The Importance of Listeriolysin O in Host Cell Invasion by Listeria monocytogenes and
its Use in Vaccine Development
Dissertation
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy
in the Graduate School of The Ohio State University
By
Christopher Phelps
Graduate Program in Microbiology
The Ohio State University
2019
Dissertation Committee
Stephanie Seveau, Advisor
Prosper Boyaka
John Gunn
Chad Rappleye
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Copyrighted by
Christopher Phelps
2019
2
Abstract
Listeria monocytogenes is a facultative intracellular pathogen and the etiological agent of the life-threatening disease listeriosis. L. monocytogenes is ubiquitous in the environment and frequently contaminates food products, including beef, poultry, vegetables, dairy products, and particularly ready-to-eat foods. Because L. monocytogenes can grow under conditions that are typically used for food preservation, including low temperatures and high salt concentrations, preventing contamination and effectively sanitizing food production equipment is both costly and difficult.
Consumption of contaminated food by otherwise healthy adults usually results in minor cases of noninvasive listeriosis that do not require treatment. However, particularly susceptible populations including pregnant women, the elderly, and those with compromised immune systems can develop severe invasive listeriosis in which the bacteria can cross the intestinal barrier to infect the liver, spleen, and ultimately the central nervous system, as well as the placenta, in the case of pregnant women.
L. monocytogenes has the ability to invade and replicate within a wide variety of phagocytic and nonphagocytic cells. L. monocytogenes has been known to use two major invasion factors to enter nonphagocytic cells, InlA and InlB. After endocytosis of the bacterium, L. monocytogenes disrupts the vacuole and enters the host cell cytosol using
ii the pore-forming toxin, listeriolysin O (LLO). In the cytosol, the bacterium replicates and spreads to adjacent cells.
Though InlA and InlB have been considered the primary invasion factors, LLO was recently identified as an additional factor in the invasion of hepatocytes. Importantly, during infection, all three of these factors are co-expressed and have the potential to act in concert with one another. In our work, for the first time, we demonstrate the relative roles and importance of LLO, InlA, and InlB during host cell invasion. LLO is an important invasin in a variety of hepatocytes, but not placental cells, or endothelial cells, and cooperates with InlA to potentiate invasion. In addition to promoting invasion of enterocytes, InlA also promotes association with human hepatocytes and placental cells.
We identified no role for InlB initially. We only observed a role for this factor when we introduced a constitutively active transcription regulator PrfA*, suggesting that InlB may not be as critical to host cell invasion in strains with properly regulated virulence regulon expression.
In light of the importance of LLO during infection, we have developed a vaccine that uses an LLO toxoid variant (LLOT) as an antigen and cholera toxin as an adjuvant.
We found that this vaccine leads to anti-LLO antibody production and the development of a Th1 type immune response. Protection in the intravenous, non-pregnant murine model appears to be independent of anti-LLO antibodies in the context of full functional
T cell response.
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Dedication
I would like to dedicate this document to my family and friends for their never-ending support throughout my education. I would, in particular, like to thank my wife, Emily
Phelps, for her love, support, and encouragement and for never failing to believe in me.
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Acknowledgments
I would like to thank members of the Seveau lab that have been supportive both in our research and in my development throughout my time as a Ph.D. student: Dr. Jonathan
Lam, Lauren Johnson, Siavash Azari, Dr. Sarika Pathak-Sharma, Dr. Joanna Marshall,
Jasneet Singh, Elizabeth Puleo, Bella Cho, and Meghan Linz.
I would like to acknowledge and thank our collaborators at Ohio State who have made many important contributions to this work. I would like to thank Dr. Prosper
Boyaka and his lab members Zayed Attia and Haley Steiner for their assistance in both the development and implementation of our vaccine project. Dr. Abhay Satoskar and his lab member Sanjay Varikuti also provided a great deal of assistance with mouse infections and the development of the vaccine project. Dr. Mikhail Gavrilin provided significant assistance and advice in developing new quantitative PCR protocols for our projects. Dr. Xiaoli Zhang and Yubo Tan provided assistance with statistical analysis of our data. I would like to acknowledge our funding source, the National Institute of
Health, NIAID.
I would also like to acknowledge my committee members, Dr. Prosper Boyaka,
Dr. John Gunn, and Dr. Chad Rappleye, for their comments and suggestions during committee meetings and their willingness to be references both for various grant applications and my job search. Last and most importantly, I would like to thank my
v advisor, Dr. Stephanie Seveau for her support, dedication, and patience as she has guided me through my doctoral training.
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Vita
2010-2013……………………………………………...Undergraduate Research Assistant
Department of Biological Sciences
Bowling Green State University
2013……………………………………………………………………..B.S. Microbiology
Magna Cum Laude
Bowling Green State University
2013-2015…………………………………………………...Graduate Teaching Associate
Department of Microbiology
The Ohio State University
2015-2018…………………………………………………...Graduate Research Associate
Department of Microbiology
The Ohio State University
2017-2019…………………………………………Delegate (Representing Microbiology)
Council of Graduate Students
The Ohio State University
2019-present…………………………………………………Graduate Teaching Associate
Department of Microbiology
The Ohio State University
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Publications
Phelps, C.C., Vadia, S., Arnett, E., Tan, Y., Zhang, X., Pathak-Sharma, S., Gavrilin,
M.A., Seveau, S. (2018) Relative Roles of Listeriolysin O, InlA, and InlB in Listeria monocytogenes Uptake by Host Cells. Infect Immun 86(10):e00555-18. https://doi.org/10.1128/IAI.00555-18. PMID: 30061379
Phelps, C.C., Vadia, S., Boyaka, P.N., Varikuti, S., Attia, Z., Dubey, P., Satoskar, A.R.,
Seveau, S. A Listeriolysin O_Toxoid-Based Vaccine Protects Mice against Listeria monocytogenes. (Manuscript in Preparation)
Fields of Study
Major Field: Microbiology
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Table of Contents
Abstract ...... ii Dedication ...... iv Acknowledgments...... v Vita ...... vii List of Tables ...... xi List of Figures ...... xii Chapter 1. Introduction ...... 1 Listeria monocytogenes ...... 1 1.1.1 Listeriosis ...... 1 1.1.2 Intracellular lifecycle of L. monocytogenes ...... 4 1.1.3 L. monocytogenes virulence factors ...... 6 1.1.4 PrfA, the regulator of virulence ...... 7 1.1.5 InlA ...... 11 1.1.6 InlB ...... 13 1.1.7 Listeriolysin O ...... 15 L. monocytogenes infection in vivo...... 21 1.1.8 Innate Immune Responses ...... 21 1.1.9 Adaptive Immune Responses ...... 24 Research and specific aims ...... 26 Chapter 2. Roles for Listeriolysin O, InlA, and InlB in Listeria monocytogenes Uptake by Host Cells ...... 28 2.1 Introduction ...... 28 2.2 Materials and Methods ...... 31 2.3 Results ...... 43 2.4 Discussion ...... 56 ix
Chapter 3. A Listierolysin O Toxoid-Based Vaccine Protects Mice Against Listeria monocytogenes ...... 62 3.1 Introduction ...... 62 3.2 Materials and Methods ...... 64 3.3 Results ...... 71 3.4 Discussion ...... 82 Chapter 4. Conclusions and Future Directions ...... 88 Bibliography ...... 92 Appendix A. Chapter 2 supplemental Figures ...... 113
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List of Tables
Table 2.1 L. monocytogenes strains used in this study ...... 31
Table 2.2 Primers used in this study ...... 33
Table 2.3 Invasion factor cooperation analysis ...... 55
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List of Figures
Figure 1.1 Route during infection by L. monocytogenes ...... 2
Figure 1.2 The intracellular lifecycle of L. monocytogenes ...... 5
Figure 1.3 The PrfA Regulon...... 8
Figure 1.4 Crystal Structure of Listeriolysin O ...... 18
Figure 1.5 Model for LLO-mediated internalization of L. monocytogenes ...... 19
Figure 2.1 inlA, inlB, and hly mRNA quantification ...... 43
Figure 2.2 InlB and LLO protein levels ...... 45
Figure 2.3 Relative roles of LLO, InlA, and InlB in L. monocytogenes invasion of human hepatocytes ...... 46
Figure 2.4 The InlB/c-Met pathway is functional in hepatocytes ...... 48
Figure 2.5 A prfA* mutation in L. monocytogenes strain 10403S leads to increased production of InlB and InlB-dependent hepatocyte invasion ...... 50
Figure 2.6 Role of LLO, InlA, and InlB in L. monocytogenes invasion of human cytotrophoblasts ...... 52
Figure 2.7 Absence of a role for LLO, InlA, and InlB in L. monocytogenes invasion of human endothelial cells...... 53
Figure 3.1 Characterization of LLOT ...... 71
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Figure 3.2 LLOT plus adjuvant CT protects mice against infection by L. monocytogenes
...... 73
Figure 3.3 LLO neutralizing antibodies are produced following immunization with LLOT plus CT ...... 74
Figure 3.4 LLOT coupled with CT but not alum protects mice from L. monocytogenes . 75
Figure 3.5 Immunzation with LLOT with either cholera toxin or alum as adjuvant lead to serum LLOT-specific IgG production with cholera toxin specifically inducing IgG2a,
IgG2b, and IgG3 isotypes ...... 77
Figure 3.6 Cholera toxin and alum adjuvants triggers balanced Th1, Th2, and Th17 responses, with significant increase in Th1 responses associated with CT compared to alum...... 79
Figure 3.7 Immunization with LLOT coupled with CT adjuvant is effective in both WT
C56BL/6 and µMT-/- mice that lack mature B cells ...... 81
Figure A.1 Validation of anti-InlB antibodies ...... 113
Figure A.2 Cooperation of LLO, InlA, and InlB in bacterial invasion of human hepatocytes ...... 114
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Chapter 1. Introduction
Listeria monocytogenes
1.1.1 Listeriosis
The genus Listeria is currently comprised of 20 recognized species of Gram- positive bacteria (1-4). Of these species, only two, L. monocytogenes and L. ivanovii, are known to be pathogens (5). While L. ivanovii is primarily known for infecting ruminants, especially sheep and cattle (6-9), L. monocytogenes has been reported to cause disease in over 40 animal species, and is the causative agent of the deadly disease listeriosis in humans (1, 5). L. monocytogenes is found ubiquitously in the environment, where it survives as a saprophyte in a wide range of niches, including soil, silage, sewage, and water sources (10, 11). The ubiquity of the bacteria, its ample exposure to farm animals that can be infected, and its ability to survive a wide range of temperatures, salt concentrations, and pH levels leads to high risks of contamination and survival in food processing plants and ready-to-eat foods (11). Additionally, without stringent cleaning and decontamination procedures, bacterial contamination can persist for months or years
(12).
In otherwise healthy adults, consumption of food contaminated with L. monocytogenes typically results in no clinical disease or in noninvasive listeriosis with relatively minor symptoms, such as gastrointestinal distress, diarrhea, and flu-like
1 symptoms (10). However, the risk for more severe disease is considerably higher in susceptible populations including pregnant women, the elderly, and those with compromised immune systems (5). In these susceptible populations, consumption of L. monocytogenes can result in life-threatening invasive listeriosis. In these cases, L. monocytogenes successfully crosses the intestinal barrier and colonizes the liver and spleen. The bacteria can enter the blood stream, cross the blood brain barrier, and, in the case of pregnant women, cross the placental barrier and infect the fetus (13, 14) (Figure
1.1).
Figure 1.1 Route during infection by L. monocytogenes L. monocytogenes enters the intestine within contaminated food and crosses the intestinal barrier. The bacteria colonize the liver and spleen, and if not cleared by the immune system, it enters the bloodstream and can cross the blood-brain barrier to infect the brain or the placental barrier. Figure adapted from Lecuit, 2007 (15).
This can lead to complications including septicemia, meningoencephalitis, premature birth, miscarriage, and severe infection of neonates. Treatment of invasive listeriosis primarily involves intravenous ampicillin or penicillin G (16). In certain cases,
2 such as infection of the central nervous system, or endocarditis, or in cases involving neonates or immunocompromised patients, gentamicin is add to the treatment regimen
(17-19). Early treatment is essential to positive outcomes. For example, when infection of the central nervous system is suspected or confirmed, delaying therapy for more than six hours is associated with increased mortality (20).
Many different strains of L. monocytogenes have been identified over the years.
These strains have been divided into four lineages (I-IV) and thirteen serotypes (21). The majority of isolates obtained belong to lineages I and II with isolates from lineages III and IV being relatively rare and coming predominately from animal sources (21).
Clinical cases of listeriosis are most commonly associated with a subset of serotypes,
1/2a (Lineage II), 1/2b (Lineage I), and 4b (Lineage I) (22). The more rarely isolated
Lineage III strains have not been linked with large-scale listeriosis outbreaks, but they are occasionally isolated from sporadic clinical cases (23). Historically, the majority of human listeriosis outbreaks have been linked with serotype 4b, but outbreaks involving serotype 1/2a and 1/2b and serotype 1/2a are becoming more common (24). Serotype 4b has also been found to have a higher association with pregnancy related listeriosis cases than non-pregnancy related cases. Indeed, in one study, serotype 4b was linked to 79.5% of pregnancy related infections and only 61% of non-pregnancy cases (25). The rate of serotypes 1/2a and 1/2b increases significantly in the non-pregnancy cases. Other work has shown similar high prevalence of serotype 4b amongst cases of meningitis (26). This study also demonstrated how uncommon meningitis is in cases of listeriosis during pregnancy. Though the risk of listeriosis during pregnancy is high, only 1 out of the 129
3 patients exhibiting meningitis was also pregnant (26). Interestingly, the three most commonly studied lab strains of L. monocytogenes, EGD, EGD-e, and 10403S all belong to Lineage II and serotype 1/2a.
1.1.2 Intracellular lifecycle of L. monocytogenes
L. monocytogenes ability to survive and thrive within a host, avoiding host immune defenses, is indispensably linked to its intracellular lifecycle (Figure 1.2). L. monocytogenes can survive within a wide variety of cell types. Infected cells include professional phagocytes such as macrophages, but also normally non-phagocytic cells, including various epithelial cells, fibroblasts, and endothelial cells (5). To successfully invade non-phagocytic cells, L. monocytogenes hijacks host signaling pathways via a number of bacterial proteins, including the surface proteins internalin (InlA) and InlB.
InlA and InlB bind host cell receptors to trigger endocytosis of the bacteria by these normally non-phagocytic cells (27, 28). To escape the endocytic vacuole, L. monocytogenes utilizes the pore-forming toxin listeriolysin O (LLO) and two phospholipases, a broad-range phospholipase C (PC-PLC) and a phosphatidylinositol specific phospholipase C (PI-PLC) (29-31). Once in the cytosol, the bacterium polymerizes host cell actin via the bacterial protein ActA to generate the motile force needed to move randomly through the cytosol (32). Upon encountering the host cell membrane, the bacterium can push on the membrane and create a protrusion that can be endocytosed by neighboring cells in a process known as cell-to-cell spread (33). Upon spreading to the new host cell, the bacterium is trapped within a double membrane 4 secondary vacuole. This secondary vacuole is disrupted by the combined activity of LLO and the phospholipases, and the intracellular lifecycle can continue (34).
Figure 1.2 The intracellular lifecycle of L. monocytogenes L. monocytogenes expresses invasion factors, InlA and InlB, which promote attachment and internalization. The bacteria then escapes the vacuole using LLO and enters the host cytosol where it can replicate. L. monocytogenes uses the surface protein ActA to polymerize host cell actin to propel itself through the cytosol and form protrusions that are endocytosed by adjacent cells. The bacteria then disrupt the secondary vacuole with LLO and the phospholipases. Figure adapted from Tilney et al., 1989 (32).
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1.1.3 L. monocytogenes virulence factors
L. monocytogenes encodes many virulence factors that facilitate a variety of critical processes, from adhesion to a variety of host cells and the induction of bacterial uptake to vacuolar escape, intracellular survival, and cell-to-cell spread. Many of the most well-studied virulence factors are clustered in two separate sections of the L. monocytogenes chromosome: the pathogenicity island (Listeria pathogenicity island – 1;
LIPI-1) (35) and the internalin operon (Figure 1.3) (36). LIPI-1 is thought to have been acquired through bacteriophage transduction in an ancestor of the Listeria genus and stabilized in what became the pathogenic species (35, 37). This region consists of five genes (plcA, hly, mpl, actA, and plcB) that work together to promote the intracellular lifecycle, and a sixth gene, prfA, which encodes the master regulator or virulence (38).
The internalin operon contains the genes inlA and inlB, which have historically been thought to be the major mediators of bacterial internalization into normally non- phagocytic cells.
The internalin family of virulence factors is a large group of L. monocytogenes proteins consisting of 27 known members (39). The characteristic feature of the internalins is the leucine-rich-repeat (LRR) motif. LRRs are binding motifs for protein- protein interactions in eukaryotes and prokaryotes (40). The internalins are classified into three categories, depending on their interaction with the bacterial cell surface: 1) LPXTG internalins that are covalently bound to bacterial peptidoglycan, 2) GW and WxL internalins that are non-covalently, via electrostatic interactions, associated with the cell wall, and 3) secreted internalins (41). The LRRs of the various internalins bind to a wide 6 variety of both extracellular and intracellular host proteins to promote many processes during infection, including attachment to host cells, bacterial internalization, cell-to-cell spread, and even specific invasion of the brain or placenta (42-45). It is thought that these highly related proteins are the result of a number of gene duplications and recombination events that have occurred throughout the evolution of the Listeria genus (46).
1.1.4 PrfA, the regulator of virulence
The major regulator of virulence genes in L. monocytogenes, including the genes of LIPI-1 and many members of the internalin family, are chiefly controlled by the master regulator PrfA as part of the PrfA regulon (Figure 1.3) (38, 47, 48). PrfA is part of the cAMP receptor protein (Crp) family of transcription factors (49). As such, functional PrfA forms a symmetrical homodimer, and each polypeptide includes an N- terminal domain that mediates monomer-monomer interactions and a C-terminal domain that consists of a DNA-binding helix-turn-helix (HTH) motif (38, 50). PrfA, and its control over the virulence factor expression of L. monocytogenes, are thought to be the critical keys for shifting the bacterium from environmental saprophyte to intracellular pathogen (51). Because precise control of the virulence gene expression is required to maximize fitness in both the environment and the host, PrfA-dependent gene expression is modulated by multiple regulatory mechanisms.
A regulatory feature of PrfA that, in part, allows for the differential expression of multiple PrfA-dependent genes is the varying affinity for the different promoters. PrfA binds to 14 bp palindromic nucleotide sequences upstream of the target gene, the PrfA 7 box. Some promoters like PplcA and Phly have completely symmetrical PrfA boxes to which PrfA binds with high affinity (38, 49). These genes are more sensitive to lower concentrations of PrfA when compared to other genes with promoters that have less than perfectly symmetrical PrfA box sequences.
Figure 1.3 The PrfA Regulon PrfA regulates numerous genes, including those of the LIPI-1 and the inlAB operon. Promoters for specific genes are indicated with “Px” and specific transcript products are indicated below each gene with a dotted line. Binding locations for PrfA are indicated by black boxes. Figure adapted from Scortti et al. 2007 (38).
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The affinity of PrfA for the various PrfA-dependent promoters gives some insight into the differential expression of genes, but it does not necessarily regulate the “on” or
“off” state of the PrfA regulon. In contrast, the concentration of PrfA in the cell and mechanisms that increase or decrease PrfA concentration also play a role in switching virulence “on” and “off”. To begin, even without being in its host niche, L. monocytogenes has a basal level of transcription of the prfA gene that is controlled by two sigma factors that bind to two of the three prfA promoters (52, 53). One of the triggers for increasing PrfA-dependent gene expression is an increase in temperature from below 30°C to above 30°C. This temperature shift is indicative of the transfer from environmental life to life within the host and is detected by a thermoswitch in the 5’ UTR of prfA transcript (54). At temperatures under 30°C, the UTR forms a hairpin that blocks access to the ribosome binding site for the gene. When temperatures rise above 30°C, the ribosomal binding site becomes available for translation to proceed, and the protein is produced (54). Once a pool of active PrfA is produced, the transcription factor can bind the PplcA promoter (55-57). This leads to the production of a bicistronic plcA-prfA transcript and, consequently, more PrfA production, in a positive feedback loop that works to increase cellular PrfA concentrations when temperatures rise above 30°C.
For many years, it was also speculated that, like other members of the Crp family,
PrfA may bind to a cofactor to increase its activity (58-60). Indeed, initial evidence that
PrfA may have a weakly activated form and a highly active form bound to a cofactor came in the form of mutants that are hyperactive and lead to constitutive overexpression of PrfA-dependent genes in non-permissive conditions (61). These mutations that lead to
9 increased affinity for target DNA sequences were designated PrfA* mutations. These
PrfA* mutations were equivalent to constitutively active Crp mutants, known as Crp* mutations, that are highly active without their activating cofactor cAMP (50, 62). The mutations also resulted in similar changes to each protein’s crystal structure. The structural similarity between PrfA and Crp and equivalent effects of these mutations supported the hypothesis that PrfA was also allosterically activated by a cofactor. Indeed, recently, it was proposed that the cofactor for PrfA is glutathione and that glutathione binding to PrfA triggers its high activity state (63, 64). Evidence suggests that extracellular glutathione, which is present within host cells, is imported, and when combined with the production of bacterial glutathione, allosterically binds to PrfA and induces enhanced PrfA-dependent gene expression (65).
Overall, when in the environment, there is a basal level of transcription of the prfA gene, but translation is limited by the thermoswitch. Upon entering a host body, the thermoswitch releases, permitting translation of the PrfA transcription factor. This causes a positive-feedback loop with PrfA activating the transcription of more prfA. At this stage, PrfA is in its weakly active state, but this activity is sufficient for the production of virulence genes that are needed to enter the intracellular niche. Once intracellular, host cell glutathione induces the production of bacterial glutathione, which acts as an allosteric activator of PrfA. This highly active PrfA then continue to promote the intracellular lifecycle.
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1.1.5 InlA
Internalin (InlA) is the prototype for the entire internalin family of L. monocytogenes virulence factors and was the first member of the family to be discovered
(28). It is a major mediator of bacterial association with host cells. The inlA gene is under the transcriptional control of PrfA (36). The protein consists of an N-terminal secretion signal sequence, an LRR domain, an inter-repeat (IR) region, and a C-terminal LPXTG motif (66). Using the N-terminal signal sequence, the protein is targeted by SecA machinery and secreted. The signal sequence is removed, and a sortase, SrtA, cleaves the
LPXTG motif between the threonine and glycine, and covalently binds the C-terminal threonine to bacterial peptidoglycan (67, 68). Once anchored to the bacterial surface,
InlA plays a significant role in both attaching L. monocytogenes to and triggering the internalization of the bacterium into normally non-phagocytic cells that express its sole receptor, E-cadherin (69). E-cadherin is a cell-cell adhesion molecule that forms homodimers via its extracellular terminal domain, forming the adherens junctions of some polarized epithelial cells (70, 71). The interaction between InlA and E-cadherin leads to the receptor clustering within cholesterol-rich lipid rafts (72). The cytoplasmic domain of E-cadherin is then phosphorylated by Src kinase and subsequently ubiquitinated by Hakai, a ubiquitin-ligase (73). These post-translational modifications lead to the recruitment of clathrin-mediated endocytic machinery, in particular, dynamin, which recruits cortactin. Cortactin is an activator of the Arp2/3 complex, which is itself one of the primary actin nucleators (74). This cascade results in actin remodeling and internalization of both the receptor and the attached pathogen into an endocytic vesicle. 11
InlA is widely regarded as being important for productive human infections.
Indeed, it has been noted that clinical strains overwhelmingly express full length versions of InlA, with very rare cases of these strains expressing the inactive truncated variants
(75). Conversely, the prevalence of strains with truncated InlA genes is disproportionately higher in isolates obtained from food samples. Other work has found a similarly high proportion of strains expressing full-length InlA in animal infections, in addition to human infections relative to food isolates, suggesting that InlA may be essential for the successful colonization of a variety of mammals (76).
Despite the apparent importance of InlA, study of its role in virulence in vivo has proven difficult, because in mice, a primary animal model for listeriosis, E-cadherin contains one amino acid change at position 16 from a proline to a glutamic acid that prevents its association with InlA (77). As such, InlA-dependent invasion pathways are not functional in wild type mice. Efforts have been made to develop “humanized” mice expressing human E-cadherin (hEcad) (78, 79), as well as to “murinize” InlA so that it can bind murine E-cadherin (80). The genetic manipulation of mice has found some success with the hEcad mice being susceptible to InlA-dependent infection of the intestine via oral inoculation. Though this particular mouse strain is not commercially available and limited work has utilized it, the strain seems to be an excellent model.
Murinization of InlA also found initial success in making the InlA-dependent infection of mice functional; however, further studies indicated that, in addition to giving InlA the ability to bind mouse E-cadherin, the InlA is also able to bind N-cadherin, which
12 significantly affects the tropism of the bacterium (81). InlA can also bind E-cadherin in gerbils, guinea pigs and rabbits (82).
E-cadherin is primarily found in epithelial cells that form cell adherens junctions, such as the cells of the intestinal lining (83). Indeed, one of the first lines of defense L. monocytogenes encounters during infection is the physical intestinal barrier. InlA is thought to play a significant role in traversing this first major barrier by binding to accessible E-cadherin on senescent enterocytes and goblet cells, which both express E- cadherin on their luminal side. It was proposed that the InlA-E-acdherin interaction mediates bacterial transcytosis from the apical to the basolateral side of the epithelium without invading the cytosol (84). This essential role in overcoming one of the initial challenges to infection may be a primary reason that strains with truncated inlA genes are so rare amongst human clinical and animal isolates (75, 76). Though they may exist and survive within the environment, they cannot make the jump to intracellular pathogen.
1.1.6 InlB
InlB is another well studied member of the internalin family of virulence factors
(39). The inlB gene is the second gene in the inlAB operon, which transcription is regulated by PrfA (85). Once transcribed and translated, similar to InlA, InlB contains an
N-terminal secretion signal peptide, an LRR domain, and 3 N-terminal repeat modules that begin with glycine (G) and tryptophan (W) amino acid residues (GW motifs) (86).
InlB is non-covalently associated with the cell wall via electrostatic interactions between the InlB C-terminal, GW motifs, and bacterial lipoteichoic acid (87). Because this 13 interaction is relatively weak, InlB exists both in association with the bacterial surface, and as released, extracellular soluble protein. Unlike InlA, which has a single receptor that is not ubiquitously expressed throughout the host, InlB has a number of widely available receptors, including the hepatocyte growth factor receptor (HGF-R) c-Met, glycosaminoglycans (GAGs), and the receptor for the globular portion of C1q complement protein (gC1q-R) (88-90).
The primary and first identified receptor for InlB is c-Met, a receptor tyrosine kinase that is expressed by many epithelial cells, but also in endothelial cells, neurons, and a variety of other cells (91). C-Met is involved in a number of important processes during embryonic development, including cell proliferation, invasive growth, and morphogenesis (92, 93). Like the interaction between InlA and E-cadherin, the binding of
InlB to c-Met, via the LRR domain, triggers post-translational modification of the receptor’s cytosolic domain (94). InlB binding leads to dimerization of the receptor leading to induction of its autophosphorylation (95). This, in turn, leads to recruitment of the ubiquitin-ligase, Cbl, and ubiquitination of the receptor (96). These modifications induce further recruitment of a number of signaling molecules and adaptor proteins, including growth factor receptor-bound protein 2 (Grb2), Grb2-associated binder 1
(Gab1), Shc, and activation of PI3K (97). With the recruitment of additional factors,
Arp2/3 is activated to induce actin cytoskeletal rearrangements and ultimately internalization of the bacterium (98, 99).
The other, less well-studied receptors for InlB are GAGs and gC1q-R. Both are recognized by the C-terminal GW domains. Data suggests that InlB binding to GAGs can
14 promote InlB-mediated bacterial invasion of host cells (89). However, data on the role of gC1q-R is controversial, with initial reports identifying the interaction as a promoter of internalization, and later reports suggesting that the interaction antagonizes InlB signaling
(88, 100).
Like InlA, InlB has some species-specific tropism. In contrast to InlA, which is known to be non-functional in mice, InlB is capable of interacting with murine c-Met, and InlB has been shown to be important for the infection of mice in vivo. InlB can also bind to gerbil, and rat c-Met However, InlB is unable to recognize c-Met in guinea pigs or rabbits (82).
1.1.7 Listeriolysin O
Listeriolysin O (LLO) is a pore-forming toxin and the major virulence factor of L. monocytogenes, which is essential for infection of mammalian hosts. Indeed, LLO deficient L. monocytogenes are completely avirulent in mice (101). LLO is a member of the largest family of pore forming toxins known as the cholesterol-dependent cytolysins
(CDCs). CDCs are expressed by numerous Gram-positive bacteria and even some Gram- negative bacteria, and, as the in case of LLO, are often associated with bacterial pathogenesis. The CDCs belong to the membrane attack complex/perforin
(MACPF)/CDC superfamily, which includes many proteins produced by eukaryotes and prokaryotes that share a common structure and are involved in pore formation (102, 103).
The CDCs are secreted as monomers that can bind to membrane-containing cholesterol. Upon binding to the host membrane, the monomers oligomerize into a
15 prepore structure that, upon subsequent conformational changes, shifts into a β-barrel transmembrane pore (104). The structures of CDC are highly conserved and are made up of 4 domains (105-109). Domain 3 (D3) is home to the pore-forming structures known as the trans-membrane β-hairpins (TMH1 and TMH2). When CDCs are secreted as monomers, these β-hairpins are in helical conformations (110, 111). Domains 1 (D1) and
2 (D2) link D3 to Domain 4 (D4). D4 has long been known as the membrane and cholesterol binding domain. The β-sandwich structure of D4 includes 3 hydrophobic loops (L1-L3) and a characteristic undecapeptide motif. It is thought that hydrophilic portions of D4 first interact with the outer leaflet of the target membrane. L1 is then inserted into the membrane, and cholesterol is bound by a conserved Threonine-Leucine pair within the loop, known as the cholesterol recognition motif (CRM) (112).
Subsequently, the other two hydrophobic loops (L2 and L3) and the undecapeptide enter the hydrophobic space of the bilayer (113). These insertions anchor the CDC to the membrane in a perpendicular orientation (114, 115). Upon successful membrane binding, conformational changes occur in D1-3 that permit oligomer formation (116, 117), which is promoted though electrostatic interactions between monomers and the new exposed amino acid residues (105, 118, 119), and formation of the prepore complex (117). When the prepore complex is complete, further conformational changes occur throughout the molecule that promote the conversion of 6 α-helices of TMH1 and TMH2 in D3 to two β- hairpins (110, 111, 120-122). Concurrently, D1-3 of each monomer collapse about 30 Å towards the membrane surface. This collapse brings the TMH into position to fully
16 disrupt the lipid bilayer (85, 114, 121, 123, 124). A representation of the LLO crystal structure and oligomerized pore complex is shown in Figure 1.4.
The pore-forming activity of LLO is essential for L. monocytogenes pathogenesis, with its earliest known and most studied role being in the process of escaping the primary and secondary endocytic vacuole or phagosome to invade the cytosol of host cells with the assistance of PC-PLC and PI-PLC, two bacterial phospholipases (29, 32, 125).
However, LLO is not required for vacuolar escape in all cells. Indeed, there are cell types in which PC-PLC is sufficient for disruption of the vacuole (30). When required for vacuolar escape, LLO forms pores on the vacuole, and this perforation of the membrane prevents full acidification of the vacuole and, in turn, delays the recruitment of LAMP-1 and full fusion with the lysosome (126, 127). It is thought that the delay in lysosomal fusion provides the environment for the PLCs to degrade the membrane, and LLO pores may even provide access to the PLC targets (128).
17
Figure 1.4 Crystal Structure of Listeriolysin O Cartoon representation of the LLO crystal structure (left). The four domains are labeled and depicted in different colors. D1 is shown in red, D2 in yellow, D3 in green, and D4 in blue. The helix bundles (HB1 and HB2) that are inserted into the membrane as trans- membrane β-hairpins are depicted in cyan. (Right) A model of an LLO pore ring is depicted with 36 LLO monomers. Adapted from Koster et al. 2014 (105)
Though LLO was thought to primarily be a phagosome-specific cytolysin, research over more than a decade has demonstrated that L. monocytogenes expresses
LLO both in the extracellular environment and in the host cytosol, and in these spaces,
LLO has a plethora of roles throughout the process of infection. In particular, our lab has shown that extracellular LLO can perforate the host cell plasma membrane and induce the internalization of bacteria and even inert beads in a Ca2+ influx and K+ efflux dependent fashion (129, 130). Indeed, it has been shown that the Ca2+ influx activates conventional protein kinase C (cPKC) which, in turn, activates the Rac1 GTPase. Rac1
18 activation ultimately induces Arp2/3-dependnet F-actin rearrangement and internalization of the bacteria (131).
Figure 1.5 Model for LLO-mediated internalization of L. monocytogenes LLO binds to cholesterol-rich membranes and forms transmembrane pores. The pore triggers Ca2+ influx which results in the activation of conventional PKC. PKC promotes the activation of Rac1, which induces Arp2/3-dependnet F-actin rearrangement that leads to L. monocytogenes internalization. Adapted from Lam et al. 2018 (131)
LLO is a unique member of the CDC family, being the only CDC secreted by an intracellular pathogen, and as such, it is particularly well adapted for use inside host cells.
Indeed, if L. monocytogenes expresses other CDCs in place of LLO, the bacteria still escapes the vacuole, but the infected host cell is subsequently killed due to the cytotoxicity (132, 133). Reciprocally, when LLO is expressed in Bacillus subtilis, the bacteria can escape phagosomes and survive within host cells (134). Due to the
19 sensitivity of the replicative niche to LLO cytotoxicity, expression and pore-forming activity are under stringent control.
Transcription of the gene encoding LLO, hly, is under the control of the master regulator of virulence in L. monocytogenes, PrfA (47, 48). As such, transcription of hly increases when ambient temperatures shift above 30°C (54). Ultimatley, hly expression is not limited to the vacuole, and other mechanisms act to reduce cytotoxicity of LLO. One of the most well-studied regulators of LLO activity is environmental pH. LLO is known to have an optimal pH of 5.5, which is in line with its primary role in the acidifying phagosome (135). Indeed, LLO is quickly denatured and forms aggregates at neutral pH when temperatures are above 30°C, and this is mediated by three acidic amino acid residues in D3 that form a pH sensor known as the acidic triad (136). The pH optimum is one control mechanism to reduce LLO’s cytotoxicity in the cytosol, but there are also mechanisms to degrade the toxin. In particular, among CDCs, LLO contains an N- terminal polyproline type II helix (105) and is rich in proline, glutamate, serine, and threonine (PEST) residues (137). These motifs were originally described as mediating protein-protein interactions and mediating protein turnover, respectively (138, 139).
Deletion of these motifs only has minor effects on hemolytic activity, but profound deleterious effects during infection of host cells. Indeed, cytotoxicity in tissue culture is significantly increased, and bacteria expressing LLO without the PEST-like sequence are
10,000 fold less virulent in mice (137). The PEST-like sequence was observed to be phosphorylated and ubiquitinated in host cells, and was thought to potentially be involved in targeted degradation of LLO, thereby reducing active protein in the host cytosol (140).
20
A recent study has suggested a different route by which the PEST-like sequence promotes LLO degradation. It was observed that LLO lacking the PEST-like sequence was associated with the plasma membrane of the host, while native LLO formed puncta in the cytosol. Data suggests that intracellular LLO binds the plasma membrane, and the
PEST-like sequence interacts with the Ap2a2 subunit of the clathrin-dependent endocytosis, promoting endocytosis of the toxin laden vesicles. LLO in these vesicle puncta localizes with ubiquitin and an autophagy adaptor protein, suggesting that these proteins are targeted for degradation in autophagosomes (141). Additionally, LLO is degraded via the ubiquitin-dependent N-end rule pathway in which LLO is recognized via its N-terminal lysine residue (142). All of these mechanisms work in tandem to limit
LLO activity once L. monocytogenes escapes into the cytosol, so as not to eliminate the bacteria’s primary niche during infection.
L. monocytogenes infection in vivo
1.1.8 Innate Immune Responses
After L. monocytogenes enters the body of a host organism, cells of the innate immune system respond to limit or prevent infection. The first steps of the infectious lifecycle involve the uptake of the pathogen by phagocytes like macrophages and dendritic cells (DCs) in the liver and spleen, as well as the invasion of epithelial cells
(143-146). These cells are initially unable to control L. monocytogenes, and neutrophils, the immune system’s “first responders,” are recruited to the site of infection where they rapidly accumulate (147). Neutrophils are recruited through a combination of host 21 produced chemokines like CXCL1 and CXCL2, which can attract neutrophils and L. monocytogenes-derived formylated peptides, which can be recognized by neutrophil formylated peptide receptors (FPRs) (148-151). They play a number of roles in the early response to infection, including killing of bacteria, recruitment of other cells, and activation of those cells. Upon arrival at the site of infection, neutrophils can interact with
L. monocytogenes through a variety of mechanisms. As professional phagocytes, neutrophils can phagocytose L. monocytogenes. This could function as a way to limit the spread of the bacteria. It has been proposed that neutrophils, once filled with bacteria, could be themselves phagocytosed by activated macrophages that can ultimately kill the bacteria (152). Alternatively, after phagocytosis, neutrophils may kill the bacteria directly. This may be mediated by the production of various reactive species including superoxide, hydrogen peroxide and hypochlorous acid (150, 153). Additionally, the normal intracellular lifecycle of Listeria can be disrupted by the secretion and action of matrix metalloprotease 8 (MMP8), which degrades LLO in the extracellular space or in the phagosome and prevents LLO-mediated phagosomal escape (154). Neutrophils can also release a variety of granules into the extracellular space that contain proteases, antimicrobial peptides, and MMPs that can be bacteriocidal (155). The importance of neutrophils in vivo is clear. Indeed, targeted depletion of neutrophils in mice prior to infection significantly increases susceptibility (147, 156-158). Though neutrophils exhibit a variety of listeriocidal properties, they are rather short-lived cells and can cause significant collateral damage in tissues when left unchecked (159). As such, they also recruit and activate a number of other immune cells to both support the existing responses
22 and set the stage for more long-term control of infection. Indeed, they are known to secrete cytokines and chemokines including MIP-1α, MIP-1β, and IFN-γ, which can recruit and activate inflammatory macrophages, natural killer cells (NKs), and dendritic cells (DCs) to the infectious foci (160-163).
Though L. monocytogenes can readily replicate within resident macrophages in the beginning stages of infection (164, 165), inflammatory macrophages that have been activated with IFN-γ are significantly more bacteriocidal due to increased production of
ROS and reactive nitrogen intermediates (166, 167). This activation is directly dependent on IFN-γ, and macrophages that cannot respond to IFN-γ are more susceptible to infection (168). Beyond their bacteriocidal roles, activated macrophages secrete cytokines to enhance the immune response. They can secrete TNF-α, G-CSF, and GM-
CSF to increase recruitment of neutrophils and inhibit their apoptosis, thereby extending their short lifespans from 6-12 h to 24-72 h (159, 169, 170). Macrophages can also secrete IL-12 which, in conjunction with TNF-α, stimulates NK cells that can also secrete
IFN-γ, which can, in turn, activate more macrophages (171). IL-12 production can also induce CD4+ T cells to undergo differentiation into Th1 type cells that also secrete IFN-γ to promote macrophage activation during the adaptive immune response (172, 173).
Similar to macrophages, DCs seem to play roles both in the establishment of and in the ultimate resolution of infection. CD8α+ DCs seem to be preferentially infected with
Listeria in the first few hours of infection before spreading to other DC subsets, monocytes, tissue-resident macrophages, and neutrophils (145, 147). Mice lacking the
CD8α+ DCs were more resistant to infection at 24 and 72 h (147). In addition, these mice
23 survived doses of bacteria that were otherwise lethal in wild type mice. However, these cells also play an important role in linking the innate immune responses to the adaptive arm of the immune system. Studies have indicated that infected CD8α+ DCs are potent antigen presenting cells (APCs) that contribute to the priming of CD8+ T cells (174-176).
Indeed, mice deficient for the CD8α+ DC chemokine receptor, Xcr1, display defects in
CD8+ T cell priming and increased bacterial burden in mice after 72 h (177). Together, this suggests that these DCs are an important early niche for L. monocytogenes, and this infection establishes an important link between initial infection and later adaptive immune responses.
1.1.9 Adaptive Immune Responses
Though innate responses to L. monocytogenes during the early stages of infection are important for initial control of bacteria, they are not sufficient to fully clear the infection. Sterilizing immunity relies on an effective adaptive immune response (178).
Indeed, mice with severe combined immune deficiency (SCID), which lack both T cells and B cells, can mount an innate response, but cannot fully resolve infection and subsequently develop chronic infection (179). It has been well established that T cells are primary mediators of adaptive immunity against L. monocytogenes (180, 181). Indeed, the adoptive transfer of T cells from previously infected mice to naïve mice confers protection against bacterial infection (179), whereas the transfer of serum antibodies is ineffective (181). As the link between innate and adaptive immunity, DCs are critical in priming T cell responses, since mice depleted of them are unable to generate a CD8+ T
24 cell response (174, 182). Listeria antigens can be presented through MHC class I and
+ + class II, and as such, both CD8 and CD4 T cells are important for the immune response
(183, 184). CD8+ T cells are thought to mediate immunity through two mechanisms.
First, they can recognize and directly lyse infected cells via perforin and granzymes to expose intracellular pathogens for engulfment by listeriocidal phagocytes (185, 186). In concert with this activity, they secrete IFN-γ to activate macrophages that kill the exposed bacteria (187). The primary T cell response peaks at 7-9 days post-infection
(188). After clearing the infection, these populations contract significantly to a small population of stable memory T cells that, upon secondary infection, can respond quickly and are a primary source of IFN-γ (189). Second, the secretion of perforin is also important during secondary infection. Mice deficient in perforin are actually able to clear primary infections but have a severe defect in clearing secondary infections (186).
T cells are clearly of critical importance to the ultimate resolution of infection.
However, B cells, the other major cell type involved in adaptive immunity, and the humoral response seem to be less important. Indeed, the adoptive transfer of serum from previously infected mice to naïve mice conferred no protection against L. monocytogenes
(181). It has been noted that there is very limited antibody production directed against L. monocytogenes antigens during infection, and it has even been observed that mice lacking mature B cells are still readily able to clear infection with the onset of T cell mediated immunity (190, 191). Despite the ability of these mice to clear infection, it was observed that the T cell populations in B cell deficient mice contract faster and to a smaller stable population than in wild type mice (190). The role of B cells seems to be
25 minor overall, and independent of antibody production. However, despite an apparent limited role for B cells and antibodies, it has been shown that the injection of monoclonal
LLO-neutralizing antibodies does offer protection against infection in naïve mice (191).
This suggests that under some conditions, antibodies may play a protective role in the adaptive immune response.
Research and specific aims
The capacity to invade and replicate within host cells is a pathogenic strategy exploited by numerous bacterial species (192). Understanding how intracellular pathogens gain entry into host cells is essential for the rational design of novel therapeutic treatments that would act by preventing the early stage of the infectious process. L. monocytogenes is a facultative intracellular pathogen responsible for listeriosis, a deadly foodborne disease in humans and a variety of animal species. In the
United States, over 90% of reported cases of listeriosis lead to hospitalization, and even with treatment the mortality rate ranges from 16-25% (193). Coupled with the high mortality associated with listeriosis, the economic toll this disease takes on the United
States economy is estimated at around two billion dollars annually (193). L. monocytogenes infects a wide variety of host cells, including professional phagocytes and normally nonphagocytic cells, including epithelial cells. Invasion of nonphagocytic cells has long been thought to be primarily promoted through the binding of two bacterial cell surface receptors, InlA and InlB, which bind to host cell receptors, E-cadherin and c-Met, respectively (39). However, it has been shown that the pore-forming toxin LLO can
26 mediate bacterial entry into host cells in vitro (129-131). Though some studies have examined the capacity for InlA and InlB to work together to potentiate bacterial internalization in some models (194-196), no work has been done to integrate LLO into our understanding of bacterial internalization in the context of these canonical invasion factors.
A major aim of this work was to establish the relative roles of LLO, InlA, and InlB in the process of host cell invasion. We hypothesized that efficient bacterial uptake requires the concerted activities of the three invasion factors, and that this may be cell type dependent. To address this, we used bacterial strains lacking combinations of the invasins and immunofluorescence invasion assays to examine the role of each factor in multiple different cell types.
The multifaceted nature of LLO with respect to the biology of infection is becoming quite clear. With its importance to various processes during infection continuing to broaden, LLO is increasingly seen as an ideal target for the development of therapeutics for listeriosis. As such, as second major aim of this work was to use LLO as a target for the development of a new vaccine candidate to combat listeriosis by eliciting both the canonical T cell responses that are known to be important for clearance in natural infections and a B cell response that leads to production of LLO-neutralizing antibodies that may also confer protection, as has been shown with the transfer of monoclonal LLO- neutralizing antibodies.
27
Chapter 2. Roles for Listeriolysin O, InlA, and InlB in Listeria monocytogenes Uptake by Host Cells
2.1 Introduction
Listeria monocytogenes is a Gram-positive, facultative intracellular bacterium responsible for the foodborne disease listeriosis. Listeriosis is a life-threatening condition for elderly and immunocompromised individuals (5). In these populations, the bacterium can propagate from the intestines to the blood and further disseminate, causing septicemia and meningoencephalitis (5, 84, 197, 198). During pregnancy, susceptibility to L. monocytogenes infection is drastically increases and the bacterium can cross the placental barrier, leading to spontaneous abortion, preterm labor, stillbirth, and severe infections of the newborn (199-201). An important virulence attribute of L. monocytogenes is its ability to infect numerous cell types, from macrophages to normally non-phagocytic cells such as intestinal and placental epithelial cells, endothelial cells, and neurons (5). The wide host cell range of this pathogen is thought to be critical for crossing the tightest barriers of the human host, i.e. the placental and blood-brain barriers.
The expression of major virulence factors that mediate the L. monocytogenes intracellular lifecycle is controlled by PrfA (47, 57, 202), which activates transcription in response to a variety of environmental signals, including temperature (54) and nutrient availability (51, 63, 203). Two of these virulence factors are the surface proteins, InlA and InlB, depicted as the major invasins responsible for L. monocytogenes uptake by 28 normally non-phagocytic cells (27, 28, 42). InlA (internalin) is covalently anchored to the peptidoglycan through its C-terminal LPXTG motif (28, 68), whereas InlB is retained non-covalently at the cell surface via electrostatic interaction between three C-terminal glycine and tryptophan (GW) repeat domains and lipoteichoic acids of the bacterial cell wall (87). The adherens junction protein E-cadherin was identified as the sole InlA receptor (69) and several host surface proteins, c-Met (or HGF receptor) (90), gC1Q receptor (88), and surface glycosaminoglycans (89), have been identified as InlB receptors. The N-terminal leucine-rich repeat (LRR) domain of InlB binds to c-Met, whereas its C-terminal moiety binds to glycosaminoglycans and gC1Q receptor in addition to being the lipoteichoic acid anchor (88, 89). InlA mediates bacterial entry only into cells expressing E-cadherin, whereas InlB is a more versatile invasin as its receptors are widely expressed. Importantly, InlA and InlB are species-specific: Humans and gerbils are permissive to both InlA and InlB while rabbits/guinea pigs and mice are only permissive to InlA or InlB, respectively (82). It has been proposed that InlB acts as a facilitator of the InlA-dependent invasion pathway in enterocytes (194, 195) and that
InlA and InlB, but not LLO, were the two most important invasion factors for crossing the intestinal barrier (84, 194, 195).
Upon ingestion by host cells, L. monocytogenes is confined within a vacuole or phagosome that is disrupted by the secreted pore-forming toxin listeriolysin O (LLO) and phospholipases to release the bacterium into the cytosol, where it divides and from which it infects other cells by cell-to-cell spreading (32, 127, 204, 205). The role of LLO in mediating vacuolar escape is certainly a major role of this toxin, as the absence of LLO
29 leads to a marked deficiency in intracellular replication of phagocytosed bacteria (205).
The role of LLO was considered to be specifically restricted to the disruption of the phagosome (206), but additional roles have been attributed to this toxin. In particular, it was shown that LLO, secreted by extracellular bacteria, perforates the host cell plasma membrane during the early stage of infection; therefore, LLO secretion and membrane perforation precedes the formation of the phagosome (130, 207). Perforation of the host cell plasma membrane activates several signaling pathways (204). One outcome of LLO- induced signaling is the internalization L. monocytogenes into epithelial cell lines
(HepG2, HeLa, and Hep2 cells) (129, 130, 208) and professional phagocytes (human neutrophils and murine bone marrow-derived macrophages) (154). However, once bacteria are opsonized, the contribution of LLO in bacterial uptake by professional phagocytes becomes negligible. In addition, LLO-mediated plasma membrane perforation by cytosolic bacteria was recently proposed to facilitate cell-to-cell spreading
(209).
Because InlA and InlB are described as the most important factors controling L. monocytogenes uptake by normally non-phagocytic cells, it was necessary to establish if the role of LLO is significant when compared to these two canonical invasins. It was also necessary to determine if LLO plays a general role in inducing L. monocytogenes internalization in all cell types. To address these questions, we used human hepatocytes and cytotrophoblasts because they are known to be infected by L. monocytogenes during listeriosis (5). It is also known that L. monocytogenes can infect endothelial cells in vitro and may infect these cells in vivo to cross the blood-brain and placental-fetal barriers
30
(210-215). As such, endothelial cells were included in this work. Though enterocytes that make up the intestinal barrier are of critical importance for the establishment of listeriosis, previous work has convincingly shown that crossing the intestinal barrier is
InlA-dependent and LLO-independent, so enterocytes were not included (84). To quantify and compare the roles of the three invasins, we used a fluorescence-based microscopy assay that directly measures the efficiency of bacterial association with host cells and the efficiency of their internalization.
2.2 Materials and Methods
Bacterial strains and culture
Escherichia coli XL1Blue and BL21(DE3) were grown in Luria-Bertani (LB) broth under agitation at 37°C. Plasmids were maintained with either ampicillin (50
µg/ml) or kanamycin (30 µg/ml), as indicated. Wild type (WT) L. monocytogenes (EGD- e) was a gift from Dr. Pascale Cossart (Pasteur Institute, Paris, France) (Table 1).
Table 2.1 L. monocytogenes strains used in this study Strain Genotype Source or reference EGD-e Wild type (216) 10403S Wild type (165) DP-L2161 10403S (Δhly) (132) DP-L4405 10403S (ΔinlA) (217) DP-L4406 10403S (ΔinlB) (217) DP-L4404 10403S (ΔinlAB) (217) SL33 10403S (ΔinlAΔhly) This Study SL40 10403S (ΔinlBΔhly) This Study SL20 10403S (ΔinlABΔhly) This Study NF-L1177 10403S (prfA G145S actA-gus-neo-plcB) (218) SL64 DP-L4406 (prfA G145S actA-gus-neo-plcB) This Study
31
WT L. monocytogenes (10403S) and isogenic mutants Δhly (DP-L2161), ΔinlA (DP-
L4405), ΔinlB (DP-L4406), and ΔinlAB (DP-L4404) were gifts from Dr. Daniel Portnoy
(U.C. Berkeley, California, USA). Strain 10403S, a member of linage II and serotype
1/2a, is a streptomycin-resistant derivative of strain 10403 (219, 220), the latter was originally isolated from a human skin lesion in 1968 (221). The triple deletion mutant,
ΔinlABΔhly, was developed previously (129). Double deletion mutants, ΔinlAΔhly and
ΔinlBΔhly, were constructed using DP-L4405 and DP-L4406, respectively by knocking out the hly gene via allelic exchange using the pKSV7 integration shuttle vector and primers in Table 2 as described previously (129, 222). The deletion of hly was confirmed by PCR using primers in Table 2. L. monocytogenes strains were grown overnight under agitation at 37°C in brain heart infusion (BHI) (BD Biosciences). For invasion assays, overnight cultures were diluted 20-fold in BHI and grown at 37°C until OD600 = 0.7-0.8.
Cells were washed three times in sterile, 37°C phosphate-buffered saline (PBS) and diluted to the indicated multiplicity of infection (MOI) in appropriate mammalian cell culture medium without serum and antibiotic.
Transduction and prfA* mutant isolation
U153 bacteriophage (223) was used to infect L. monocytogenes strain NF-L1177
(prfA* G145S actA-gus-neo-plcB), and the phages were recovered and used to transduce the prfA* (leading to G145S) actA-gus-neo-plcB to target strains, WT 10403S and ΔinlB, as previously described (U153 bacteriophage and strain NF-L1177 were gifts from Dr.
Nancy Freitag (University of Illinois Chicago, Illinois, USA)) (224, 225). Transductants
32
Table 2.2 Primers used in this study All probes have a 5’ 6-FAM reporter dye and a 3’ Iowa Black FQ quencher. Constructs Oligonucleotide Sequence (5’-3’) Reference Construction of Forward – GGG AAT TCA ATT GTT GAT ACA (132) Δhly strains ATG ACA TC Reverse – GGC TGC AGG GTC TTT TTG GCT TGT GTA T Primers to Forward – CCG TCG GAT CCA TGA AAA AAA (132) amplify the hly TAA TGC TAG TTT TTATTACAC ORF Reverse – ATC CGC GCT GCA GTT CGA TTG GAT TAT CTA CTT TAT TAC pET29b- Forward – AAC GTG CAT ATG GAG ACT ATC This Study inB6His ACC GTG CCA ACG (bp 106 - 1890) Reverse – ATT CTC GAG TTT CTG TGC CCT TAA ATT AGC TGC Sequencing Forward – CTA TCT GTT GCA GCT CTT CTT GG This Study prfA mutants Reverse – CAG CTA ACA ATT GTT GTT ACT GCC Confirm gus- Forward – GCA GTC AAT TAA TAT GCC GAG This Study neo insertion CC Reverse - CGG ACC AAC TAA GTT TAT (prfA* mutants) GTG G Hydrolysis primers and probes for qPCR for gene target Forward – GGC AAA GAA ACA ACC AAA GAA inlA G This Study Reverse – GGG CAT CAA ACC AAC CAA This Study Probe - AT TGA CTG AAC CAG CTA AGC CCG T This Study inlB Forward – CCG AGC ACT TAA CAC ATT CTA C This Study Reverse – TTA TCT GCT ACC GGG ACT TTA T This Study Probe - ATG TCA GCG CCA ATA AAG CTG GC This Study hly Forward – CTG GTT TAG CTT GGG AAT GG This Study Reverse – ATT TCG GAT AAA GCG TGG TG This Study Probe - TGA TGA CCG GAA CTT ACC ACT TGT GA This Study gapDH Forward – TCA CAG CGC AAG ACA AAG This Study Reverse – ACT GTT TCA GTT CCG TCT AAT G This Study Probe - TG TTA TCT CCG CTC CAG CAA CTG G This Study rpoB Forward – TGT AAA ATA TGG ACG GCA TCG T (226) Reverse – GCT GTT TGA ATC TCA ATT AAG TTT GG (226) Probe - CT GAT TCG CGC AAA ACT TCT ACG CG (226)
33 were selected by plating the phage/bacteria mixture on BHI/agar plates (5 µg/ml neomycin) for 2 days at 37°C. Neomycin resistant mutants were further screened by plating on BHI/agar plates containing 5 µg/ml neomycin plus 50 µg/ml 5-bromo-4- chloro-3-indolyl-ß-D-glucuronic acid (x-gluc) to confirm the prfA* mutation and the downstream actA-gus-neo-plcB transcription fusion. The actA-gus-neo-plcB insertion was then confirmed by PCR and the prfA G145S mutation was confirmed by sequencing using primers described in Table 2.
RNA Purification, Reverse Transcription, and RT-qPCR
For RNA purification, L. monocytogenes was cultured in BHI under agitation at
9 37°C to an OD600 of 0.7 to 0.8. RNA was purified from 10 bacteria and subsequently treated with RNase-free DNase as described previously (203). RNA concentration and purity were measured via a NanoDrop ND-1000 spectrophotometer. RNA integrity was determined on a 1.2% agarose gel. Reverse transcription was performed using a High-
Capacity RNA-to-cDNA kit (Applied Biosystems) according to the manufacturer’s instructions. Duplicate reactions lacking the reverse transcriptase enzyme were performed in parallel, and these samples were used in RT-qPCR to test for residual DNA contamination. RT-qPCR was performed using a CFX96 Real-Time System and C1000
Thermal Cycler (Bio-Rad). All reactions were performed in 96-well plates using 1.5 ng of converted cDNA, iQ Supermix (Bio-Rad), forward and reverse oligonucleotide primers, and hydrolysis probes (Table 2). No reverse transcriptase (NRT) samples were used as negative controls. inlA, inlB, and hly gene expression was normalized to
34 housekeeping genes gapDH and rpoB. Fold-changes in gene expression are relative to
WT L. monocytogenes. Primer and probe concentrations were optimized by testing a concentration gradient of all oligonucleotides as described previously (227). All primer/probe sets yielded reaction efficiencies ~100%. Samples were analyzed in triplicate by RT-qPCR.
InlB Purification and Generation of anti-InlB Rabbit polyclonal Antibodies
The inlB gene, excluding the signal sequence (bp 106 - bp 1890), was amplified from genomic DNA of L. monocytogenes strain EGD-e using primers (Table 2) that contain NdeI and XhoI restriction sites. This DNA fragment was ligated into the pET29b expression vector upstream of the C-terminal 6His-Tag sequence. The resulting expression vector pET29b-inlB was transformed into Escherichia coli strain BL21(DE3).
For expression of recombinant protein, this strain was grown at 37°C until OD600 = 0.6 and expression of recombinant InlB-6His was induced by addition of 1 mM IPTG (228).
After 5 h of induction, bacteria were pelleted and suspended in binding buffer (5 mM imidazole, 500 mM NaCl, and 50 mM HEPES, pH 7.9) and lysed by French press. The crude lysate was centrifuged and the supernatant incubated with Ni-NTA Agarose
(Qiagen). After washes, the protein was eluted and dialyzed overnight. Purified rInlB was sent to GenScript (Piscataway, NJ, USA) to generate rabbit anti-InlB polyclonal antibodies. To immunize rabbits, rInlB and Complete Freund’s Adjuvant was administered via subcutaneous injection. After the primary immunization, 3 boosts were performed over the course of 66 days. InlB-specific IgG antibodies were purified from
35 serum by affinity chromatography using Sepharose 4B gel coupled to rInlB. Specificity of the antibodies was ensured by western blotting analysis of WT and inlB-deletion mutant L. monocytogenes strains (Figure. A.1).
Mammalian cell culture
The human hepatocyte cell line HepG2 (HB-8065) was purchased from ATCC.
The human hepatocyte cell lines Hep3B (HB-8064, ATCC), PLC5 (CRL-8024, ATCC), and Huh7 (Health Science Research Resources Bank, Osaka, Japan; JCRB0403) were gifts from Dr. Ching-Shih Chen (The Ohio State University, Ohio, USA). HepG2,
Hep3B, and PLC5 cells were grown in minimal essential medium (MEM) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS; Atlanta Biologicals), 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). Huh7 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% HI-FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. The human choriocarcinoma cell line BeWo (ATCC CCL-98) was a gift from Dr. John Mitchell Robinson (The Ohio State University, Ohio, USA). BeWo cells were grown in DMEM/F12 (1:1) medium supplemented with 10% HI-FBS, 100U/ml penicillin, and 100 µg/ml streptomycin. Human Umbilical Vein Endothelial Cells
(HUVECs, ScienCell Research Laboratories, San Diego, CA, USA) were cultured in endothelial cell medium (ECM) with 5% HI-FBS, endothelial cell growth supplement
(ECGS, ScienCell), 100 U/ml penicillin, and 100 µg/ml streptomycin. All plates and
36 flasks used for HUVEC culture were coated with 2 µg/cm2 human fibronectin (BD
Biosciences).
Western blotting (LLO, InlB, c-Met)
Bacterial lysates were loaded at several dilutions (8x107, 4x107, and 2x107 bacteria loaded for LLO, and 3.2x108, 1.6x108, and 8x107 bacteria loaded for InlB) and subjected to SDS-PAGE and western blot analysis using PVDF membranes and anti-LLO
(Rabbit polyclonal from Abcam), anti-InlB (rabbit polyclonal from Genscript), and secondary anti-rabbit IgG conjugated to horseradish peroxidase (Cell Signaling) antibodies. For detection of InlB production in prfA* mutants, 1.6x108 cells were used.
We also probed for p60 as a loading control (Adipogen). Signal detection was performed using Amersham ECL Select Reagent Kit (GE Healthcare) and a ChemiDoc XRS
Imaging System (Bio-Rad). Densitometry analysis was performed by enclosing each protein band within a region of standard size, and the intensity of each band was measured using Image J gel analysis. Results were the average intensities calculated from
3 independent experiments. All intensities were set relative to WT bacterial lysates. For detection of c-MET, hepatocytes were grown to 80% confluence in the same experimental conditions as for invasion assays. Cell lysates were subjected to SDS-PAGE and western blot analysis using PVDF membranes with anti-c-MET (4F8.2, Millipore) antibodies and secondary anti-mouse IgG antibodies conjugated to horseradish peroxidase (Cell Signaling). Signal detection was performed as described above.
37
Measuring Bacterial association and internalization
HepG2 (105 cells/well), Hep3B (0.75x105 cells/well), PLC5 (0.75x105 cells/well),
Huh7 (0.75x105 cells/well), and HUVECs (2x104 cells/well) were cultured in 24-well tissue culture plates on glass coverslips at 37°C in 5% CO2 atmosphere for 48 h before infection. BeWo cells (0.85x104 cells/well) were cultured in 24-well tissue culture plates on glass coverslips coated in 0.2% gelatin for 72 h before infection. The hepatocyte cell lines were infected with L. monocytogenes at a multiplicity of infection (MOI) 20;
HUVECs at a MOI 5; BeWo were infected with 106 bacteria/well. Infection of hepatocytes with prfA* bacterial strains were performed at MOI 5 to avoid toxicity of
LLO in prfA*. Plates were centrifuged for 5 min (500 x g) at room temperature and incubated for 30 min at 37°C. Cells were washed 3 times with PBS, fixed with 4% paraformaldehyde (PFA) in PBS for 15 min at room temperature, and blocked for 1 h in
0.1 M glycine and 10% HI-FBS in PBS, pH 7.4. Extracellular bacteria were labeled with anti-L. monocytogenes rabbit polyclonal antibodies (GeneTex) and with anti-rabbit secondary antibodies conjugated to Alexa Fluor 488 (Molecular Probes). Samples were permeabilized with 0.5% Triton X-100 and total (extracellular and intracellular) bacteria were labeled with anti-L. monocytogenes antibodies and secondary antibodies conjugated to Alexa Fluor 568 (Molecular Probes). Slides were mounted in ProLong Gold Antifade containing DAPI (4’,6-diamidino-2-phenylindole; Molecular Probes) to stain nuclei. To quantify the number of cells, images (phase-contrast, DAPI, Alexa Fluor 488, and Alexa
Fluor 568) were automatically acquired for each experimental condition using the 20X objective. MetaMorph analysis software was used to enumerate the total number of
38 bacteria (Nt), extracellular bacteria (Ne), and mammalian cells (Nc) (229). The efficiency of bacterial internalization was calculated as: Internalization = ((Nt – Ne)/Nt)X 100. The efficiency of bacterial association was calculated as: Association = Nt/Nc. For each experimental condition, a minimum of 100 bacteria were counted (this applies to bacterial mutants with the lowest association efficiency) and a minimum of 150 mammalian cells (this applies to Hep3B, which are the largest cells and the cells with which L. monocytogenes associates the most effectively). The average numbers of WT bacteria and corresponding mammalian cells counted in each experiment is indicated in the figure legends. Because BeWo cells clustered in a fashion that made individual cell nuclei challenging to enumerate, we quantified the cell surface area by tracing plasma membrane outlines in MetaMorph and determined the surface area in µm2. We then calculated the efficiency of bacterial association as: Association = Nt/Cell surface area
(µm2).
Polystyrene bead coating with recombinant InlB and invasion assay
Blue fluorescent carboxylate-modified latex beads (1 µm diameter, Molecular
Probes) were coated covalently with a mixture of recombinant InlB (5 mg/ml) and BSA
(5 mg/ml) according to the manufacturer’s instructions. Control, BSA-coated beads were prepared with 10 mg/ml BSA under the same conditions. The beads were then washed 3 times with 0.33X PBS pH 7.4 and stored at 4°C. To assess the capacity for InlB-coated beads to be ingested by hepatocytes, HepG2 cells were seeded in 24-well plates on coverglasses for 48 h, as described for bacterial invasion assays. Cells were washed with
MEM, and InlB/BSA-, or BSA-coated beads were added to the wells at MOI 5. Plates
39 were centrifuged for 3 min at 500 x g and incubated for 30 min at 37°C in 5% CO2. Cells were washed with PBS, fixed with 4% paraformaldehyde (PFA) in PBS for 15 min at room temperature, and washed and blocked for 1 h in 0.1 M glycine and 5% Blotting- grade blocker (Bio-Rad) in PBS, pH 7.4. Extracellular beads were labeled with rabbit anti-BSA antibodies (Sigma-Aldrich, B1520) followed by anti-rabbit secondary antibodies conjugated to AlexaFluor 488. Slides were mounted in ProLong Gold Antifade containing DAPI to stain nuclei. The percentage of intracellular beads was determined by fluorescence microscopy. The percentage of intracellular beads was calculated as the number of intracellular beads divided by the total number of beads, multiplied by 100.
Live cell imaging to assess hepatocyte response to InlB
Hepatocytes were seeded (HepG2- 4x105 cells/dish; Hep3B, PLC5, and Huh7 –
3x105 cells/dish) in 35 mm diameter imaging dishes (Matek, P35G-1.5-10-C) and cultured at 37°C in 5% CO2 for 48 h. Cells were placed on the 37°C microscope stage and incubated with cell imaging medium without phenol red. Differential interference contrast (DIC) images were acquired with the 63X objective every 20 s for 15 min. 5 min after the start of imaging, rInlB was added to the cell culture medium to a final concentration of 1 nM. In control condition, the cells were imaged for 15 min without
InlB.
40
Western blotting analysis of Akt phosphorylation
Hepatocytes were seeded (HepG2 - 5 x 105 cells/dish; Hep3B, PLC5, and Huh7 -
3 x 105 cells/dish) in 35 mm diameter cell culture dishes and cultured for 48 h. For exposure to recombinant InlB, cells were washed and incubated for 30 min in serum-free medium and were incubated with, or without, 1.25 nM InlB for 5 min at 37°C. Cells were then washed with cold PBS and lysed with cold lysis buffer (150 mM NaCl, 20 mM
Tris/HCl, 2 mM EDTA, 1% NP-40, 3mM sodium orthovanadate, 50 mM sodium fluoride, and 1x EDTA-free protease inhibitor cocktail (Roche)). To assess the effect of
InlB produced by L. monocytogenes, cells were washed with medium without serum and infected with WT or InlB-deficient bacteria at MOI 20 for 30 min at 37°C (same experimental conditions as the invasion assay). Cells were then washed, lysed. Cell lysates were subjected to western blot analysis using PVDF membranes and anti-Akt or anti-phospho-Akt (Ser473) antibodies (Cell Signaling) and secondary anti-rabbit IgG antibodies conjugated to horseradish peroxidase (Cell Signaling).
Microscope equipment
Images were acquired on a motorized, inverted, wide-field fluorescence microscope (Axio Observer D1, TempModule S, Heating Unit XL S; Zeiss) equipped with a PZ-2000 XYZ automated stage, 20X Plan Neofluar (numerical aperture = 0.5),
40X Plan Neofluar (NA = 1.3), and 63X Plan ApoCHROMAT (NA = 1.4) objectives, a high-speed Xenon fluorescence emission device (Lambda DG-4, 300 W; Sutter
Instrument Company), a Lambda 10-3 optical emission filter wheel for the fluorescence
41 imaging, a Smart shutter to control the illumination for phase-contrast and DIC imaging
(Sutter Instrument Company), a back-illuminated, frame-transfer electron-multiplying charge-coupled device (EMCCD) camera (Cascade II 512; Photometrics), and an ORCA-
Flash 4.0 sCMOS camera (Hamamatsu). The filter sets for fluorescence were purchased from Chroma Technology Corporation and were as follows: DAPI (49000), Alexa Fluor
488 (49002), Alexa Fluor 568 (49005), and Cy5 (49006). Images were acquired and analyzed using MetaMorph imaging software (Molecular Devices).
Statistical Methods
All experimental work involved at least three biological replicates, each performed on different days. Data obtained each day include different treatment conditions, which are considered as a cluster. Data within the same cluster are more correlated to each other than to data from clusters obtained on different days. Linear mixed effects models were used to account for the correlation among observations from a same cluster. Linear mixed effects models were used to analyze data from invasion assays (bacterial entry and association), studies of the interaction between invasion proteins, RT-qPCR, and quantitative western blot analyses. For RT-qPCR and western blot, data were first normalized to internal controls or loading standard to reduce variation before analysis. Holm’s procedure was used to adjust for multiple comparisons such as comparing each L. monocytogenes deletion mutant to WT. SAS 9.4 was used for all analyses (SAS Institute, Inc., NC). Although normalized data were presented in some
42 figures for a clearer visualization of results, all statistical analyses were performed on raw data before normalization.
2.3 Results
LLO, InlA, and InlB expression levels in single and double deletion mutants
To ensure that deletion of the virulence genes hly, inlA and inlB, in the single and double deletion L. monocytogenes 10403S mutants, does not affect the expression of the others, the levels of mRNA and proteins of the 3 invasion factors were measured.
Bacteria were grown in the same experimental conditions as for the cell invasion assay and reverse transcription quantitative realt-time PCR (RT-qPCR) was used to measure hly, inlA, and inlB mRNA levels. As expected, deletion of one or two virulence genes does not significantly affect the expression of the other genes when compared to the WT strain (Figure 2.1). We the measured the protein expression levels by western blotting,
Figure 2.1 inlA, inlB, and hly mRNA quantification Reverse transcription quantitative real-time PCR (RT-qPCR) was performed to measure inlA, inlB, hly, gapDH, and rpoB transcripts from L. monocytogenes WT and isogenic deletion mutants. The housekeeping genes, gap and rpoB, were used to normalize the expression of (A) inlA, (B) inlB, and (C) hly. Results are the average fold-change in gene
43 expression ± SEM relative to WT (n ≥3). Statistical differences compared to WT (n.s. = non-statistically significant).
which required antibodies against LLO, InlB, and InlA. Anti-LLO antibodies are commercially available, but not anti-InlA and anti-InlB. Therefore, we cloned inlA and inlB genes (without signal peptides) into an expression vector (pET29b), purified the recombinant proteins, and obtained purified polyclonal rabbit anti-InlB and -InlA. The anti-InlB antibodies could efficiently detect InlB (Fig. A.1), but we were not successful with the anti-InlA antibodies. We then measured LLO and InlB protein expression levels by western blotting. For rigorous evaluation, we analyzed serial dilutions of cell lysates and performed densitometry analyses of the corresponding bands. As expected, single and double deletions of the inlA, inlB, or hly genes do not significantly affect the expression levels of LLO or InlB (Figure. 2.2).
44
Figure 2.2 InlB and LLO protein levels (A and B) L. monocytogenes cell lysates, undiluted and at the dilutions ½ and ¼, were subjected to western blot analysis using anti-InlB and anti-LLO antibodies. (C and D) Densitometry analysis was performed using the ImageJ software. Representative western blots are shown. Results are the mean ± SEM relative to WT (n ≥ 3). Statistical differences compared to WT using data prior to normalization (n.s. = non-statistically significant).
InlA and LLO, but not InlB, control L. monocytogenes uptake by human hepatocytes
To establish the relative roles of the three virulence factors in L. monocytogenes uptake by human hepatocytes, we used four human hepatocyte cell lines (HepG2, Hep3B,
PLC5, and Huh7) to rule out any cell line-specific phenotype and draw conclusions that can generally apply to hepatocytes. Hepatocytes were incubated with L. monocytogenes
(WT, Δhly, ΔinlA, ΔinlB, ΔinlAB, ΔinlBΔhly, ΔinlAΔhly, or ΔinlABΔhly) for 30 min at
37°C and were processed for fluorescence microscopy analysis. Full data sets, including 45 association and internalization efficiencies of the eight bacterial strains into the four cell lines, are presented in Figure A.2. We first focused on analyzing data obtained with the single and triple deletion mutants in comparison to WT L. monocytogenes (Figure 2.3).
Figure 2.3 Relative roles of LLO, InlA, and InlB in L. monocytogenes invasion of human hepatocytes HepG2, Hep3B, PLC5, and Huh7 cells were infected with WT, InlA-deficient (ΔinlA), InlB-deficient (ΔinlB), LLO-deficient (Δhly), or InlAB/LLO-deficient (ΔinlABΔhly) bacteria (MOI 20) for 30 min at 37°C. Cells were washed, fixed and labeled with fluorescent antibodies and DAPI. (A) Bacterial association efficiency was calculated as the total number of bacteria associated per host cell. The average bacterial association values for the WT strain before normalization was: HepG2 – 0.14, Hep3B – 3.13, PLC5 – 1.34, Huh7 – 0.77. (B) Bacterial internalization efficiency was calculated as the percentage of intracellular bacteria. The average percentage of internalization for the WT strain before normalization was: HepG2 – 26.45%, Hep3B – 38.77%, PLC5 – 18.29%, Huh7 – 33.12 %. The minimum number of host cells counted was: HepG2 – 1000, Hep3B – 150, PLC5 – 600, Huh7 – 2000. The average number of WT bacteria counted per experiment was: HepG2 – 600, Hep3B – 4,000, PLC5 – 2,000, Huh7 – 3,000 with a minimum count of 100 bacteria being required for any mutant with reduced association efficiency. Results are expressed as the mean ± SEM relative to WT (n ≥ 3). Statistical analyses compared each deletion strain to the WT strain and were performed on raw data before normalization (*, p <0.01, ** p < 0.001).
46
Data show that LLO does not promote L. monocytogenes association with hepatocytes. In one of the hepatocyte cell line (Hep3B), LLO even significantly decreases bacterial association. On the contrary, InlA is the only factor that promotes bacterial association with hepatocytes, in three out of the four cell lines. The decrease in association of the inlA single deletion and the triple deletion mutants were similar in all hepatocyte cell lines, confirming that among the three factors, InlA is the only adhesin. LLO and InlA, but not InlB, promote internalization of L. monocytogenes, although the role of LLO was more prominent in that function than InlA. In one cell line (Hep3B), single deletion mutants had no internalization phenotype, whereas the triple (ΔinlABΔhly) and double
(ΔinlAΔhly, Figure A.2) deletion mutants display a significant decrease in internalization.
This latter result shows that LLO and InlA can exert a redundant role in L. monocytogenes internalization. To our surprise, no role for InlB was detected in L. monocytogenes association and internalization into the four hepatocyte cell lines when single, double, and triple deletions mutants were considered (Figure 2.3 and Figure A.2).
This prompted us to clarify this result.
InlB-mediated L. monocytogenes internalization is dependent on InlB expression levels
The absence of a role for InlB led us to verify that its receptor, c-Met, was expressed and functional in the hepatocyte cell lines used in these studies. As expected, c-
Met was expressed in all tested hepatocyte cell lines (Figure 2.4A). Previous studies established that InlB activates c-Met-dependent Akt phosphorylation and F-actin 47 remodeling (97, 230, 231). As expected, cell exposure to recombinant InB induced a significant increase in Akt
Figure 2.4 The InlB/c-Met pathway is functional in hepatocytes (A) HepG2, Hep3B, PLC5, and Huh7 cell lysates were subjected to western blot analysis using anti-c-Met and anti-actin (loading control) antibodies. (B) Cells were exposed, or not, to 1.25 nM InlB for 5 min and cell lysates were subjected to western blot analysis using anti-Akt and anti-phospho-Akt antibodies. A representative western blot is presented (n = 3). (C) HepG2 cells were incubated with BSA- or BSA/InlB-coated beads for 30 min at 37°C (MOI 5). Results are expressed as the average percent internalization ± SEM (n = 4, * p < 0.01, ** p < 0.001). (D) After infection with WT or ΔinlB bacteria (MOI 20) for 30 min, HepG2 cells were lysed and lysates were subjected to western blot analysis using anti-Akt and anti-phospho-Akt antibodies. A representative western blot is shown (n = 3).
48 phosphorylation in all cell lines (Figure 2.4B). As a second approach, live cell imaging showed that hepatocytes exposed to InlB formed dynamic membrane ruffles, which were not observed in the absence of InlB (movies S1 to S4). Finally, to evaluate if hepatocytes could undergo InlB-dependent phagocytic uptake, we exposed cells to polystyrene beads
(1 µm diameter) that were covalently coated with saturating concentrations of InlB or bovine serum albumin (BSA), used as negative control. As shown in Fig. 4C, 80% of
InlB-coated beads were internalized by hepatocytes. We then established if InlB produced by L. monocytogenes could stimulate c-Met. HepG2 cells were incubated with
WT and ΔinlB 10403S strains for 30 min at MOI 20, as performed in the invasion assays.
As shown in Figure 2.4D, WT but not InlB-deficient bacteria induced Akt phosphorylation. Together, these results demonstrate that the hepatocyte cell lines express a functional c-Met and that InlB from 10403S is expressed in sufficient amount to activate c-Met signaling. However, InlB produced by 10403S failed to induce significant bacterial entry. We then tested the hypothesis that InlB was not produced in sufficient amounts by 10403S strain to promote bacterial uptake. This hypothesis was based on the fact that the bead surface was coated with saturating amount of recombinant
InlB and the fact that laboratory strains used to show a role for InlB in bacterial internalization express high levels of InlB (27, 87, 90, 97, 211, 212, 220, 228, 232-234).
Indeed, the commonly studied lab strain EGD expresses a constitutively active variant of the transcriptional regulatory factor PrfA, known as a PrfA* (G145S) variant, which is responsible for high production levels of InlB and other PrfA-regulated virulence factors
(50, 51, 61, 220, 225, 235-237). To test if an increase in InlB production in the
49 background 10403S would result in InlB-mediated internalization of L. monocytogenes, we generated prfA* and ΔinlB prfA* strains in the 10403S background by phage transduction (61, 220, 224). We compared the production of InlB between 10403S WT and prfA* and report a marked increase in InlB production, as expected (220) (Figure
2.5A).
Figure 2.5 A prfA* mutation in L. monocytogenes strain 10403S leads to increased production of InlB and InlB-dependent hepatocyte invasion (A) Bacterial lysates (1.6 x 108 cells) were subjected to western blot analysis using anti- InlB and anti-p60 (loading control) antibodies. A representative western blot is shown (n = 3). (B and C) PLC5 cells were infected with WT, ΔinlB, WT-prfA*, or ΔinlB-prfA* bacteria (MOI 5) for 30 min at 37°C. Cells were washed, fixed, and labeled with fluorescent antibodies and DAPI. (B) The bacterial association efficiency was calculated as the total number of bacteria associated per host cell. (C) The bacterial internalization efficiency was calculated as the percentage of intracellular bacteria. (B and C) A minimum of 2000 bacteria were counted per condition and a minimum of 500 host cells were counted per condition. Results are expressed as the mean ± SEM (n = 4, * p < 0.01, ** p < 0.001, n.s. = non-statistically significant).
50
The replacement of WT prfA with prfA* led to a 5-fold increase in bacterial association
(Figure 2.5B) and a 7–fold increase in bacterial entry into host cells (Figure 2.5C).
Comparison between prfA* and ΔinlB prfA* showed that, in the prfA* background, InlB plays a significant role in bacterial entry (Figure 2.5C) while comparison of the WT and
ΔinlB strains show no difference in either bacterial association or bacterial entry (Figure
2.5B and C). Collectively, these data show that a bacterial strain such as 10304S produces enough InlB to activate c-Met, but this amount is not sufficient to affect L. monocytogenes internalization.
Only InlA, but not InlB and LLO, controls L. monocytogenes uptake by human cytotrophoblasts
We next determined the role of LLO, InlA, and InlB in L. monocytogenes uptake by human cytotrophoblast-like BeWo cells. Cytotrophoblasts are cells of fetal origin located at the interface between maternal and fetal tissues. Invasion of the placenta requires traversal of the cytotrophoblast barrier. No role for LLO in L. monocytogenes association and entry was detected in BeWo cells. Two other cytotrophoblast-like cells
Jeg-3 and JAR were also tested, leading to the same conclusion (data not shown). Only
InlA plays a major role in L. monocytogenes association with BeWo cells, but does not affect the efficiency of internalization (Figure 2.6). Finally, no role for InlB was observed in invasion of BeWo cells, as previously reported by others using the same bacterial strain (217).
51
Figure 2.6 Role of LLO, InlA, and InlB in L. monocytogenes invasion of human cytotrophoblasts BeWo cells were infected with WT, LLO-deficient (Δhly), InlA-deficient (ΔinlA), or InlB-deficient (ΔinlB) bacteria (106 bacteria/well) for 30 min at 37°C. Cells were washed, fixed and labeled with fluorescent antibodies and DAPI. (A) The bacterial association efficiency was calculated as the number of cell-associated bacteria per unit surface area (µm2). The average association for the WT strain before normalization was 0.0015 bacteria/µm2. (B) The bacterial internalization efficiency was measured as the percentage of intracellular bacteria. The average internalization for the WT strain before normalization was 13.82%. The average number of WT bacteria counted per experimental condition was 5,000 with a minimum count of 100 bacteria being required for any mutant with reduced association efficiency. Results are expressed as the mean ± SEM relative to WT (n ≥ 3). Statistical analyses compared each strain to the WT strain and were performed on raw data before normalization (* p < 0.01, ** p < 0.001; n.s. = non-significant).
Uptake of L. monocytogenes by HUVEC is independent of the three invasion factors
We next assessed the role of LLO, InlA, and InlB in uptake of L. monocytogenes by human umbilical vein endothelial cells (HUVECs). We used a low multiplicity of infection (MOI 5) because HUVEC were severely damaged at higher MOI due to LLO activity, as we we have observed and as recently reported (214). Data show no role for
52
InlA, InlB, or LLO in the invasion of HUVECs (Figure 2.7). This is congruent with the most recent report in the literature regarding L. monocytogenes strain 10403S and
HUVECs that support the notion that bacterial uptake is largely independent of InlA,
InlB, and LLO (214).
Figure 2.7 Absence of a role for LLO, InlA, and InlB in L. monocytogenes invasion of human endothelial cells HUVECs were infected with WT, LLO-deficient (Δhly), InlA-deficient (ΔinlA), or InlB- deficient (ΔinlB) bacteria (MOI 5) for 30 min at 37°C. Cells were washed, fixed and labeled with fluorescent antibodies and DAPI. (A) The bacterial association efficiency was calculated as the number of cell-associated bacteria per human cells. The average association for the WT strain was 0.13 bacteria/host cell. (B) The bacterial internalization efficiency was measured as the percentage of intracellular bacteria. The average internalization efficiency for the WT strain was 13.23%. The average number of WT bacteria counted per experiment was 500 and a minimum of 2000 host cells were counted per condition. Results are expressed as the mean ± SEM relative to WT (n ≥ 3). Statistical analyses compared each strain to the WT strain and were performed on raw data before normalization (n.s. = non-significant).
53
Establishing cooperation between LLO and InlA in L. monocytogenes invasion of hepatocytes
Hepatocyte infection data indicated an important role for both LLO and InlA in L. monocytogenes host cell invasion. This infection model was therefore appropriate to establish if LLO and InlA cooperate to potentiate the efficiency of host cell invasion.
The biological expectation for positive cooperation between the two proteins, also referred to as synergism, is that the biological response when both proteins are expressed
(when both genes are present) will be greater than the sum of their individual response
(when one of the corresponding genes is deleted) (238). To establish if InlA and LLO display positive cooperation in bacterial association with host cells on entry into host cells, we established four groups: InlA and LLO are both expressed (WT strain), LLO alone (ΔinlA strain), InlA alone (Δhly strain), and neither of the two proteins are expressed (ΔinlAΔhly double deletion mutant). A linear mixed effects model was used to test this hypothesis (µboth-µneither)>( µA- µneither)+(µB- µneither), i.e., µboth- µA - µB +
µneither>0, where both is the WT, neither is the double deletion mutant, and µ is the mean outcome for each group (238). If the p-value for this test is significant, we claim that there is significant synergistic interaction (positive cooperation) between the two proteins. We used this analytical method to test whether InlA and LLO work synergistically to affect bacterial association and internalization of Listeria monocytogenes. Similar analyses were performed to test for potential positive cooperation between InlB and LLO and InlB and InlA. Estimates and accompanying
54 statistics are included in Table 3. In the process of bacterial association, no pattern of positive cooperation was observed (Table 2.3).
This is consistent with InlA being the sole contributor to association among the tested invasins. In the process of bacterial internalization, no synergistic effect was observed between InlB and the two other invasins (Figure A.2 and Table 2.3), confirming that InlB does not affect the uptake of L. monocytogenes (strain 10403S) into human hepatocytes. Only LLO and InlA interact in a synergistic manner to potentiate L. monocytogenes internalization into HepG2 and PLC5 cells.
Table 2.3 Invasion factor cooperation analysis The estimate is the result of the interaction tests described in Results. Statistically significant P values (<0.05) indicate positive cooperation. SE, standard error. Cooperation in internalization Cooperation in association Invasion factor combinations Cell line Estimate SE P value Estimate SE P value InlA/LLO HepG2 13.996 5.9587 0.0232 -0.0404 0.0379 0.2917 Hep3B -16.86 9.9539 0.0995 -1.1548 0.6499 0.0848 PLC5 6.5958 2.9439 0.0332 -0.3497 0.4422 0.4357 Huh7 7.0611 6.8443 0.3114 0.01674 0.2237 0.9409
7.6389 6.0995 0.2168 -0.0271 0.03911 0.4928 InlA/InlB HepG2 -2.8172 10.1881 0.7838 -0.4551 0.6875 0.5126 Hep3B 3.645 3.3279 0.2827 -0.6872 0.4973 0.1779 PLC5 -1.0749 7.8543 0.8922 -1.0749 7.8543 0.8922 Huh7
8.3178 5.4892 0.1365 -0.0721 0.03502 0.0452 InlB/LLO HepG2 -3.0958 9.4808 0.746 0.4169 0.6232 0.5082 Hep3B 0.9946 2.9439 0.738 0.04456 0.4422 0.9205 PLC5 Huh7 2.6579 7.1474 0.7129 2.6579 7.1474 0.7129
55
2.4 Discussion
This work focused on establishing the relative roles of LLO, InlA, and InlB in L. monocytogenes (strain 10403S) association with and internalization into normally non- phagocytic human cells. The data show that LLO activity is cell-type dependent, as LLO plays a significant role in L. monocytogenes internalization into hepatocytes, but not into cytotrophoblasts or endothelial cells. InlA and LLO are the two virulence factors that significantly contribute to invasion of human hepatocytes, with InlA playing a significant role as an adhesin and LLO as an invasin. To our surprise, no role for InlB was detected unless the prfA gene was replaced by a constitutively active prfA* mutant, indicating that higher expression levels of InlB are required for InlB-mediated bacterial internalization.
Studies that identified the L. monocytogenes virulence factors controlling host cell invasion have traditionally used the gentamicin survival assay. This assay robustly measures bacterial intracellular survival but presents some limitations. First, it indiscriminately and collectively reports the efficiencies of bacterial association and internalization. Second, host cell perforation by LLO allows for diffusion of gentamicin and potential targeting of intracellular bacteria (130). Finally, this assay generally involves long incubation times, which can be sufficient for intracellular bacterial division or killing. Because of these limitations, we analyzed cells infected for only 30 min at low
MOI and in the absence of gentamicin, using a fluorescence microscopy approach (229).
Microscope automation allows for rapid acquisitions of a high number of images and software-assisted analytical tools considerably decrease the time for analysis.
56
Importantly, this approach specifically quantifies with sensitivity and accuracy the efficiencies of bacterial attachment and association with host cells (229).
No role for InlB was initially detected in the present work. This result was unexpected because numerous studies report that InlB promotes host cell invasion (27,
90, 97, 212, 228, 232). Using the hepatocyte model, we showed that the InlB receptor, c-
Met, was expressed and functional. In addition, the amount of InlB produced by L. monocytogenes 10403S in our experimental conditions was sufficient to activate c-Met- dependent signaling, but not bacterial internalization (Figure 2.3 and 2.4). Furthermore, hepatocytes could massively internalize polystyrene beads coated with high concentrations of recombinant InlB (Figure 2.4C). Studies that characterized the role of
InlB in host cell invasion mostly used the strain EGD, which carries a mutation in the gene coding for the master regulator of the virulence genes prfA (designated prfA*), leading to high expression levels of InlB among other virulence factors (220). Among all sequenced L. monocytogenes strains analyzed, the prfA* mutation is very rarely observed
(220). When the prfA* mutation was introduced into EGD-e, inlB transcription was increased over 40-fold (220). This led us to hypothesize that the strain used in our study,
10403S, may not produce enough InlB for productive bacterial internalization. To test this hypothesis, we replaced the WT prfA allele with a prfA* allele in the 10403S background and consequently observed a marked increase in InlB production and a statistically significant role for InlB in bacterial internalization. Together, these data support the idea that the level of expression of InlB is critical for bacterial internalization.
Therefore, it is reasonable to extrapolate that any conditions, including different bacterial
57 cell growth conditions or environmental conditions, that substantially increase InlB expression would favor InlB-dependent internalization. For example, the transcription level of inlB in strain EGD-e is increased in human blood and the murine intestine (239).
One should also consider that the role of InlB observed at later time points of infection may be related to bacterial intracellular survival and/or multiplication, and not to bacterial internalization.
As expected, InlA promotes invasion of cells that express its receptor E-cadherin
(69). Importantly, the role of InlA was substantial even in strain 10403S expressing wild type prfA. Few studies have focused on distinguishing the role of E-cadherin in anchoring the bacterium to the host cell surface from its role in stimulating bacterial internalization.
It was initially proposed that InlA/E-cadherin interaction promotes both anchoring and internalization, since the intracellular domain of E-cadherin and its association with the
F-actin cytoskeleton were necessary for InlA-dependent L. monocytogenes uptake by fibroblasts (240). More recent work studying L. monocytogenes invasion of MDCK epithelial cells expressing wild-type E-cadherin or glycosylphosphatidylinositol (GPI)- anchored extracellular domain of E-cadherin concluded that InlA/E-cadherin interaction anchors the bacterium to the host cell surface, but is dispensable for F-actin-dependent internalization of the bacterium (241). Our results are in accordance with both studies.
We report that the primary function of the InlA/E-cadherin interaction is to anchor the bacterium to the host surface, but this interaction can also control the efficiency of bacterial internalization in some, but not all, cell lines.
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LLO plays a critical role in L. monocytogenes internalization into hepatocytes.
Other studies established that the formation of LLO pores on the plasma membrane activates the following signaling cascade: influx of extracellular Ca2+, activation of Ca2+- dependent conventional protein kinase C upstream from the Rho GTPase Rac1, and
Arp2/3-dependent formation of F-actin-rich membrane projections that promote internalization of the bacterium (129-131). Because LLO targets all membranes that contain cholesterol, it was expected that LLO would activate bacterial internalization in all animal cells, including cytotrophoblasts and HUVECs, but to our surprise this was not the case. However, hepatocytes are not the only cells thus far identified to undergo LLO- dependent L. monocytogenes internalization, as this was also reported in HeLa cells,
Hep2 cells, human neutrophils, and macrophages (129). Furthermore, LLO-dependent internalization has been demonstrated for L. monocytogenes strains 10403S, L028, and
EGD (129, 208). The difference in host cell response to LLO should be investigated further to understand what makes some cell types permissive to the LLO-dependent entry pathway. This would be useful for understanding how pathogens can generally take advantage of plasma membrane perforation to gain entry into host cells (242, 243).
We report that InlA and LLO cooperate in an additive or synergistic fashion depending on the cell line. Though the mechanism by which LLO and InlA cooperate is still unknown, two non-mutually exclusive hypotheses can be envisioned. First, by anchoring L. monocytogenes to the host cell, InlA increases local LLO concentration and thereby LLO-dependent internalization. Along this line, InlA likely served as the adhesin and LLO promoted the signaling cascade for bacterial internalization into MDCK cells
59 expressing GPI-anchored E-cadherin (241). Second, LLO- and InlA-induced signaling cascades may potentiate the activation of common transducers for the remodeling of F- actin and bacterial engulfment (74, 131).
Most studies that addressed the roles of InlA, InlB, and LLO utilize the laboratory strains EGD, EGD-e or 10403S, which all belong to serovar 1/2a. EGD is derived from the strain of L. monocytogenes isolated from guinea pigs in 1926 (244). EGD-e is thought to be a derivative of strain EGD (216, 220). 10403S is a derivative of the strain 10403, a strain initially isolated from a human skin lesion (219). Of these strains, EGD-e is the most virulent in mice and was shown to express high levels of some of the PrfA- stimulated genes despite the absence of the prfA* allele (220). L. monocytogenes strains associated with clinical cases and outbreaks of listeriosis predominantly belong to serovar
1/2a, 1/2b, and 4b, with greater than 50% of isolates belonging to serovar 4b (25, 245).
Characterization of virulence factors in clinical strains seems to be lacking. A role for
InlA in the invasion of Caco-2 cells has been demonstrated with a clinical isolate
(ScottA, serotype 4b) from an outbreak of listeriosis in Massachusetts in 1983 (246, 247).
One epidemiological study reported that 96% of clinical isolates, and only 65% of food isolates, express full-length InlA (75), and other studies have similarly found a higher prevalence of full-length InlA in strains associated with human and animal infection with more strains expressing truncated InlA in food isolates (76, 248, 249). Other work has found that LLO and InlB encoding genes are highly prevalent in clinical strains (250).
However, these studies only emphasize the importance of InlA, InlB, and LLO as virulence factors, but do not directly inform on their mechanism of action in vivo. In vivo
60 studies using animal models also established a role for these three virulence factors. Of the three factors, LLO is the most important for virulence as LLO-deficient strains are avirulent, so dissecting its role in vivo is challenging. In mice infected with 10403S or
EGD-e, InlB does not affect liver and spleen colonization or the LD50 (251, 252). One recent study infecting E-cadherin-humanized mice and gerbils with EGD (prfA*) showed that neither InlA nor InlB affected infection of the liver (78). The same study also showed that InlA is important for infection of the intestines, colon, and cecum, and that both InlA and InlB contribute to infection of the placenta and fetus.
In conclusion, to successfully cross the host barriers and invade multiple tissues,
L. monocytogenes uses a collection of virulence factors that collectively facilitate bacterial anchoring to host cells and successive internalization. It appears that InlA is the major adhesin, while InlA, LLO, and InlB can stimulate bacterial internalization alone or in concert with InlA. Collectively the three factors are conserved among clinical strains, but their roles likely vary in a tissue- and strain-dependent fashion.
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Chapter 3. A Listierolysin O Toxoid-Based Vaccine Protects Mice Against Listeria monocytogenes
3.1 Introduction
Listeria monocytogenes is a foodborne pathogen and the causative agent of the life- threatening disease listeriosis. The risk and severity of listeriosis are significantly increased among pregnant women, the elderly, infants, and individuals with a compromised immune system (35). Listeriosis clinical manisfestations include septicemia, meningitis, encephalitis, miscarriage, stillbirth, and severe infection of neonates (35, 197-201) with an associated mortality rate ranging from 16-25% despite treatment (193).
Although the food industry has rigorous standards for prevention and surveillance of Listeria contamination, the reported incidence of listeriosis in the US more than doubled from 2007-2014 (54). With increasing incidence of listeriosis and its associated high mortality rate, a vaccine targeting L. monocytogenes could offer an effective preventative measure to reduce the risk of this deadly disease in the susceptible populations. In particular, the aging population representing 80% of listeriosis patients is constantly increasing (253). Towards the goal of vaccine development, live-attenuated L. monocytogenes strains have shown promise in the lab, in providing protection against secondary L. monocytogenes infection (181, 254-256) and in the development of anti-
62 cancer vaccines, with the latter currently tested in clinical trials (257). However, the potential dangers of L. monocytogenes live-attenuated strains in immunocompromised individuals have been documented (258). Given that populations at higher risk for listeriosis are immunocompromised, or are characterized by a weak or altered immunity, a bacteria-free vaccine would be best to prevent the risk of vaccine-related infections while providing the benefits of vaccination for these populations. As such, protein-based vaccines that utilize important L. monocytogenes virulence factors, including listeriolysin
O, GAPDH, and p60 have been developed (259-262). Most of these vaccines induce strong T cell responses (CD4+ and CD8+), which are known to be essential for the acquisition of sterilizing immunity against L. monocytogenes (259-262).
Among the L. monocytogenes virulence factors, the pore-forming toxin listeriolysin O (LLO) is indispensable for host cell invasion and pathogenesis, with LLO- deficient strains being avirulent (101). Indeed, LLO plays an essential role in the intracellular lifecycle of L. monocytogenes by promoting phagosomal escape of the bacterium into the host cell cytosol, where the bacterium establishes its replicative niche.
In addition to its role as a virulence factor, LLO has been shown to constitute a major source of CD4 and CD8 T cell antigens during the adaptive immune response to L. monocytogenes in mice (183, 263). LLO is a member of the largest family of bacterial pore-forming toxins, the cholesterol-dependent cytolysins (CDCs) (104), a hallmark of which is the formation of large oligomeric pores in the cholesterol-rich membranes of nucleated cells and erythrocytes. CDC binding to cholesterol is indispensable for the prepore-to-pore transition of the toxin, and the cholesterol-binding domain was identified
63 as a conserved Theonine-Leucine pair located in their C-terminal domain (112) . Thus, we generated a non-hemolytic LLO variant by substituting the Theonine-Leucine pair with Glycine residues. We expected this LLO variant to be non-hemolytic and an antigen that can be used to develop a vaccine against L. monocytogenes infection.
3.2 Materials and Methods
Generation of LLO variants
The gene coding for six-His-tagged LLOT with the substitutions T515G and
L516G was generated by PCR-based site-directed mutagenesis using the pET29b plasmid harboring wild type hly (the gene coding LLO) as a template and mutagenic primers
(Forward - 5-gaa ata tct cca tct ggg gca ccg ggg gtt atc cga aat ata gta ata aag-3 and
Reverse - 5-ctt tat tac tat att tcg gat aac ccc cgg tgc ccc aga tgg aga tat ttc-3) as described previously (129). The gene coding for six-His-tagged LLOW492A was also generated using the same strategy and the mutagenic primers (Forward – 5-ggt tta gct tgg gaa tgg gcg aga acg gta att gat gac cgg-3 and Reverse – 5-ccg gtc atc aat tac cgt tct cgc cca ttc cca agc taa acc-3) (264). The gene coding for the six-His-tagged truncated listeriolysin O
LLO (LLO D1-3) was amplified by PCR from the wild type sequence of hly using the
Forward – 5’-aac gtg cat atg gat gca tct gca ttc aat aaa G-3’ and Reverse – 5’-att ctc gag tgt ata agc ttt tga agt tgt-3’(265) and cloned into pET29b using NdeI and XhoI restriction enzymes. Purification of LLO variants was performed as previously described (129,
131). LLO variants were aliquoted in 50 mM phosphate, pH 6, 1 M NaCl and stored at -
80C until used. Endotoxin measurements were performed as directed by the manufacturer
64 using the Chromogenic Endotoxin Quant Kit (Pierce), and LLOT was used when endotoxin levels were below the recommended limit of 36 EU/ml (266).
SDS-PAGE
1 µg of recombinant LLO, LLOW492A, LLOT, or LLOD1-3 was diluted 1:5 in
5x Laemmli sample buffer with β-mercaptoethanol and denatured by heating at 95°C for
5 min. Samples were then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) in 10% polyacrylamide gels. Gels were stained with coomassie blue and imaged using a ChemiDoc XRS imaging system (Bio-Rad).
Cholesterol Binding Assay
Spots (2 µl) of a serially diluted ethanol-cholesterol solution were deposited onto a PVDF membrane and air-dried. The membrane was blocked in a 20 mM Tris buffer
(TBS) with 4% nonfat milk and 0.2% Tween 20 at pH 7.4. LLO and LLOT (20 µg/ml) were incubated at 4° for 3 h in TBS with 0.2% Tween 20. After washes, rabbit anti-LLO
Abs (Abcam) were incubated for 1 h in TBS with 0.1% Tween 20, followed by washes and incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies in
TBS-Tween 0.1%. LLO was detected with ECL Western Blotting Detection Kit
(Amersham). We verified, by western blotting, that the rabbit anti-LLO antibodies recognize LLO and LLOT with similar efficiency (data not shown).
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Hemolysis Assays
Human blood was drawn in heparinized Vacutainer tubes from healthy adult volunteers with approval of the Ohio State University Institutional Review Board. After centrifugation of blood on Polymorphprep (Axis-Shield, Oslo, Norway), erythrocytes were collected from the lower cell layer and were washed with Alsever’s solution and stored at 4°C. The concentration of toxin leading to 50% hemolysis (EC50) was determined by performing a hemolysis assay between purified human erythrocytes and recombinant LLO and its variants as follows. Erythrocytes were washed three times with phosphate buffered saline (PBS) and diluted to a concentration of 4 x 107 cells/ml.
Duplicate serial dilutions of native LLO, LLOW492A, LLOD1-3, and LLOT were made at 4°C in a round bottom 96-well plate, and 160 µl of cold erythrocytes suspension were added in each well. Concentration ranges tested were: native LLO (100 nM – 0.1 nM),
LLOW492A (3,000 nM – 1.5 nM), LLOT (10,000 nM – 5 nM), LLOD1-3 (6,000 nM – 3 nM). Plates were then incubated for 30 min at 37°C, centrifuged, and 150 µl of the supernatants were transferred to a flat bottom 96-well plate for reading their absorbance
(540 nm) in a spectrophotometer. For a positive control, erythrocytes were treated 0.1%
Triton X-100 (considered 100% hemolysis) and erythrocytes were treated with PBS alone as a negative control. Percent hemolysis was determined for each toxin by comparing to the positive control. The concentration of toxin leading to 5-% hemolysis (EC50) was determined by polynomial regression using Graph Pad Prism 7 software (GraphPad
Software Inc, La Jolla,CA)
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Bacterial Cell Culture
Wild type L. monocytogenes (strain DP10403S) were grown overnight at 37° C in brain heart infusion (BHI). For infections, overnight cultures were diluted 1/20 in BHI and grown at 37°C until OD600 = 0.7-0.8. Bacteria were washed three times and diluted in injectable grade phosphate-buffered saline (PBS).
Immunization procedure
All animal protocols were approved by The Ohio State University’s Institutional
Laboratory Animal Care and Use Committee. Seven to eight week-old C57BL/6 or
C57BL/6-Igh-6tm1Cgn (B cell-deficient, also known as µMT-/-) (267) mice, purchased from
The Jackson Laboratory (Bar Harbor, ME), were housed in the university for one week before starting immunization. Mice were immunized on days 0, 7, and 14 by intraperitoneal injection of 100 µl of PBS containing various combinations of the following adjuvants and/or toxoids: control PBS, 20 µg LLOT, 20 µg LLOT plus 1 µg cholera toxin (List Biological Laboratories, Inc, Campbell, CA), 20 µg LLOT adsorbed on 40 µg alum (ThermoFisher Scientific, Waltham, MA), 1 µg cholera toxin, or 40 µg of alum. For the preparation of alum plus LLOT, LLOT was adsorbed to alum via gentle mixing for 45 min at 4°C. Blood was collected from mice via submandibular cheek bleed during the immunization procedure at days 14, 21, and 28. Serum was obtained after the clotted blood was centrifuged at 1,500 x g for 15 min at 4°C. Larger volumes of blood for IgG isolation were obtained via cardiac puncture immediately after sacrifice of the animals.
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Mouse Infection
Mice were inoculated by tail vein injection with L. monocytogenes (2 x 104 bacteria in 100 µl PBS) on day 28 after immunization. After 72 h, mice were euthanized and livers, spleens, and blood were collected. Organs were homogenized in PBS and homogenates were serially diluted, plated on BHI agar plates and incubated at 37°C for
48 hours. Bacterial colonies were enumerated to determine the colony forming units
(CFUs).
Evaluation of LLO-specific antibody titers
To determine the LLO-specific antibody titers, ELISA was performed with LLO- coated plates. Briefly, microtiter plates were coated with 100 µl of LLOT (5 µg/ml in
PBS) at 4°C overnight. Plates were washed three times with cold PBS and blocked for 5 h with 1% BSA in PBS. Plates were washed three times with cold PBS, sera were serially diluted in PBS 1% BSA and added to the plates and incubated at 4°C overnight. The
LLOT-binding antibodies were detected with HRP-conjugated anti-mouse IgG antisera
(1:3000 dilution) (Southern Biotech Associates Inc., Birmingham, AL). Alternatively, to measure IgG subclass responses, biotin-conjugated rat anti-mouse IgG1, IgG2a/c, IgG2b, or IgG3 monoclonal Abs and HRP-conjugated streptavidin (BD Biosciences, San Jose,
CA) were used (0.5 µg/ml). The reactions were revealed by addition of the water-soluble
HRP substrate ABTS (2,2'-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]- diammonium salt, Sigma-Aldrich), and the Ab titers were determined as the last dilution
68 of samples with an absorbance of >0.1 above that of control samples from negative control mice that received PBS immunizations.
Evaluation of the production of LLO-neutralizing antibodies
For LLO neutralization assays, IgGs were purified from serum using protein G- agarose (Pierce) according to the manufacturer’s instructions. LLO (5 nM) in PBS and purified serum IgGs were pre-incubated on ice in a 96 well plate for 15 min before the addition of erythrocytes at 4 x 107 cells/ml. Triton X-100 (0.1%) and PBS served as positive and negative controls for hemolysis, respectively. Samples were transferred to a spectrophotometer at 37°C and the absorbance at 700 nm wavelength was measured every minute for 30 min.
Analysis of LLOT-specific T helper cell cytokines responses
Spleens were aseptically removed from mice 38 days after initial immunization and minced by pressing through a cell strainer. Red blood cells were removed by incubation in 0.84 % ammonium chloride and, following a series of washes in RPMI
1640, spleen cells were resuspended in RPMI 1640 supplemented with 2 mM L- glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% fetal calf serum. The cell concentration was adjusted to 5 × 106 cells/ml, and 100 µl cell suspensions were added to each well (3 wells per spleen) of a
96-well micro-titer plate and cultured either alone or in the presence of 5 μg/ml LLOT for
5 days at 37 °C in a 5% CO2 atmosphere. These cells were used to determine the
69 frequencies of T helper cell cytokine responses. Cells were restimulated with PMA and
Ionomycine (BD-Pharmangen, NJ, US) and incubated for 1 h at 37 °C in a 5% CO2 atmosphere. The golgi function was blocked by Golgistop, (BD-Pharmangen, NJ, US), and cells were incubated at 37 °C in a 5% CO2 atmosphere for 5 h. Cells were then collected and washed twice with FACS buffer (PBS, 2% BSA, 0.01% NaN3). For extracellular T-cells lineage markers staining, cells were incubated with Alexa Fluor
700 anti-CD3 and Alexa Fluor 750 anti-CD4 antibodies (Biolegend, San Diego, CA) for
30 minutes at 4 C, then washed twice with FACS buffer. For intracellular cytokine staining, cells were incubated with Fixation-Permeabilization Buffer (BD-Pharmagen,
NJ, US) for 20 minutes at 4° C and washed twice with permeabilization buffer (BD-
Pharmangen, NJ, US). Cells were then labeled with Th1, Th2, Th17, and Tfh cytokine- specific antibodies (Alexa Fluor 488-IFNγ, PerCP Cy5.5-TNFα, PE-IL-5, Alexa Fluor
647-IL-21, PECy7 IL-10, Brilliant Violet 650 IL-17, Brilliant Violet 605 IL-4
(Biolegend, Sandiego, CA)) for 30 min at 4°C. Cells were washed twice with the
Permabilization buffer and then washed twice with the FACS buffer. Cells were suspended in FACS buffer for flow cytometry analysis with an Attune NxT flow cytometer (Thermo Fisher Scientific, Waltham, MA). The data were analyzed by triple gating as (CD3+CD4+Cytokines+). Statistical differences were determined by one-way
ANOVA, and significant differences were considered at p ≤ 0.05 (*). Statistical tests were performed using Graph Pad Prism7 (GraphPad Software Inc, La Jolla,CA).
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3.3 Results
Generation of a full-length non-hemolytic listeriolysin O toxoid (LLOT)
We generated a full length LLO toxoid (LLOT) by substitution of the cholesterol- binding threonine-leucine pair with glycines (T515G/L516G) (112). Because LLO binding to cholesterol is absolutely required for formation of the toxin pore, this LLO variant was expected be non-toxic (non-hemolytic), while retaining its antigenic properties.
Figure 3.1 Characterization of LLOT (A) Recombinant LLO, LLOT (T515G/L516G), LLO W492A, and LLOD1-3 (1 µg loaded) were subjected to SDS-PAGE and stained with coomassie blue. (B) Recombinant LLO and LLOT were incubated on a PVDF membrane pre-coated with a serial dilution of a cholesterol solution (20 to 0.15 mg/ml cholesterol in EtOH). LLO and LLOT binding to cholesterol was measured by adding primary anti-LLO antibodies and HRP-conjugated secondary antibodies, followed by chemiluminescent detection. A T representative experiment, of 2, is shown. (C) The EC50 of LLO, LLO W492A, LLO
71
(T515G/L516G), and LLO D1-3 was measured from four independent hemolytic assays, each performed in duplicate. P values were calculated using a two-tailed Student’s t-test (* = P, <0.05; ** = P<0.01; *** = P<0.001).
As expected, LLOT is a full length peptide 56 kDa which is equivalent to native LLO as assessed by SDS-PAGE (Figure 3.1A) Using a dot blot assay, we confirmed that unlike native LLO, LLOT was unable to bind cholesterol (Figure 3.1B). We then compared the hemolytic activity of LLOT to native LLO, a truncated LLO (D1-3) variant devoid of the host cell binding domain D4, and a full-length LLO variant with the amino acid substitution W492A in domain 4 that was previously reported as non-hemolytic (LLO
W492A) (264). As presented in figure 1B, LLOT hemolytic activity was markedly decreased when compared with native LLO and LLO W492A by 3,500-fold and 80-fold respectively. As expected, LLOT hemolytic activity was nearly as low as the truncated
LLO D1-3 variant (Figure 3.1C).
Immunization with LLOT plus cholera toxin protects mice against L. monocytogenes
To determine if LLOT could promote immunization of mice against L. monocytogenes, LLOT was administered via intraperitoneal injections at weekly intervals for 3 weeks, in the presence or absence of cholera toxin (CT), used as an adjuvant (268).
Mice that received PBS only were used as negative control. At day 28 after initial immunization, mice were challenged with 2 x 104 L. monocytogenes by tail vein injection and bacterial burden was determined by CFU enumeration in the spleen and liver three days post-infection. As shown in Figure 3.2, mice immunized with LLOT plus CT were
72 significantly protected against L. monocytogenes when compared to the groups treated with only LLOT, CT, or PBS.
Figure 3.2 LLOT plus adjuvant CT protects mice against infection by L. monocytogenes Mice were immunized at weekly intervals for 3 consecutive weeks by intraperitoneal injection of PBS (negative control), cholera toxin (CT) (1 mg) used as an adjuvant, LLOT (20 mg), or LLOT (20 mg) plus cholera toxin (1 mg) (LLOT+CT). At day 28, mice were intravenously inoculated with 2 x 104 wt L. monocytogenes and sacrificed after 72 h to collect blood and enumerate bacterial colony forming units (CFUs) in the liver and spleen. (A and B) CFUs were enumerated in organ homogenates. Results are expressed as CFUs/organ and medians are presented. Data are from two independent experiments, each including five female mice per experimental condition. Statistical significance was calculated using a two-sided Mann-Whitney test, ** P < 0.01.
Because injection of monoclonal LLO-neutralizing antibodies have been shown to protect mice against L. monocytogenes (191), and because CT is known to potentiate antibody production, we next measured the production of anti-LLOT IgG titers in the various groups of animals. Data showed that mice treated with LLOT or with LLOT + CT developed anti-LLOT IgG, but the adjuvant greatly enhanced the production of LLOT- specific IgG including IgG1 and IgG2a (Figure 3.3A). Furthermore, mice treated with
73
LLOT or LLOT + CT produced more IgG1 than IgG2a, a profile previously seen when CT was administered with inert antigens such as ovalbumin (269). We then determined if these antibodies could neutralize LLO. IgGs purified from mice treated with LLOT + CT, but not from control mice treated with PBS or with CT alone, efficiently neutralized LLO
(Figure 3.3B). IgGs purified from mice treated with LLOT alone could also neutralize
LLO, but to a lower extent (data not shown), consistent with the lower concentration of anti-LLO antibodies produced in this group of animals (Figure 3.3A).
Figure 3.3 LLO neutralizing antibodies are produced following immunization with LLOT plus CT (A) The titers of LLOT-specific IgG, IgG1 and IgG2a were determined by ELISA from sera collected from mice treated with LLOT and LLOT plus cholera toxin. Results are expressed as Log2 values of serum dilution titers of one immunization experiment. Statistical significance was calculated using a two-sided Student’s t- test, * P < 0.05, ** P < 0.01. N = titers from 5 mice (LLOT) or 4 mice (LLOT + CT). Horizontal bars indicate the mean values. (B) IgGs were purified from pooled serum isolated from mice treated with cholera toxin alone (CT) or LLOT plus cholera toxin (LLOT + CT) and tested for
74 their ability to inhibit hemolysis of native LLO over 30 min at 37°C. As negative and positive controls, erythrocytes were incubated with PBS or Triton X-100, respectively.
Immunization with LLOT and alum as adjuvant does not protect against L. monocytogenes
In order to further examine the possibility that anti-LLO antibodies alone could play a sufficient role in the protection of mice as was observed with a monoclonal anti-
LLO neutralizing antibody (191), we then chose to test the effectiveness of alum, a widely used vaccine adjuvant that predominantly induces strong antibody responses to antigens, and Th2 responses (270).
Figure 3.4 LLOT coupled with CT but not alum protects mice from L. monocytogenes Mice (4 male and 4 female mice/experimental condition) were immunized at weekly intervals for 3 consecutive weeks by intraperitoneal injection of PBS (negative control), cholera toxin (1 µg) used as an adjuvant, alum (40 µg) used as an adjuvant, LLOT (20 mg), LLOT (20 mg) plus cholera toxin (1 mg), or LLOT (20 mg) plus alum (40 µg). At day 28, mice were intravenously inoculated with 2 x 104 wt L. monocytogenes and 75 sacrificed after 72 h to collect blood and enumerate bacterial colony forming units (CFUs) in the liver and spleen. (A and B) CFUs were enumerated in organ homogenates. Results are expressed as CFUs/organ and medians are presented. Statistical significance was calculated using a two-sided Mann-Whitney test, ** P < 0.01.
After a similar immunization procedure in which we compared the adjuvant activity of alum and CT, we observed that mice that received LLOT + Alum were not protected against L. monocytogenes (Figure 3.4).
Mice treated with LLOT + Alum presented higher LLOT-specific IgG1 production than LLOT alone, but to a lesser extent than LLOT + CT (Figure 3.5A). In contrast to CT, alum did not induce higher levels of IgG2a isotypes relative to LLOT alone (Figure 3.5A).
We also tested for the production of antigen-specific IgG2b and IgG3 isotypes, and as with IgG2a, LLOT alone or with alum led to similar antibody titers, and the addition of
CT substantially increased production of these isotypes.
To establish if antibodies produced in response to the LLOT + Alum injection could neutralize LLO, we purified serum IgGs from immunized animals. As observed previously, IgGs from mice treated with LLOT + CT efficiently neutralized the hemolytic activity of LLO however, despite increased IgG production after LLOT + Alum treatment, IgGs from these animals were unable to neutralize LLO (Figure 3.5B).
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Figure 3.5 Immunzation with LLOT with either cholera toxin or alum as adjuvant lead to serum LLOT-specific IgG production with cholera toxin specifically inducing IgG2a, IgG2b, and IgG3 isotypes (A)The titers of LLOT-specific IgG, IgG1 and IgG2a, IgG2b, and IgG3 were determined by ELISA in serially diluted (1:2) sera from mice immunized with LLOT alone, LLOT+CT or LLOT+Alum. Serum dilution Ab titers were determined as the last dilutions of sera that gave an absorbance > 0.1 above that of control sera from naïve mice. Results are expressed as Log2 values of serum dilution titers. Statistical significance was calculated using a one-way ANOVA, * P < 0.05, ** P < 0.01. N = titers from 8 mice for each group. (B) IgGs were purified from pooled serum isolated from mice treated with PBS, LLOT + CT or LLOT + Alum and tested for their ability to inhibit hemolysis of native LLO over 30 min at 37°C. As negative and positive controls, erythrocytes were incubated with PBS or Triton X-100, respectively.
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The protective immunization with cholera toxin elicits a pronounced increase in Th1 type responses to LLO
To establish the nature of the T helper responses elicited by the protective
(LLOT+CT) and non-protective (LLOT+Alum) treatments, we isolated and cultured splenocytes from the different groups of mice. After restimulation of splenocytes with recombinant LLOT, splenocytes were labeled with fluorescent anti-CD3 and anti-CD4 antibodies to denote CD4+ T helper cells, and colabeled with additional fluorescent antibodies to identify Th1 (IFN-γ, and TNF-α)-, Th2 (IL-5, IL-4, and IL-10)-, Th17 (IL-
17A)-, and Tfh (IL-21)-type cytokines, and were subsequently analyzed by flow cytometry.
Th1 cells and their characteristic cytokines (IFN-γ and TNF-α) promote cell- mediated immunity, including cytotoxic CD8+ T cells and the activation of macrophages, both of which are important for protection against intracellular pathogens, including L. monocytogenes (179). Flow cytometry analysis of CD3+CD4+ cells after restimulation with LLOT showed that immunization with LLOT+CT led to a significant increase in
IFN-γ producing T helper cells when compared to all other treatments (Figure 3.6A).
LLOT+Alum treatment led to a significant increase in IFN-γ producing T helper cells over Alum, or CT control groups; however, this increase was still significantly lower when compared to LLOT+CT (Figure 3.6A). Treatment with LLOT in the presence or absence of adjuvants led to a significant increase in TNF-α producing T helper cells when compared to the PBS negative control (Figure 3.6A).
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Figure 3.6 Cholera toxin and alum adjuvants triggers balanced Th1, Th2, and Th17 responses, with significant increase in Th1 responses associated with CT compared to alum Spleens were isolated from immunized mice, homogenized into a cell suspension and cultured for 5 days in the presence of 5 µg/ml LLOT. The frequencies of LLOT-specific Th1 (CD3+CD4+IFN-γ+ and CD3+CD4+TNF-α+) (A), Th2 (CD3+CD4+IL-5+, CD3+CD4+IL-4+, and CD3+CD4+IL-10+) (B and D), Th17 (CD3+CD4+IL-17A+) (C), and Tfh (CD3+CD4+IL-21+) (E) cells were determined by flow cytometry after intracellular staining with the corresponding anti-cytokine antibodies. Data were expressed as mean % positive cells ± standard deviation. Statistical differences were determine by one-way ANOVS and significant differences were considered at (* p ≤ 0.05)
79
Th2 cells are known to produce cytokines (IL-5, IL-4, and IL-10) that support the production of antibodies. The main products of Tfh cells (IL-21) and Th17 cells (IL-17A) also facilitate antibody production and their affinity maturation. We found that for all of these cytokines, immunization with LLOT plus Alum or CT led to significant increases in cytokine producing T helper cells after restimulation with LLOT in vitro when compared to the PBS control group and the groups given the adjuvants alone. Additionally, of these cytokines, immunization with LLOT alone led to increases in IL-5 producing T helper cells (Figure 3.6B-E).
Taken together, the results indicate that both the non-protective alum and the protective CT adjuvants lead to increased Th1, Th2, and Th17 responses. However, the protective immunization with LLO plus CT leads to the most pronounced increase in
IFN-γ responses when compared to all other conditions, including the condition in which the LLO alone (which is non-protective) was used for immunization.
LLOT plus the cholera toxin adjuvant protects mice lacking mature B cells against
L. monocytogenes
The protective immunization regimen (LLOT + CT) was characterized by both increased LLO-specific antibody production (Figure 3.5) and Th1 responses (Figure 3.6) when compared to the non-protective (LLOT + Alum) treatment. To establish if the production of anti-LLO antibodies was playing a significant role in protection against L. monocytogenes in the immunized group, we then repeated the immunization procedure using mice that lack mature B cells (µMT-/-) in comparison to wild type mice. We
80 observed that regardless of treatment, there was a significant reduction in bacterial burden in µMT-/- mice compared to WT mice, suggesting that compensatory mechanisms may have limited infection in µMT-/- mice. As expected, we did not detect LLO-specific
IgGs in µMT-/- mice; whereas LLO-specific IgGs were being induced by treatment with
LLOT+CT as previously observed (data not shown). Despite the lack of LLO-specific antibody production in µMT-/- mice, LLOT+CT could still induce significant protection against L. monocytogenes (Figure 3.6). Together, our data establish that LLOT is a promising toxoid that can elicit protective immunity against L. monocytogenes in the presence of a Th1 adjuvant.
Figure 3.7 Immunization with LLOT coupled with CT adjuvant is effective in both WT C56BL/6 and µMT-/- mice that lack mature B cells WT mice and µMT-/- mice (8 mice/experimental condition) were immunized at weekly intervals for 3 consecutive weeks by intraperitoneal injection of PBS (negative control), LLOT (20 mg), LLOT (20 mg) plus cholera toxin (1 mg), or LLOT (20 mg) plus alum (40 µg). At day 28, mice were intravenously inoculated with 2 x 104 wt L. monocytogenes 81 and sacrificed after 72 h to collect blood and enumerate bacterial colony forming units (CFUs) in the liver and spleen. (A and B) CFUs were enumerated in organ homogenates. Results are expressed as CFUs/organ and medians are presented. Statistical significance was calculated using a two-sided Mann-Whitney test, * P <0.05, ** P < 0.01.
3.4 Discussion
We generated a full length LLO toxoid to be used as a tool for vaccine development. In this study, we report that immunization with LLOT with the adjuvant cholera toxin protects mice against L. monocytogenes infection.
Much is known regarding the key players that mediate sterilizing adaptive immune response to L. monocytogenes. These include CD4+ Th1 cells producing IFN-γ, known to activate macrophages, and CD8+ cytotoxic T cell responses (179, 181, 271,
272). Some studies suggested that, unlike the robust T cell responses targeting L. monocytogenes, B cell responses and the production of antibodies against L. monocytogenes were limited (191). However, the passive transfer of monoclonal LLO- neutralizing antibodies protected naïve mice against sub-lethal and lethal doses of L. monocytogenes (191). This led to the hypothesis that a L. monocytogenes vaccine that could elicit the production of LLO-neutralizing antibodies, in addition to the typical T cell responses, would offer extensive protection against L. monocytogenes. To this end, we sought to generate a full length LLO toxoid to be used as a tool for vaccine development. To construct a LLO toxoid, we substituted the Thr-Leu (T515G/L516G) pair in domain 4, known as the cholesterol recognition motif (CRM), that is essential for cholesterol binding in PFO, streptolysin O (SLO), pneumolysin (PLY), and
82 intermedilysin (ILY) and thought to be conserved amongst the CDC family (112, 273).
We designated our LLO toxoid LLOT. As expected, LLOT has significantly reduced hemolytic activity when compared to the native toxin, and even compared to the LLO
W492A variant toxin. In fact, it appears to be only slightly more hemolytic than the LLO variant lacking the entire membrane-binding domain (LLO D1-3) (Fig. 1). In addition to the safety of LLOT, the threonine-leucine substitutions in the toxoid do not overlap with the known LLO T cell or B cell epitopes (274, 275). The significant reduction in hemolytic activity and maintenance of epitope integrity makes LLOT an ideal candidate for use as a toxoid for the development of vaccines.
Because Th1 type immune responses and passive transfer of neutralizing LLO antibodies have been shown separately to be effective in reducing bacterial infection, we sought to use our toxoid to elicit both responses (191). To accomplish this, we used cholera toxin, an adjuvant that is known to elicit a balanced and robust Th1, Th2, and
Th17 type immune response (268). In line with our expectations, the addition of cholera toxin to our LLOT immunization significantly increased production of IgG1 and IgG2a isotypes (269) (Figure 3.3A), with IgG2a isotype class switching being known to be driven by IFN-γ (276). Importantly, these antibodies neutralized LLO activity (Figure
3.3B). Finally, inoculation of LLOT plus the cholera toxin adjuvant significantly protected mice against L. monocytogenes (Figure 3.2).
To interrogate the potential role of LLO-neutralizing antibodies in the protective response, we also used a common adjuvant, alum, which is known to induce strong antibody production without concurrently inducing strong Th1 T cell responses (270).
83
We found that, unlike the immunization including the antigen and cholera toxin, the inclusion of alum with the antigen did not result in reduced infectious burden in mice
(Figure 3.4). To establish differences in the immune responses elicited by the protective
LLOT+CT vaccine and the non-protective LLOT+Alum vaccine, we compared the titers of IgG, their neutralizing capability, and the various subclasses and the cytokine profile of antigen-specific CD4+ T cells. Even though alum is well known for generating strong antibody responses, we found that it was slightly less efficient than cholera toxin in eliciting antigen-specific total IgG and IgG1. In addition, the levels of IgG2a, IgG2b, and
IgG3 were substantially lower with alum than CT (Figure 3.5). The IgG2a, IgG2b, and
IgG3 isotypes are thought to be the chief complement-fixing and opsonizing isotypes in mice (277, 278). Additionally, we found that IgGs from mice treated with LLOT + Alum vaccine did not neutralize LLO, while IgGs from mice treated with LLOT + CT were neutralizing. Based on the titers of the different isotypes, it is possible that, at least in part, a combination of the IgG2a, IgG2b, and IgG3 isotypes may mediate the LLO- neutralizing activity. Interestingly, IgGs from the mice treated with only LLOT also had neutralizing capacity. This suggests that, in addition to the potential role for the CT specific IgG isotypes, the addition of alum to the vaccine may be leading to changes in the affinity maturation that reduces the neutralizing capacity of the IgGs produced.
We also compared the ability of the two adjuvants to elicit LLO-specific T helper responses. After immunization, splenocytes were isolated and re-exposed to LLOT in vitro. The data showed that the only distinction of the LLOT+CT vaccine is the significantly increased percentage of IFN-γ+ CD4+ T cells relative to the LLOT+Alum
84 vaccine, indicating a Th1 response. This is in agreement with the critical role of IFN-γ in the activation of CD8+ T cells and macrophages in the clearance of L. monocytogenes.
IFN-γ is also known to be pivotal for Ig class switching to the IgG2a isotype, with the associated Th1 response being important for IgG2b and IgG3 (278). To determine if the
Th1 response, which is implicated in protection, is sufficient in the absence of LLO- specific antibodies, we immunized µMT-/- mice, which lack mature B cells. There was a significant reduction in bacterial CFUs in the livers and spleens of mice immunized with
LLOT+CT compared to all other groups of animals (Figure 3.7). This indicates that, in the context of other immune responses elicited by the CT coupled vaccine, the antibody responses are dispensable for protection. However, we cannot rule out that the production of neutralizing antibodies may have a yet undefined role in protective immunity.
Taken together, the data suggests that the elicitation of a strong Th1 response is important for the effectiveness of the LLOT+CT vaccine. Future work will define the specific cell types that are mediating the protective effects of the vaccine and the molecular mechanisms responsible. The populations in which a listeriosis vaccine would be beneficial are those that are predominantly susceptible to disease, including pregnant women and the elderly. There have been studies attempting to immunize mice to reduce the effects of L. monocytogenes during pregnancy with seemingly conflicting results
(254, 279, 280). Two studies using gold-nanoparticles conjugated to GAPDH or LLO peptides have shown some promise in reducing adverse effects on neonates (279, 280). In another study, researchers attempted to vaccinate mice with an attenuated L. monocytogenes strain to protect against infection during pregnancy. They found that
85 although the vaccine strain conferred protection in non-pregnant mice, in the case of allogenic pregnancy, immunization effectiveness was significantly reduced (254). They did find protection during syngeneic pregnancy, which is in line with the former two studies. However, the data in allogenic pregnancy is critical, given that it is a more realistic model for human pregnancies. Despite this data, testing our vaccine in pregnant mice is of interest. Though a T cell response may not be sufficient for protection during an allogenic pregnancy, our vaccine also elicits strong anti-LLO antibody responses.
Because IgGs can pass the placental barrier in humans (281, 282) and the placental yolk sac in mice (283), it may be that in the context of pregnancy, where T cell responses are insufficient, that our additional antibody responses may have a role in protection.
Given the success of this LLO toxoid in our vaccine strategy, similar toxoids derived from other CDCs of other human pathogens by targeting the CRM could potentially be combined with the cholera toxin adjuvant for the development of other novel vaccines. In support of this strategy, vaccines against other Gram-positive pathogens have been developed using toxoids of their respective CDCs that have been detoxified through different methods (284-288).
LLO is known to have adjuvant properties, and given the reduced toxicity and increased safety of our toxoid, determining if LLOT could function as an adjuvant in a variety of vaccines could be of great interest. Here, we show that LLOT alone has some immunogenic properties, as it elicits an antibody response, including production of all
IgG isotypes, and significantly increases TNF-α and IL-5 positive CD4+ T cells,. When used as an adjuvant with a dengue virus antigen, another LLO variant containing
86 mutations targeting the undecapeptide was found to elicit increased dengue virus envelope protein-specific IgG1 and IgG2a (289). This particular LLO variant has also been shown to be effective as an adjuvant for tumor immunotherapy in mice (290). Given the immunogenicity of LLOT it could potentially have similar effects, both as adjuvant for other antigens as a part of a vaccine, and in cancer treatments.
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Chapter 4. Conclusions and Future Directions
L. monocytogenes is a foodborne intracellular pathogen and the etiological agent of human listeriosis. Though many healthy adults can be exposed to L. monocytogenes with only relatively minor non-invasive listeriosis eliciting symptoms such as gastroenteritis and flu-like symptoms, susceptible populations, including pregnant women, the elderly, and those with compromised immune systems, may develop invasive listeriosis (5, 10). Listeriosis is a deadly disease with a mortality rate from 16-25% in reported clinical cases. This disease also carries incredibly high economic costs associated with hospitalization, surveillance, recalls of contaminated goods, and prevention in food production facilities (193).
Successful infection of the host by L. monocytogenes involves the bacterium establishing an intracellular niche in not only professional phagocytes like macrophages and dendritic cells but also in normally nonphagocytic cells, including a variety of epithelial cells (5). To potentiate entry into nonphagocytic cells, L. monocytogenes uses an arsenal of invasion factors that include the major canonical invasins, InlA and InlB, as well as the pore-forming toxin LLO (27, 28, 42, 129-131). Each of these invasion factors induces their own separate signaling pathways that ultimately lead to actin cytoskeletal rearrangement and internalization of the bacterium. Though these invasins are co- expressed during infection, there has been limited work describing their importance when
88 all three are considered. Indeed, to date, studies have only begun to examine how InlA and InlB may work together to potentiate the internalization of bacteria in specific circumstances (194-196), and no other work has addressed the importance of LLO as an invasion factor when in the context of InlA and InlB.
In this work, we have addressed, for the first time, the role of the pore-forming toxin LLO during bacterial invasion of nonphagocytic cells in the context of the two canonical L. monocytogenes invasins, InlA and InlB. Using L. monocytogenes strain
10403S, we found that LLO and InlA are the major invasins for infection of hepatocytes, with LLO specifically inducing internalization of the bacterium and InlA primarily promoting bacterial attachment to the host cells (Figure 2.3). In placental cells we found that LLO plays no role, whereas InlA acts specifically to promote adhesion to host cells
(Figure 2.6). In endothelial cells, we found no role for any individual invasion factor, which is consistent with other recent work (214). Interestingly, we initially found no role for InlB in any cell type tested. This was a surprise because InlB was originally described as being important for the invasion of hepatocytes (27). Our data suggests that the lack of a detected role for InlB is related to its expression level, which is PrfA-dependent. Strain
10403S encodes a wild-type version of PrfA, whereas the primary strain used for InlB studies is a stain encoding a PrfA* variant that is constitutively active, leading to overexpression of virulence factors, including InlB (220). Indeed, when we introduced the same mutation in the prfA gene of strain 10403S, we observed a significant increase in InlB production. Subsequently, in invasion assays we finally observed a role for InlB in facilitating bacterial internalization. We also observed significant increases in bacterial
89 association with the host cells, which is likely caused by increases in the other factors, including the major adhesin, InlA. While we have also identified that LLO and InlA are working in a greater than additive fashion to potentiate internalization of bacteria into hepatocytes, future work will be needed to address the mechanism by which the invasins cooperate.
LLO has become known as a multifaceted virulence factor with a variety of roles during the process of L. monocytogenes infection (204). Because of its many roles and its essential nature during infection, it is a prime target for the development of new therapeutics for combating listeriosis. To this end, we developed a LLO toxoid (LLOT) based vaccine strategy to protect against L. monocytogenes infection in mice. We found that when administered with the cholera toxin (CT) adjuvant, bacterial burdens in mice were significantly reduced. This immunization also resulted in the production of LLO- neutralizing IgGs. We also identified Th1 responses, and particularly the production of
IFN-γ, as potentially important for protection. Indeed, mice that were given a non- protective vaccine with LLOT and alum as adjuvant produced LLO-specific antibodies and similar CD4+ T cell responses with the sole exception of IFN-γ. Though we hypothesized neutralizing antibodies may be important for protection, we found that the vaccine that was protective in wild type mice was also protective in mice lacking mature
B cells and antibodies. Thus the Th1 response most likely provides the protection in this model. Future work will be required to determine what other cells are involved in the clearance of bacteria. Given the production of IFN-γ, and based on the literature, we anticipate that CD8+ cytotoxic T cells activity and the activation of macrophages are
90 chiefly responsible. However, we cannot completely rule out the possibility of neutralizing antibodies playing some role in response to infection in different contexts. In humans, the primary susceptible populations in which a prophylactic vaccine would be of interest include pregnant women and the elderly. In particular, a vaccine that generates
LLO-neutralizing antibodies may be of particular interest during pregnancy because some classes of IgG can cross the placental barrier whereas T cells do not cross the barrier and are less responsive in the placenta due to the establishment of fetal tolerance. It is possible that LLO-specific IgGs could then have an effect on limiting bacterial invasion and growth in the placenta. Testing our vaccine candidate in a pregnant mouse model in the future is of great interest.
91
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Appendix A. Chapter 2 supplemental Figures
Figure A.1 Validation of anti-InlB antibodies Cell lysates of WT and InlB-deficient (ΔinlB) L. monocytogenes (3.2x108 bacteria), and 5 ng of InlB (used as a positive control) were subjected to western blot analysis using the anti-InlB polyclonal rabbit antibodies.
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Figure A.2 Cooperation of LLO, InlA, and InlB in bacterial invasion of human hepatocytes Hepatocyte cell lines were infected with WT, LLO-deficient (Δhly), InlA-deficient (ΔinlA), InlB-deficient (ΔinlB), InlA/LLO-deficient (ΔinlAΔhly), InlB/LLO-deficient (ΔinlBΔhly), InlAB-deficient (ΔinlAB), and InlAB/LLO-deficient (ΔinlABΔhly) bacteria (MOI 20) for 30 min at 37°C. Cells were washed, fixed and labeled with fluorescent antibodies and DAPI. (Left Column) Total bacteria/host cell. The average bacterial association for the WT strain before normalization were: HepG2 – 0.14, Hep3B – 3.13, PLC5 – 1.34, Huh7 – 0.77. (Right Column) Percent intracellular bacteria. The average internalization values for the WT strain before normalization were: HepG2 – 26.45%, Hep3B – 38.77%, PLC5 – 18.29%, Huh7 – 33.12 %. The minimum number of host cells counted was: HepG2 – 1000, Hep3B – 150, PLC5 – 600, Huh7 – 2000. The average number of WT bacteria counted per experiment was: HepG2 – 600, Hep3B – 4,000, PLC5 – 2,000, Huh7 – 3,000 with a minimum count of 100 bacteria being required for
114 any mutant with reduced association efficiency. Results are expressed as the mean ± SEM relative to WT (n ≥ 3). Statistical analyses compared each strain to the WT strain and were performed on raw data before normalization (* p < 0.01, ** p < 0.001).
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