

4.3 CDI System-Mediated Competition and Cooperation by the Burkholderia cepacia

Complex

In Chapter 2 of this dissertation, I describe three CDI systems produced by the Bcc pathogen B. dolosa strain AU0158 (BdAU0158) (22). This pathogen caused a devastating epidemic at Boston Children’s Hospital from 1998-2005, killing many of the infected CF patients

(23, 24). Each CDI system of BdAU0158 has a unique toxin-immunity pair, characteristic of polymorphic toxin-antitoxin systems. A recent investigation has also demonstrated CDI- mediated interbacterial competition by B. multivorans strain CGD2M (BmCGD2M), another Bcc pathogen (25). Certain Bcc strains are known to colonize CF patients infected with another Bcc strain and displace the preexisting strain (26), and perhaps CDI systems provide competitive advantages to these superinfecting strains. Since CDI appears to function only against closely related bacterial cells (27), Bcc pathogens may use these systems for intragenus competition within the CF respiratory tract. Until the outer membrane (OM) receptors for BcpA proteins are identified and characterized for Burkholderia-type CDI systems, as well as any inner membrane

(IM) translocators and permissive factors promoting BcpA-CT toxicity, the phylogenetic range of susceptible target cells will remain unknown. Nevertheless, receptors and permissive factors can be shared across different species, as the BdAU0158 Bcp-1 CDI system induces CDS in B. thailandensis strain E264 (BtE264) (28), and BmCGD2M-BtE264 interspecies CDI occurs (25).

124 The genes encoding one of the BdAU0158 CDI systems (the Bcp-3 system) were not expressed under standard laboratory conditions, though an active internal promoter allowed constitutive expression of this system’s immunity-encoding gene (22). CDI system-encoding genes studied to date are often under intricate regulation. The BtE264 bcpAIOB genes are expressed stochastically, with only approximately one in 1,000 cells expressing these genes; however, these bcpAIOB-active cells express the genes at a high level (29). The E. coli strain

93 (EC93) CDI system has typically been studied by artificially expressing this strain’s cdiBAI operon in a laboratory strain of E. coli to promote maximal CDI-mediated competition (30). It is possible the immunity protein-encoding genes of these operons, as well as those of other CDI system-encoding operons under complex regulation, are not strictly regulated at the level of transcription. Constitutive immunity protein production would provide a bet-hedging strategy in case a cell entered a new environment or came into contact with a CDI system-producing cell where the immunity protein would provide protection. Such environmental transition could include transmitting from the external environment to a CF patient’s respiratory tract colonized by another Bcc strain; however, this potential immunity protein-based bet-hedging strategy would only confer competitive fitness if the invading Bcc strain produced an analogous CDI system to that of the infecting strain. Regardless of regulatory complexities, two of the

BdAU0158 bcpAIOB operons were expressed under standard laboratory conditions and their encoded systems mediated strong interbacterial competition (22), suggesting BdAU0158 uses

CDI systems for competition (and possibly additional behaviors) within microbial communities.

My thesis research also provides additional information on the distribution of CDI systems among related bacteria. Despite slight differences in the amino acid sequences of the

BcpA-CT’s and BcpI’s of the nearly identical CDI systems shared between BdAU0158 and

BtE264, these toxin-immunity pairs function in the heterologous species (i.e., the immunity proteins from each strain protect against CDI in the other strain) (22). This “shared” CDI system is present in many Burkholderia spp.; however, homologous genomic regions do not flank the

125 operons encoding this system, indicating the genes are not in synteny across the different species. This brings into question how these and other CDI system-encoding genes are shared among bacteria. The BtE264 bcpAIOB genes are located on a mobile genetic element (31), and genomic analyses predict other Burkholderia-type CDI system-encoding genes to be present on genomic islands (32, 33). Despite these facts, genes encoding the “shared” system of

BdAU0158 must not have disseminated via horizontal gene transfer, as the operons are not syntenous, or must have disseminated far back in evolutionary time to allow for dramatic diversification of the genomic regions flanking these bcpAIOB genes. Rationale exists for horizontal transfer of CDI system-encoding genes, as these genes, and other toxin-antitoxin- encoding genes, provide competitive advantages to their hosts, consistent with the selfish gene hypothesis (34). Given sequence conservation of bcpB and bcpA (at least the 5’ end of bcpA)

(29, 35), homologous recombination of entire bcpAIOB operons may promote diversification of

Burkholderia-type CDI systems by swapping in new sequences encoding distinct BcpA-CT/BcpI protein pairs. Although E. coli-type CDI systems are hypothesized to diversify via homologous recombination-mediated replacement of cdiA-CT/cdiI nucleotide sequences with “orphan” cdiA-

CT/cdiI pairs (36), the bcpAIOB operon structure would inhibit such diversification of

Burkholderia-type CDI systems (as bcpO and bcpB would be excised from the genome during such a process).

Despite my research only identifying a competitive function for the BdAU0158 CDI systems, intercellular cooperation mediated by these systems (i.e., CDS) may occur. The

BtE264 CDI system induces global gene expression changes and cooperative behaviors among kin cells by delivering a functional BcpA-CT “toxin” that, hypothetically, forms a signaling complex with BcpI to promote expression changes and intercellular cooperation (27, 28, 37).

Such cooperative behaviors include intercellular aggregation, production of an uncharacterized pigment, binding of the dye Congo red, and biofilm formation. Assays developed to examine these cooperative behaviors did not identify BdAU0158 CDS; however, these assays may be

126 specific to BtE264 and not applicable to BdAU0158. Comparing the transcriptomic profiles of

CDI-proficient BdAU0158 to those of bcpAIOB mutant BdAU0158 strains (either deletion mutations or mutations abrogating BcpA-CT toxicity) would reveal whether CDI systems induce global gene expression changes in this pathogen. CDS could play a role during Bcc infection of the CF respiratory tract. Although these pathogens do not form biofilms in the airways and typically reside within macrophages and epithelial cells (18), intercellular communication may be important during host cell infection. Bcc pathogens reside within vacuoles and the endoplasmic reticulum during macrophage infection, where they can replicate to high numbers (38, 39), and thus behaviors important for intracellular growth and survival may be coordinated by CDS.

4.4 T6SS-Mediated Burkholderia cepacia Complex Colonization and Infection of the

Cystic Fibrosis Respiratory Tract

In Chapter 3 of this dissertation, I characterize the T6SS of B. cenocepacia strain

AU1054 (BcAU1054), an epidemic Bcc strain, and describe potential human colonization dynamics mediated by the T6SS in the context of competition against P. aeruginosa. Like CDI systems, T6SSs are widespread among Gram-negative bacteria, with ~25% of all published

Gram-negative genomes containing predicted T6SS-encoding gene clusters (40). Notably, and unlike CDI systems, T6SSs target a wide range of prey cells, including bacteria from different classes of Proteobacteria (41), fungi (42), amoebae (43), and human cells (44). Given its promiscuity, T6S may provide a competitive advantage to several bacterial pathogens of the polymicrobial CF respiratory tract, including P. aeruginosa (45), Bcc species (46), and

Achromobacter xylosoxidans (47). P. aeruginosa is a T6SS-proficient bacterium, having three separate T6SSs (48), and often being extremely aggressive in co-cultures with non-kin bacteria

(at least in experiments using laboratory strains) (41, 49, 50). Since P. aeruginosa can predominate in the CF airways (2, 3, 5), mechanisms of interbacterial competition like the T6SS

127 may provide this pathogen a strong competitive advantage; however, my research shows this is not the case for all P. aeruginosa infecting the CF respiratory tract.

Genomic techniques have improved our understanding of the diversification of P. aeruginosa within CF patients (51-53), as well as the diversity among individual laboratory isolates of the reference strain PAO1 (54). Therefore, we cannot attribute all findings from experiments using reference strains to the true P. aeruginosa isolates infecting patients. My studies demonstrated a Bcc pathogen (BmCGD2M) was able to efficiently kill the T6SS- proficient P. aeruginosa reference strain PAO1, and thus PAO1 must not be invincible in competitions against other bacterial pathogens. In experiments using P. aeruginosa strains more relevant than PAO1, specifically recently-collected isolates from CF patients, I showed that host adaptation can render P. aeruginosa T6SS-deficient, and thus highly susceptible to being killed by the BcAU1054 T6SS. P. aeruginosa is known to evolve within its human host over many years to transition to a chronic infection lifestyle (51, 52), which is beneficial for the pathogen; however, this host adaptation could be detrimental if the P. aeruginosa population loses T6SS activity and a Bcc pathogen invades the microbial community of the CF respiratory tract, as the Bcc pathogen may use its T6SS to kill resident P. aeruginosa and establish an infection. These evolutionary and competitive dynamics may explain why Bcc infections are limited to older CF patients, since many years are required for P. aeruginosa to adapt to the host. Therefore, host adaptation by P. aeruginosa would not only be detrimental to CF patients by promoting chronic infection by this pathogen, but by potentially also rendering the patient susceptible to Bcc superinfections.

By using previously unstudied Bcc strains with regards to T6S (BcAU1054, BmCGD2M, and BdAU0158), I showed that T6SS-mediated competition likely provides ecological advantages to many Bcc pathogens. The T6SS-1 of B. cenocepacia strain H111 (analogous to the T6SSs I studied of BcAU1054, BmCGD2M, and BdAU0158) confers a modest competitive advantage to this strain (46), whereas BcAU1054, BmCGD2M, and BdAU0158 all showed

128 strong T6SS-mediated interbacterial competition in my research. Perhaps the B. cenocepacia strain H111 T6SS is less active than the T6SSs of other strains due to regulatory reasons

(either transcriptional, post-transcriptional, or post-translational), or the conditions and/or target strains used in the previous study influenced the extent of T6SS-mediated competition by that strain. The prevalence of T6SS-encoding gene clusters among Bcc species indicates these bacteria use T6S during intercellular interactions in their natural environments, but also brings into question how T6SSs are distributed among species. In the Bcc, the T6SS-1 is the most prevalent system and likely arose from a common ancestor, but certain species also contain additional T6SSs (46). BmCGD2M and BdAU0158 have two and three T6SSs, respectively, though only one system of each (the T6SS-1) mediated interbacterial competition in my studies.

Conversely, BcAU1054 and B. cenocepacia strain J2315 (BcJ2315) have only one T6SS each.

By comparing the gene clusters encoding the T6SSs of BcAU1054 and BcJ2315, I showed that diversification of individual T6SSs can occur, as the BcAU1054 cluster contains additional elements compared to that of BcJ2315. Although it is difficult to determine which T6SS gene cluster of these two strains originated first, regions of the BcAU1054 cluster that are missing from the BcJ2315 cluster include genes predicted to be involved in phage and other mobile genetic elements; thus BcAU1054 may have gained these additional sequences via horizontal gene transfer.

Although I identified four BcAU1054 antibacterial T6SS effectors and the cognate immunity proteins to these effectors, the fact that BcAU1054 ∆9E-I (which lacks all nine predicted effector-immunity (E-I) pairs) still efficiently killed target bacteria shows there are unidentified E-I pairs of this strain. Since T6SS E-I pairs are examples of polymorphic toxin- antitoxin pairs, the amino acid sequences of E-I proteins are dramatically diverse among different bacterial strains and species (55), making it difficult to identify genes encoding these proteins solely by genome browsing. The development of bioinformatic pipelines to predict

T6SS E-I pairs is in its infancy (56), and the use of such pipelines may identify the additional

129 BcAU1054 T6SS E-I pair(s). Alternatively, proteomics comparing the proteins secreted by

T6SS-active and T6SS-inactive BcAU1054 may identify the additional effector(s); similar techniques have been used to identify effectors delivered by the T6SSs of Vibrio proteolyticus and Vibrio cholerae (57, 58).

Complex regulation of T6SS assembly and activity has been characterized in P. aeruginosa, though outstanding questions remain regarding the regulation of T6SSs in Bcc bacteria. AtsR is a sensor kinase-response regulator hybrid of B. cenocepacia with slight amino acid sequence identity (19%) to RetS of P. aeruginosa, which inhibits T6SS protein production by P. aeruginosa (59, 60); likewise, a plasmid disruption in atsR increases T6SS activity by B. cenocepacia (59). I show the BcAU1054, BmCGD2M, and BdAU0158 T6SSs are strong antibacterial weapons without manipulation of these strains’ atsR genes, and thus there must be conditions where AtsR is not repressing T6SS activity by Bcc bacteria. Derepression of T6SS protein production by AtsR may occur during growth on solid surfaces, or during co-culture with competitor bacteria. Alternatively, AtsR may not repress T6SS activity in the Bcc strains used in my research, as this AtsR function was elucidated in B. cenocepacia strain K56-2 (59).

An additional intriguing T6SS regulation question emanating from my studies centers on the enhanced ability of BcAU1054 to kill BdAU0158 and P. aeruginosa target cells compared to

E. coli target cells. Experiments using P. aeruginosa strain PAO1 have shown that this bacterium is more T6SS-active when co-cultured with antagonistic bacteria, like V. cholerae,

Acinetobacter baylyi, and BtE264 (all of which are T6SS-proficient) compared to co-cultures with less aggressive bacteria like E. coli (41, 49). The authors of these studies propose two explanations for this behavior: T6SS-dueling, where PAO1 retaliates to T6SS attack from a neighboring cell by launching a T6SS counterstrike (49), and the P. aeruginosa response to antagonism (PARA), where lysed PAO1 cells stimulate T6SS activity by their viable kin cells

(41). T6SS-dueling and PARA require activity by GacSA, Fha1, PppA (as well as other proteins), and I found the genes encoding these proteins were mutated in host-adapted P.

130 aeruginosa isolates that were susceptible to the BcAU1054 T6SS. Therefore, P. aeruginosa cells within teenage/adult CF patients may not be able to retaliate to attack by Bcc pathogens, thus allowing Bcc pathogens to invade these microbial communities and establish infections.

Given that BcAU1054 better outcompeted BdAU0158 and P. aeruginosa than it outcompeted E. coli, this strain (and other Bcc pathogens) may show increased T6SS activity against antagonistic neighboring cells, analogous to T6SS activity by PAO1. Nevertheless, BcAU1054 showed the strongest T6SS-mediated competitive advantage in my studies against T6SS- defective host-adapted P. aeruginosa isolates, indicating that T6SS attack by P. aeruginosa is not required for T6SS activity by BcAU1054. Perhaps BcAU1054 and other Bcc pathogens have evolved to sense commonly encountered competitor bacteria, like P. aeruginosa, and activate

T6S upon detecting these competitors.

Interbacterial competition is not the only role of T6S in Bcc pathogens. Several studies have described the B. cenocepacia T6SS in the context of macrophage infection. The T6SS- delivered effector TecA promotes B. cenocepacia infection of macrophages, but also stimulates pyroptosis by infected macrophages (38, 59, 61-63), which may exacerbate airway inflammation in CF patients. Although TecA is not conserved throughout the Bcc (63), additional T6SS effectors (or additional T6SSs in species harboring multiple systems) may be important for host cell infection. In addition to T6SS-mediated competition and virulence by Bcc pathogens, the

T6SS may promote cooperative behaviors by these bacteria, similarly to CDI/CDS. Although my research focused solely on T6SS-conferred competitive advantages of Bcc pathogens, this secretion system may coordinate intercellular communication and cooperation. It has been proposed that T6SS effectors delivered between Proteus mirabilis kin cells mediate cooperative behaviors (64, 65). Similar T6SS-mediated kin cell cooperation may occur in Bcc bacteria, as well as other T6SS-proficient CF pathogens. Like CDI toxins, some T6SS effectors are nucleases, and in the cytoplasm of kin cells that are immune to each other’s effectors, nuclease effectors could associate with their cognate immunity proteins to form a signaling complex that

131 acts on DNA, mRNA, or cyclic nucleotide global regulators like cyclic adenosine monophosphate and cyclic diguanylate. Within the CF respiratory tract, T6SS-mediated communication and cooperation among Bcc bacteria may promote host cell infection or extracellular lifestyles upon escape from host cells, and thus be important for establishing long- term infections in these patients.

4.5 Future Investigations for the Burkholderia cepacia Complex Sociomicrobiology Field

My thesis research has characterized two mechanisms of cell contact-dependent interbacterial competition by Bcc pathogens: CDI and T6S. Given the prevalence of genes predicted to encode CDI systems and T6SSs within Bcc species (22, 25, 29, 46), as well as within strains of P. aeruginosa (45, 66-68), intercellular interactions mediated by these systems may play a role during CF infections. However, these interactions would require direct cell-cell contact, which may be precluded by the architecture of the respiratory tract. In order to investigate potential impacts of CDI and T6S on Bcc colonization and infection of CF patients, there needs to be drastic improvements in animal models to study chronic infections caused by

CF pathogens (69, 70). To date, experimental infection of mouse models with Bcc pathogens has typically involved acute infections (71-73). To investigate long term infections caused by

Bcc pathogens, chronic infection models must be developed. Moreover, to examine Bcc T6SS- mediated invasion of P. aeruginosa-infected respiratory tracts, chronic infection models need to be developed for P. aeruginosa. The best model for chronic respiratory infection by P. aeruginosa involves embedding the bacteria in agar beads (74, 75). Despite prolonged infection in these models, agar beads would likely inhibit Bcc-P. aeruginosa interactions, especially during experiments where P. aeruginosa-colonized animals are subsequently inoculated with

Bcc pathogens. Animals genetically manipulated to exhibit CF pathophysiology, such as the b-

ENaC mouse model (76), as well as ferret and pig models (77, 78), may provide useful for studies of Bcc sociomicrobiology during CF infection.

132 In addition to improving our animal model repertoire, longitudinal human studies certainly would improve our understanding of Bcc infections. Genomic experiments have been conducted to assess the evolution of P. aeruginosa within CF patients (51), and similar studies would illuminate the evolutionary capabilities of Bcc pathogens. A few longitudinal experiments have been conducted on Bcc-infected CF patients, though more are required to gain comprehensive understanding of host adaptation by these pathogens (79-82). Two of these studies analyzed longitudinal isolates from the B. dolosa epidemic at Boston Children’s Hospital (79, 80); intriguingly, the bcpA-2 gene, which encodes the BcpA-2 exoprotein of one of the CDI systems produced by B. dolosa, was found to be under parallel adaptive evolution in CF patients.

Therefore, this CDI system, and potentially other mechanisms of interbacterial competition within the Bcc (in addition to the B. cenocepacia T6SS), are important for human infection. My research detailing T6SS-mediated interactions between Bcc pathogens and P. aeruginosa could also be followed up on by longitudinal human studies. If Bcc pathogens can only colonize a P. aeruginosa-infected CF respiratory tract if the resident P. aeruginosa have evolved to lose T6SS activity, longitudinal genomic analyses should detect T6SS-abrogating mutations in P. aeruginosa within patients before they acquire Bcc superinfections. Once Bcc infections commence, genomic analyses could also assess the presumed genetic maintenance of T6SS gene clusters in Bcc pathogens (or at least the lack of potential T6SS-abrogating mutations in

Bcc isolates). Expanding longitudinal studies of Bcc-infected CF patients would complement the in vitro-based studies of my dissertation.

Improved animal models and human studies will increase our knowledge on the importance of interbacterial interactions during Bcc infection of the CF respiratory tract. The

T6SS is already known to be important for B. cenocepacia murine infection, and if data emerge suggesting T6SSs, and/or CDI systems, of several pathogens are important for CF infection, drugs should be developed to disrupt these systems. T6S is an attractive candidate for therapeutic intervention of CF infections as it likely plays a role in virulence and interbacterial

133 competition by Bcc bacteria, if not other pathogens as well. Additionally, T6SS-mediated interbacterial competition could be harnessed to treat P. aeruginosa infections if the resident P. aeruginosa population has become T6SS-inactive during host adaptation. Such strategies could include generating non-pathogenic T6SS-proficient probiotic bacteria to outcompete T6SS- deficient P. aeruginosa within CF airways. Altogether, our collective understanding of the social lives of microorganisms has expanded over time, and sociomicrobiology can be applied to many facets of infectious disease, including CF, to improve human health.

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141 APPENDIX

In addition to my research detailed in this dissertation, I contributed to the following studies during my graduate work:

Garcia EC, Perault AI, Marlatt SA, Cotter PA. (2016) Interbacterial signaling via Burkholderia contact-dependent growth inhibition system proteins. Proc Natl Acad Sci USA. doi: 10.1073/pnas.1606323113. PMID: 27335458. For this publication, I generated strains that were used to assess whether contact- dependent growth inhibition systems could promote interbacterial signaling between different bacterial species. I wrote portions of the Materials and Methods and helped edit the paper.

Bone MA, Wilk AJ, Perault AI, Marlatt SA, Scheller EV, Anthouard R, Chen Q, Stibitz S, Cotter PA, Julio SM. (2017) Bordetella PlrSR regulatory system controls BvgAS activity and virulence in the lower respiratory tract. Proc Natal Acad Sci USA. doi: 10.1073/pnas.1609565114. PMID: 28167784. I contributed to this publication during my rotation in the Cotter Lab. I conducted mouse infection experiments to test if the PlrSR regulatory system is important for global virulence regulation by Bordetella during infection of the respiratory tract.

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