2021 Molecular Basis of Infectious Diseases (MBID) Research Retreat

Schedule &

Abstract Booklet

January 8, 2021, 10 AM

Sponsors

The Department of Microbiology & Molecular Genetics, McGovern Medical School The Molecular Basis of Infectious Diseases (MBID) Training Program

Organizing Committee Theresa M. Koehler, Ph.D., Professor & Chair, Microbiology & Molecular Genetics, McGovern Medical School Michael Lorenz, Ph.D., Professor, Microbiology & Molecular Genetics, McGovern Medical School Heidi B. Kaplan, Ph.D., Associate Professor, Microbiology & Molecular Genetics, McGovern Medical School Shelby Simar, MBID Fellow, UTHealth School of Public Health/Epidemiology, Human Genetics & Environmental Sciences Ahn Kim Trinh Nguyen, MBID Fellow, Microbiology & Molecular Genetics, McGovern Medical School 0 Suhyeorn Jane Park, MBID Fellow, Pharmacology & Chemical Biology, Baylor College of Medicine

8:45 AM Career Development Panel Jennifer Dale, Ph.D. Research Scientist, Minnesota Department of Health Ph.D. with Theresa Koehler, MDAnderson UTHealth Graduate School

Alejandra King, Ph.D. Manager, Strategic Alliances, Regeneron Pharmaceuticals Ph.D. with Ambro van Hoof, MDAnderson UTHealth Graduate School

Fahd Majiduddin, Ph.D. Associate Director - Patent Counsel, Teva Pharmaceuticals Ph.D. with Timothy Palzkill, Baylor College of Medicine MDAnderson UTHealth Graduate School

Shrenik Mehta, Ph.D. Scientist, Genentech Ph.D. with Timothy Palzkill, Baylor College of Medicine MDAnderson UTHealth Graduate School

Elke Saile, Ph.D. Microbiologist, Centers for Disease Control and Prevention National Center for Emerging and Zoonotic Infectious Diseases Postdoc with Theresa Koehler, McGovern Medical School

10:00 Welcome Theresa Koehler, Ph.D.

10:15 Keynote 1 Alina Baum, Ph.D. Senior Staff Scientist, Regeneron Pharmaceuticals “Development of REGN-COV2 antibody combination for COVID-19”

11:15 Session 1 Kathryn Patras, Ph.D. Assistant Professor, Baylor College of Medicine “Group B Streptococcus and the vaginal microenvironment”

Kathryn Brink Graduate Student, Rice University “High-throughput identification of human antimicrobial peptide activators of Salmonella PhoQ”

1 Alexandra Powell Graduate Student, Texas A&M HSC “Classical complement pathway evasion in Lyme disease spirochetes, Borrelia miyamotoi, and tick-borne relapsing fever Borrelia”

John Taylor Graduate Student, Texas A&M Institute of Biosciences & Technology “The Type VII secretion system is a virulence determinant of Streptococcus gallolyticus subspecies gallolyticus”

12:15 PM Lunch Break 12:45 Poster Session, Zoom Breakout Rooms

2:00 Keynote 2 Scott C. Weaver, M.S., Ph.D. John Sealy Distinguished University Chair in Human Infections and Immunity Chair, Department of Microbiology & Immunology Director, Institute for Human Infections & Immunity Scientific Director, Galveston National Laboratory Professor, Microbiology & Immunology and Pathology, UTMB Health “Mechanisms of urban arbovirus emergence”

3:00 Session 2 Christian Perez, Ph.D. Associate Professor, McGovern Medical School, Microbiology and Molecular Genetics “Mammalian host colonization by the fungus Candida albicans” Justin Clark, Ph.D., Postdoctoral Fellow, Baylor College of Medicine “Comparative pathogenomics of to identify vaccine targets from the virulome” Max Odem, Ph.D. Postdoctoral Fellow, McGovern Medical School, Microbiology and Molecular Genetics “A high-throughput, open-source pipeline/workflow for the measurement and analysis of gut motility in larval zebrafish” Austen Terwilliger, Ph.D. Laboratory Director, Baylor College of Medicine, Microbiology & Molecular Genetics “Wastewater trends of SARS-COV-2 as a predictive measure of clinical trends for a major metropolis”

4:00 Awards and Closing Remarks

2 BREAKOUT ROOM/ POSTER TITLE PRESENTER

GS1 - Carter, Hannah Cytotoxic effects of atypical enteroaggregative E. coli suggests an adherence- independent mechanism of disease

GS2 - Corsi, Ileana AtxA-controlled small RNAs of virulence pXO1 regulate gene expression in trans

GS3 - Cristy, Shane Virulence and formation in Candida albicans are inhibited by short peptide subunits of EntV

GS4 - Ewan, Jellisa Investigating the uncharted functions of Dcp2 decapping enzyme

GS5 - Flores, Erika Zebrafish (Danio rerio) as a model host to study the role of mucin sulfation in adherent-invasive E. coli infection

GS6 - Jelinski, Joseph Best 3 out of 5: Progress towards a heme-based vaccine for anthrax

GS7 - Kang, Alex An in vitro cell culture model for siderophore-mediated virulence

GS8 - Martinez, Melissa Characterizing the antagonistic interaction between Candida albicans and Enterococcus faecalis

GS9 - Nguyen, April Dynamics of Enterococcus faecalis cardiolipin synthase gene expression reveal compensatory roles in daptomycin resistance

GS10 - Odo, Chioma Identifying molecular mechanisms underlying emergence of emm4 GAS

GS11 - Park, Suhyeorn Discovery of inhibitors of the KPC-2 carbapenemase using a focused DNA-encoded library

GS12 - Salazar, Keiko Pandemic ExPEC phage-resisters are predictable and attenuated

GS13 - Simar, Shelby The role of the accessory in enterococcal bacteremia: Results from the vancomycin-resistant enterococcal bacteremia Outcomes Study (VENOUS)

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GS14 - Skrobarczyk, Characterization and comparison of human and equine rotavirus isolates for Jill therapeutic development

GS15 - Trinh Nguyen, Characterizing the role of intestinal epithelial extrusion and immune Anh surveillance during gastrointestinal infection using zebrafish (Danio rerio) as a model host

GS16 - Zulk, Jacob Evaluating phage therapy for the treatment of urinary tract infection

PD1 - Curtis, Michael Characterizing the roles of putative lipoproteins during tick-borne relapsing fever Borrelia vector competence

PD2 - Guha, Shantanu Development of novel antifungals against Candida based on an antifungal peptide produced by E. faecalis

PD3 - Sanchez, Belkys Development of with anti-biofilm properties as novel treatment for catheter-associated urinary tract infections

S1 - Hunter, Robert The pathogenesis of tuberculosis: The Koch phenomenon reinstated

S2 - Krishnavajhala, Diversity and distribution of the tick-borne relapsing fever spirochete Borrelia Aparna turicatae

S3 - Mehta, Heer Evolutionary leapfrogging leads to the failure of a promising antimicrobial strategy

4

CYTOTOXIC EFFECTS OF ATYPICAL ENTEROAGGREGATIVE E. COLI SUGGESTS AN ADHERENCE-INDEPENDENT MECHANISM OF DISEASE

Hannah Carter*, Anubama Rajan, Lily Carlin, Pablo Okhuysen, Anthony Maresso (Breakout Room, GS1)

*Graduate Student, Baylor College of Medicine/Microbiology & Molecular Genetics

Cause of pediatric diarrhea in developing nations as well as infections in travelers and immunocompromised patients globally. These diverse pathogenic E. coli have been categorized into “pathotypes” based on disease presentation, adherence and aggregation patterns, and the presence of certain toxins. Yet, many E. coli strains meet the criteria for multiple pathotypes and some pathotypes, such as Enteroaggregative E. coli (EAEC), are heterogeneous and fail to account for varying shades of pathogenicity. Molecular tools and new models, like human intestinal organoids (HIEs) allow the opportunity to re-define diarrheagenic E. coli in ways that are more clinically relevant. Typical EAEC is defined by a characteristic “stacked brick” aggregation pattern on Hep2 cells and the presence of a number of genes known to drive this phenotype. However, many E. coli have been found which demonstrate this adherence pattern but lack the genes known to facilitate it. These are termed atypical EAEC. Previous outbreaks and data from invertebrate models suggest that atypical EAEC can cause disease in humans, but their pathogenicity is still debated. Emerging data suggests that enteric are a larger problem in US clinical populations than previously thought. Thus, we isolated E. coli strains from stools of immunocompromised patients at M. D. Anderson with ongoing diarrhea which tested negative for other enteropathogens. Strains were pathotyped according to current standards and confirmed to be E. coli using MALDI TOF. Adherence ability and patterning were analyzed on colonoid HIE monolayers following a 6-hour infection. We have found that typical EAEC strains show far greater adherence and aggregation to human colonoids than atypical strains, but atypical EAEC strains have greater adherence and aggregation than protype strains of four other major pathotypes of diarrheagenic E. coli. Using whole genome , we’ve found that atypical EAEC strains indeed lack adhesions, toxins, and other virulence factors associated with typical EAEC. We have isolated several strains of atypical EAEC which are cytotoxic to HIEs and are currently investigating the mechanism of these cytotoxic effects. We hypothesize that these strains contain undescribed virulence factors which can elucidate the pathogenic mechanisms of atypical EAEC.

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ATXA-CONTROLLED SMALL RNAS OF BACILLUS ANTHRACIS VIRULENCE PLASMID PXO1 REGULATE GENE EXPRESSION IN TRANS

Ileana D. Corsi*, Soumita Dutta, Ambro van Hoof, Theresa M. Koehler (Breakout Room, GS2)

*Graduate Student, McGovern Medical School/Microbiology & Molecular Genetics, MDAnderson UTHealth Graduate School

Small regulatory RNAs (sRNAs) are short transcripts that base-pair to mRNA targets or interact with regulatory proteins. sRNA function has been studied extensively in Gram- negative ; comparatively less is known about sRNAs in Firmicutes. Here we investigate two sRNAs encoded by virulence plasmid pXO1 of Bacillus anthracis, the causative agent of anthrax. The sRNAs, named “XrrA and XrrB” (for p¬XO1-encoded regulatory RNA) are abundant and highly stable primary transcripts, whose expression is dependent upon AtxA, the master virulence regulator of B. anthracis. sRNA levels are highest during culture conditions that promote AtxA expression and activity, and sRNA levels are unaltered in Hfq RNA chaperone null-mutants. Comparison of the transcriptome of a virulent Ames-derived strain to the transcriptome of isogenic sRNA- null mutants revealed multiple 4.0- to >100-fold differences in gene expression. Most regulatory effects were associated with XrrA, although regulation of some transcripts suggests functional overlap between the XrrA and XrrB. Many sRNA-regulated targets were chromosome genes associated with branched-chain amino acid metabolism, proteolysis, and transmembrane transport. analysis revealed complementarity between XrrA and the 5’ UTR of seven mRNA transcripts whose expression is affected by the sRNA, suggesting base-pairing interactions. A translational fusion of one of these targets, the secreted metalloprotease inhA1, to GFP suggests XrrA- mediated regulation of protease translation. Finally, in a mouse model for systemic anthrax, the lungs and livers of animals infected with xrrA-null mutants had a small reduction in bacterial burden, suggesting a role for XrrA in B. anthracis pathogenesis. XrrA and XrrB are the first reported sRNAs of B. anthracis. Future work will focus on the molecular basis for sRNA function, including investigations of potential RNA and/or protein interacting partners of XrrA and XrrB.

6 VIRULENCE AND BIOFILM FORMATION IN CANDIDA ALBICANS ARE INHIBITED BY SHORT PEPTIDE SUBUNITS OF ENTV

Shane A. Cristy*, Danielle A. Garsin, Michael C. Lorenz (Breakout Room, GS3)

*Graduate Student, McGovern Medical School/Microbiology & Molecular Genetics, MDAnderson UTHealth Graduate School

Candida albicans, the most clinically relevant fungal , commonly causes oral cavity and urogenital tract infections and is the third most common cause of bloodstream infections. Invasive infections are most common amongst patients with compromised innate immune responses, such as those receiving chemotherapy or recovering from surgery. C. albicans can also form difficult to treat on implanted medical devices. C. albicans exists as a member of the commensal flora of the skin and gut where many complex polymicrobial interactions occur, some of which potentiate or inhibit virulence. C. albicans virulence is modulated by a peptide, EntV, produced by the bacterial gastrointestinal commensal species Enterococcus faecalis. The active 68 amino acid EntV peptide inhibits biofilm formation in vitro and attenuates fungal virulence in a Caenorhabditis elegans infection model and a murine oral candidiasis model. Based on structural information, we hypothesized that the activity of EntV could be localized to a single helix of the mature peptide. In this study, we report that smaller peptides derived from this helix ranging from 12 to 16 amino acids have equal to improved efficacy in inhibiting C. albicans biofilm formation. These smaller peptides inhibit initial adhesion to abiotic surfaces and reduce the size of mature biofilms measured by confocal microscopy. Further trimming of these peptides to fewer than 11 amino acids reduces and eventually eliminates activity. These data indicate that EntV derived peptides warrant further investigation as potential non-fungicidal additives to medical devices and antifungal therapeutics.

7 INVESTIGATING THE UNCHARTED FUNCTIONS OF DCP2 DECAPPING ENZYME

Jellisa Ewan*, Minseon Kim, Ambro van Hoof (Breakout Room, GS4)

*Graduate Student, McGovern Medical School/Microbiology & Molecular Genetics, MDAnderson UTHealth Graduate School

In yeast, the major mRNA decay pathway involves deadenylation of the 3’ poly-A tail, followed by either decapping of the 5’ m7Gppp cap and subsequent 5’ to 3’ degradation or 3’ to 5’ degradation. This is carried out by Pan2/Pan3 and Ccr4-Not deadenylases, Dcp2 decapping enzyme, and Xrn1 and the RNA exosome exoribonucleases respectively. A dcp2∆ is lethal, but this lethality can be suppressed by several other . These mutations improve growth in dcp2∆ yeast strain without improving mRNA decay. This suggests Dcp2 may perform functions irrespective of mRNA decay. I hypothesize that Dcp2 performs functions outside of conventional mRNA decay. Dcp2 possesses a small amino-terminal containing its catalytic domain and a large unstructured carboxy-terminal known to facilitate interactions with multiple proteins, including the enhancer of decapping (Edc3). I will investigate which functions of Dcp2 are essential and which are impacted by suppressor mutations. Site directed mutagenesis was used to investigate the essential functions of Dcp2 by generating point mutations in the catalytic, cap recognition and mRNA binding site of Dcp2, along with various C-terminal truncations. Secondly, two suppressors, KAP123 and tL(GAG)G, were investigated in a dcp2∆ yeast strain. These studies revealed that Dcp2 carboxy-terminal is dispensable for growth under normal conditions. It was also noted that different suppressors may influence dcp2∆ in a different manner. Further investigations are required to determine the function of the carboxy- terminal and to understand the mechanisms by which suppressor influence dcp2∆.

8 ZEBRAFISH (DANIO RERIO) AS A MODEL HOST TO STUDY THE ROLE OF MUCIN SULFATION IN ADHERENT- INVASIVE E. COLI INFECTION

Erika Flores*, Diana Pereira Vaz, Anne-Marie Krachler (Breakout Room, GS5)

*Graduate Student, McGovern Medical School/Microbiology & Molecular Genetics, MDAnderson UTHealth Graduate School

The intestinal mucus layer forms a protective barrier between luminal bacteria and the underlying epithelium. Inflammatory bowel disease (IBD) is a multifactorial chronic disorder that affects millions of patients worldwide. Although patient genetics, environmental factors, and intestinal microbiota are all known disease modifiers, the cause of IBD is unknown. A hallmark of IBD is a compromised mucus barrier that allows direct contact between luminal bacteria and the intestinal epithelium, leading to exacerbated inflammation. Adherent-invasive E. coli (AIEC) is a pathobiont frequently found in IBD biopsies that strongly adheres to and invades intestinal epithelial cells. AIEC survives and replicates inside macrophages, inducing a proinflammatory response that promotes IBD progression. However, the mechanism by which the healthy mucosa confers protection against AIEC during homeostasis, and how this balance is disrupted in IBD, is poorly understood. We show that AIEC harbors a sulfoglycan-binding adhesin that mediates retention of AIEC by sulfomucin and prevents translocation of the mucosa during homeostasis. Mucin-desulfation decreases E. coli retention and enhances translocation of E. coli through the mucin layer. A decrease in mucin glycosylation and in mucin sulfoglycans has been linked to loss of mucosal homeostasis, and inflammation has been shown to decrease mucin-sulfation. To study the interplay between mucin- sulfation and AIEC in vivo, we established a zebrafish larval model of AIEC infection. Using this model, we study how pre-existing inflammation affects mucosal barrier and AIEC infection. We investigate how AIEC impacts inflammation, and how the sulfatase- producing commensal, Bacteroides thetaiotaomicron modulates barrier function and AIEC infection.

9 BEST 3 OUT OF 5: PROGRESS TOWARDS A HEME-BASED VACCINE FOR ANTHRAX

Joseph Jelinski*, Austen Terwilliger, Anthony Maresso (Breakout Room, GS6)

*Graduate Student, Baylor College of Medicine/Microbiology & Molecular Genetics

B. anthracis is the causative agent of anthrax disease, presents with high mortality, and has been at the center of bioweapon efforts. The only currently U.S. FDA approved vaccine to prevent anthrax in humans is Anthrax Vaccine Adsorbed (AVA), which is protective in several animal models and induces neutralizing antibodies against Protective Antigen (PA), the cell-binding component of anthrax toxin. However, AVA requires a five course regimen to induce immunity, along with an annual booster, and is composed of undefined culture supernatants from a PA-secreting strain. In addition, it appears to be ineffective against strains that lack anthrax toxin. Here, we investigated a vaccine formulation consisting of recombinant proteins from a surface-localized heme transport system containing near-iron transporter (NEAT) domains and its efficacy as a vaccine for anthrax disease. Our findings were as thus: 1) A five NEAT domain cocktail was protective against a lethal challenge of inhaled spores at both three and twenty-eight weeks after vaccination. 2) Reduced formulation with three NEATs (IsdX1, IsdX2 and Bslk) was as effective as the five NEAT cocktail. 3) The human approved adjuvant alum was as protective as Freund’s Adjuvant. 4) Protective vaccination correlated with increased anti- NEAT antibody reactivity and reduced bacterial levels in organs. 5) Passive transfer with anti-NEAT antisera reduced mortality and disease severity of infected mice, suggesting the protective component is comprised of antibodies. Collectively, this research provide evidence that a vaccine based upon recombinant NEAT proteins should be considered in the development of a next generation anthrax vaccine.

10 AN IN VITRO CELL CULTURE MODEL FOR SIDEROPHORE-MEDIATED VIRULENCE

Donghoon Kang*, Natalia V. Kirienko (Breakout Room, GS7)

*Graduate Student Rice University/BioSciences

Pseudomonas aeruginosa is a multidrug-resistant, opportunistic pathogen that utilizes a wide-range of virulence factors to cause acute, life-threatening infections in immunocompromised patients, especially those in intensive care units. It also causes debilitating chronic infections that shortens and worsens the quality of life for cystic fibrosis patients. One of the key virulence factors in P. aeruginosa is the siderophore pyoverdine, which provides the pathogen with iron during infection, regulates the production of secreted toxins, and disrupts host iron and mitochondrial homeostasis. These roles have been characterized in model organisms such as Caenorhabditis elegans and mice. However, an intermediary system using cell culture to investigate the activity of this siderophore has been absent. We developed the first in vitro cell culture model for pyoverdine-mediated virulence where murine macrophages are treated with pyoverdine- rich filtrates from P. aeruginosa. The production of pyoverdine and pyoverdine-regulated virulence factors were responsible for substantial filtrate cytotoxicity against cells, and the inhibition of pyoverdine biosynthesis (genetic or chemical) was sufficient to mitigate virulence. Furthermore, pyoverdine was able to translocate from the extracellular milieu into early endosomal compartments in macrophages and disrupt host mitochondrial homeostasis, most likely by chelating intracellular iron sources. Most importantly, we observed a strong correlation between pyoverdine production and virulence in a panel of P. aeruginosa clinical and environmental isolates, reaffirming pyoverdine’s utility as a promising target for therapeutic intervention.

11 CHARACTERIZING THE ANTAGONISTIC INTERACION BETWEEN CANDIDA ALBICANS AND ENTEROCOCCUS FAECALIS

Melissa Martinez*, Melissa R. Cruz, Danielle Garsin, Michael Lorenz (Breakout Room, GS8)

*Graduate Student, McGovern Medical School/Microbiology & Molecular Genetics, MDAnderson UTHealth Graduate School

The human body is teeming with a diverse set of microbes. How polymicrobial communities within the body achieve balance has not been well elucidated. The antagonistic relationships some of these microbes exhibit have the potential to be exploited and used as a basis for antimicrobial therapies. Enterococcus faecalis is a gram-positive bacterium that is becoming increasingly resistant to the antibiotic treatments available to patients today. This bacterium poses a serious threat to those whose immune systems have been compromised by chronic illness. Using a C. elegans infection model, it was previously demonstrated that co-infection with the opportunistic fungal pathogen, Candida albicans, attenuates E. faecalis virulence. We have shown that supernatants isolated from dual-species biofilms of C. albicans and E. faecalis reduces the ability of E. faecalis to form biofilms and kill C. elegans. Supernatants from single species C. albicans or E. faecalis lacks this activity. Based on these data and preliminary characterization of the active compound, we hypothesize that C. albicans, upon exposure to E. faecalis, produces a small peptide that hinders the virulence of the bacterium. To understand the fungal mechanisms driving bacterial inhibition, we screened a C. albicans mutant library for strains that failed to generate this activity when grown in the dual-species biofilm. Detailed characterization of several C. albicans mutants that are unable to repress E. faecalis biofilm formation is ongoing to identify the peptide that governs this inhibition, from which we will then determine its mechanism of action. This peptide has the potential to become a novel or adjunctive therapy in the treatment of E. faecalis infections.

12 DYNAMICS OF ENTEROCOCCUS FAECALIS CARDIOLIPIN SYNTHASE GENE EXPRESSION REVEAL COMPENSATORY ROLES IN DAPTOMYCIN RESISTANCE April H. Nguyen*, Vinathi Polamraju, Truc T. Tran, Diana Panesso, Ayesha Khan, Eugenia Mileykovskaya, Heidi Vitrac, Cesar A. Arias (Breakout Room, GS9)

*Graduate Student, McGovern Medical School/Microbiology & Molecular Genetics, MDAnderson UTHealth Graduate School

Background: Daptomycin (DAP) is a lipopeptide antibiotic targeting membrane anionic phospholipids (APLs) at the division septum, and resistance (DAP-R) has been linked to mutations in genes encoding i) the LiaFSR stress response system or its effector LiaX, and ii) cardiolipin synthase (Cls). Activation of the E. faecalis (Efs) LiaFSR response is associated with DAP-R and redistribution of APL microdomains away from the septum. Cardiolipin is predicted to be a major component of these microdomains. Efs harbors two cls genes, cls1 and cls2. While changes in Cls1 have been implicated in DAP-R, the exact roles of each enzyme in DAP-R are unknown. We aim to characterize the contributions of Cls1 and Cls2 in the development of DAP-R. Methods: cls1 and cls2 were deleted singly and in tandem from Efs OG117 and DAP-R Efs OG117∆liaX (with an active LiaFSR response). Mutants were characterized by DAP minimum inhibitory concentration (MIC) using E-test and localization of APL microdomains with 10-N-nonyl-acridine orange. Quantitative PCR (qRT-PCR) was used to study gene expression profiles of cls1 and cls2 in Efs OG117∆liaX relative to Efs OG117. Results: cls1 and cls2 show differential expression profiles associated with DAP-R. cls1 was highly upregulated in stationary phase concurrent with a decrease in cls2 expression. However, independent deletion of cls1 or cls2 in the DAP-R background resulted in no significant changes. Further studies revealed that cls2 expression is upregulated upon deletion of cls1, suggesting a potential compensatory role for Cls2. Double deletion of both cls genes in the DAP-R strain decreased DAP MIC restored the septal localization of APL microdomains. Conclusions: Cls1 is the major and predominant enzyme involved in cell membrane adaptation associated with the development of DAP-R in E. faecalis. However, we describe a novel compensatory and overlapping role for cardiolipin synthases to ensure bacterial survival upon attack from daptomycin.

13 IDENTIFYING MOLECULAR MECHANISMS UNDERLYING EMERGENCE OF EMM4 GAS

Chioma M. Odo*, Brittany J. Shah, Misu A. Sanson, Sruti DebRoy, Samuel A. Shelburne, Anthony R. Flores (Breakout Room, G10)

*Graduate Student, MDAnderson UTHealth Graduate School

BACKGROUND: The major human pathogen Group A Streptococcus (GAS) is classified based on sequence variability of the emm gene. We recently identified a temporal clonal emergence among emm4 GAS beginning in 1999-2001 leading to an almost total replacement by 2016. Previous studies have found that novel, hypervirulent GAS clones result from horizontal gene transfer (HGT) and recombination events leading to increased production of the key cytotoxin streptolysin O (SLO). This study sought to determine mechanisms driving clonal emergence in emm4 GAS. METHODS: The transcriptomes of the replaced vs. the emergent emm4 strains were compared with RNAseq. Virulence was compared using a mouse model of intraperitoneal infection. Laboratory growth characteristics were determined using standard growth curve methodologies whereas ex vivo growth was analyzed in human blood. SLO levels were determined via western blot analysis. RESULTS. The transcriptomes of the emergent and historic strains were distinct. Surprisingly, the emergent strains had significantly lower transcript levels of genes encoding multiple GAS virulence determinants. Conversely, increased transcript levels of genes encoding cell wall modifying and oxidizing enzymes were identified in the emergent strains. Although the emergent strains grew slower under laboratory conditions, the emergent strains grew better in human blood relative to the historic strains. Similarly, the emergent strains were significantly more virulent in the mouse challenge model. Contrary to previous reports, there was no significant increase in SLO production in the emergent strains. CONCLUSIONS: We have identified dramatic phenotypic differences in the emergent emm4 strains that stand in stark contrast to previous paradigms of GAS clonal emergence. Ongoing investigation will focus on identifying mechanisms responsible for the hypervirulence observed in these emergent strains.

14 DISCOVERY OF INHIBITORS OF THE KPC-2 CARBAPENEMASE USING A FOCUSED DNA-ENCODED LIBRARY

Suhyeorn Park*, Srinivas Chamakuri, Ying-Chu Chen, Nihan Ucisik, Huang-Chi Du, John Favor, Murugesan Palaniappan, Martin Matzuk, Timothy Palzkill (Breakout Room, GS11)

*Graduate Student, Baylor College of Medicine/Pharmacology and Chemical Biology

The CDC has reported at least 2 million infections and 23,000 deaths related to antibiotic resistance every year in the US. The most common method of resistance against β- lactam antibiotics is enzymatic inactivation by β-lactamases. Klebsiella pneumoniae carbapenemase-2 (KPC-2) is a plasmid-encoded β-lactamase enzyme expressed in the periplasmic space of some Gram-negative bacteria. KPC-2 can hydrolyze most currently available β-lactam antibiotics including carbapenems, which are normally kept as a last resort. As a class A β-lactamase, KPC-2 uses a serine residue in the active site to trap and hydrolyze β-lactam antibiotics, rendering them ineffective. There are several effective inhibitors that were developed as an effort to inactivate β-lactamases such as KPC-2, including avibactam, relebactam, and vaborbactam. However, a KPC-2 mutant identified in a clinical isolate can confer resistance to avibactam. Since most of the available inhibitors share a common core structure of a β-lactam ring or similar derivatives, it is a growing concern that bacteria will rapidly evolve to become resistant to the inhibitors. Thus it is important to find a novel core structure to slow the development of resistance. This project will discover new small molecule inhibitors of KPC-2 using DNA-encoded library technology (Dec-Tec). Unlike conventional high-throughput drug screening, this technology uses diversity oriented synthesis and allows screening of a wider chemical space. This novel technique will be a fast and efficient way of discovering a non-β- lactam β-lactamase inhibitor for KPC-2. A β-lactamase focused library will be synthesized to increase the probability of discovering a potent inhibitor. In addition, we have screened existing libraries and have identified several hit molecules. These hit compounds were synthesized and tested for their potency against KPC-2, and will further be analyzed for potential optimization and structural-activity relationship studies.

15 PANDEMIC EXPEC PHAGE-RESISTERS ARE PREDICTABLE AND ATTENUATED

Keiko Salazar*, Li Ma, Sabrina I. Green, Justin R. Clark, Austen L.Terwilliger, Jacob J. Zulk, Robert F. Ramig, Anthony W. Maresso (Breakout Room, GS12)

*Graduate Student, Baylor College of Medicine/IMBS/MVM

Extraintestinal pathogenic E. coli (ExPEC), often multidrug-resistant (MDR), is a leading cause of urinary and systemic infections. The crisis of emergent MDR pathogens has led some to propose as a therapeutic. However, bacterial resistance to phage is a crucial blockade that we must understand to overcome. Here we demonstrate that E. coli sequence type (ST) 131, a currently circulating pandemic strain of E. coli, rapidly develops resistance to ϕHP3, a phage often employed in phage efficacy and animal infection studies. Whole genome sequencing of the resisters revealed truncations in genes involved in LPS biosynthesis, the outer membrane transporter ompA, or both; implicating them as receptors for this phage. Interestingly, resisters were attenuated in blood and demonstrated decreased virulence in a murine model of E. coli systemic infection. We also developed a novel chemostat system which we used to generate an evolved phage isolate with restored infectivity in all LPS-truncated resisters. Our findings suggest that although resistance of pathogenic E. coli to our phage is inevitable, it comes at a fitness cost in its mammalian host, and new phage variants can be readily isolated by directed evolution.

16 THE ROLE OF THE ACCESSORY GENOME IN ENTEROCOCCAL BACTEREMIA: RESULTS FROM THE VANCOMYCIN-RESISTANT ENTEROCOCCAL BACTEREMIA OUTCOMES STUDY (VENOUS)

Shelby Simar*, Blake Hanson, German Contreras, Katherine C. Reyes, Pranoti Sahasrabhojane, Helina Misikir, An Q. Dinh, Catherine Liu, Yohei Doi, Fernanda Barberis, Lilian Abbo, Maria de los Angeles Spencer, Marcus J Zervos, Samuel L. Aitken, David van Duin, Samuel A. Shelburne, Truc T. Tran, Jose M. Munita, Cesar A. Arias (Breakout Room, GS13)

*Graduate Student, UTHealth School of Public Health/Epidemiology, Human Genetics & Environmental Sciences

Background: Vancomycin-resistant enterococci (VRE) are a major cause of nosocomial bloodstream infections. Enterococci exhibit remarkable genomic plasticity and can recombine through the acquisition of genetic material, including antimicrobial resistance (AMR) genes, via mobile genetic elements (MGEs). The accessory genome plays a major role in the evolution of enterococci within the human host and in the environment. Thus, examining the entire genome (pan-genome) is of vital importance to characterize the population structure of enterococci causing disease. Methods: VENOUS is an ongoing prospective, observational study of adults with enterococcal bacteremia. From September 2016-March 2018, E. faecalis (Efs) and E. faecium (Efm) were collected in 11 hospitals that included a major cancer center in Houston, TX, and 10 general hospitals in both Houston and in Detroit, MI. Short-read whole genome sequencing was performed with Illumina MiSeq and HiSeq 4000 platforms, and long-read sequencing utilized the Oxford Nanopore Technologies GridION X5. A custom pipeline was utilized for genome assembly and further analyses. Results: Short-read sequencing of 146 Efs and 86 Efm isolates was performed. The mean proportion of core (shared) genes in each genome was 64.9% (53.0-74.2) and 49.1% (45.4-54.6) for Efs and Efm, respectively. The vanA operon was identified in 4.1% (6/146) of Efs and 58.1% (50/86) of Efm. Of note, all vanA-harboring Efs possessed aac(6’)-le- aph(2”)-la conferring high-level aminoglycoside resistance, and the majority belonged to ST6. Long-read sequencing of vanA-harboring from a subset of VRE revealed that the vanA cluster was carried by a variety of plasmid types, with sizes ranging from 20.7-132.3 kb. Although the vanA cluster was fairly conserved in both Efm and Efs, insertions of MGEs and sets of other AMR genes were identified in the intergenic regions of vanS/vanH and vanX/vanY. Furthermore, a variety of MGE insertions mediated integration of the vanA operon, including IS1216. Conclusions: Accessory genes, including AMR genes, comprise a significant proportion of the pan-genome of enterococci, indicating major genetic plasticity in these organisms that is not captured with traditional, core gene-focused surveillance methods. Acquired resistance genes appear to have a high degree of recombination, and they play a substantial role in the expansion of the genomic repertoire in clinical isolates.

17 CHARACTERIZATION AND COMPARISON OF HUMAN AND EQUINE ROTAVIRUS ISOLATES FOR THERAPEUTIC DEVELOPMENT

JW Skrobarczyk*, CL Martin, S Pillai, N Cohen, LR Berghman (Breakout Room, GS14) *Texas A&M College of Agriculture and Life Sciences/Poultry Science/Immunology

Rotavirus is the leading cause of severe gastroenteritis in humans and a variety of animal species, including horses. are classified into 10 groups based on the gene sequence of the inner capsid protein, VP6. Group A rotaviruses (RVA) encompass both human and animal strains, including equine viruses. RVA are genotyped according to the nucleotide sequence of the G (VP7) and P (VP4) viral proteins. These capsid proteins contain epitopes that are recognized by neutralizing antibodies. In a previous study, we developed high-titered, neutralizing egg yolk antibodies against the human rotavirus (HRV) strain Wa, a G1P[8] genotype. The aim of this study was to characterize the genotype of equine rotavirus (ERV) isolates to determine potential efficacy of therapeutic antibodies developed against HRV in horses. Fecal swab and stool samples collected from foals were generously provided by farms in New York and Kentucky. was isolated by serial passage of the fecal suspensions on monolayers of MA104 cells in the presence of trypsin. Only those isolates that exhibited cytopathic effect on the 3rd passage were used in this study. Viral RNA was isolated from culture and stool suspensions using the Viral RNA Isolation Kit (Zymo Research) and cDNA was generated using the Qscript cDNA Supermix (QuantaBio). Gene-specific primers were designed according to the published sequence of the 2 most prevalent equine rotavirus VP7 genotypes (G3 and G14) and the 2 most common VP4 genotypes (P12 and P18). ERV genes were amplified by PCR using gene specific primers. All isolates were identified as the genotype G14P[12]. Substantial sequence homology (~70%) can be observed when comparing ERV VP7 and VP4 sequences to those of the HRV Wa G1P[8] genotype, indicating that HRV-neutralizing antibodies might provide cross-protection in foals. This is important because rotavirus infections remain a major health problem for foals and effective ERV therapeutics are lacking.

18 CHARACTERIZING THE ROLE OF INTESTINAL EPITHELIAL EXTRUSION AND IMMUNE SURVEILLANCE DURING GASTROINTESTINAL INFECTION USING ZEBRAFISH (DANIO RERIO) AS A MODEL HOST

Anh K Trinh Nguyen*, Max A Odem, George T Eisenhoffer, Anne-Marie Krachler (Breakout Room, GS15)

*Graduate Student, McGovern Medical School/Microbiology & Molecular Genetics, MDAnderson UTHealth Graduate School

The gastrointestinal mucosa forms a protective barrier against pathogens and other noxious stimuli. In order to maintain barrier homeostasis, the intestinal epithelium eliminates dying or crowded cells by extrusion. Previous observations in cultured cells have shown that hyper-activation of epithelial extrusion leads to disruption of cell-cell contacts, gaps in the epithelial layer, and loss of barrier integrity. Therefore, we hypothesize hyper-activation of epithelial extrusion could potentially disrupt mucosal homeostasis, by allowing infiltration of the epithelium by luminal bacteria and small molecules, and immune cell infiltration. The interactions between intestinal epithelial cells and luminal bacteria represent some of the earliest events during infection, but their dynamic interplay together with immune cells are poorly understood in vivo. An in vivo model of intestinal cellular extrusion suitable for dynamic high-resolution imaging has not been established. To overcome these limitations, we have developed a drug- inducible in vivo epithelial extrusion model using larval zebrafish (Danio rerio), along with a foodborne gastrointestinal infection assay. Using this system, we interrogate the pathogen response to transient loss of intestinal barrier function during excessive epithelial extrusion. The objective of this study is to understand how compromised barrier function drives bacterial infiltration and inflammation in the intestinal epithelium. Our preliminary results show excessive intestinal epithelial extrusion and gastrointestinal infection synergize to drive host pathogenesis. This approach also revealed increased neutrophil recruitment at sites of excessive extrusion and epithelial damage. Together, this research will uncover novel mechanisms of how pathogens take advantage of excessive cell extrusion to promote infection, and how the intestinal epithelium works in concert with immune cells to sense and respond to pathogens during intestinal epithelial damage.

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EVALUATING PHAGE THERAPY FOR THE TREATMENT OF URINARY TRACT

Jacob Zulk*, Katy Patras, Anthony Maresso (Breakout Room, GS16)

*Graduate Student, Baylor College of Medicine/Immunology and Microbiology

The estimated 7 million urinary tract infections (UTIs) that occur annually in the United States represent the most common cause of outpatient antibiotic prescriptions. The majority of UTIs are caused by uropathogenic E. coli (UPEC) and resolve with standard antibiotic treatment. However, a subset of those treated will have recurrence, requiring additional antibiotic treatment. Because antibiotics negatively impact the healthy microbiota and can contribute to the development of antibiotic resistance, alternative treatments are needed. Although bacteriophages (phages), viruses that selectively infect bacteria, are an appealing targeted therapy for many bacterial pathogens, the efficacy of phage therapy in treating UTIs has not been well-defined. Classically, phage-bacteria interactions have been studied in bacteriologic media, or host environments such as the blood. Because of this, our objective was to characterize interactions between phage and bacteria in the unique pH, salinity, and nutrient conditions present in urine. We screened a variety of phages against UPEC isolates, altering culture media and concentration of phage. Phage-mediated killing was measured via optical density over an 18-hour timeframe. Our results demonstrate that urine is broadly inhibitory towards phage- mediated UPEC killing and that phage resistance develops in several UPEC strains within the first six hours of infection leading to an “OD rebound” observed by 18 hours. However, in vivo, these mutations appear to negatively impact the bacteria’s ability to colonize and cause inflammation in the bladder of infected mice. Ongoing research seeks to classify the genetic bases underlying resistance to individual phages and to further define the inhibitory nature of urine. These findings will identify phages capable of being used in vivo and will contribute to the development of phage-phage and phage- antibiotic cocktails for treating and preventing UTI.

20 CHARACTERIZING THE ROLES OF PUTATIVE LIPOPROTEINS DURING TICK- BORNE RELAPSING FEVER BORRELIA VECTOR COMPETENCE

Michael W Curtis*, Aparna Krishnavajhala, Clay D Jackson-Litteken, Jon S Blevins, Job E Lopez (Breakout Room, PD1)

*Postdoctoral Fellow, Baylor College of Medicine/Pediatrics

Tick-borne relapsing fever (TBRF) is a neglected disease despite being a significant cause of morbidity and mortality in the developing world. Spirochetes from the Borrelia genus are the etiological agents of TBRF and cause recurring episodes of acute fever, pregnancy termination, and/or potential death if the infection is not treated. Soft ticks transmit the majority of the TBRF Borrelia species. To be maintained in nature, TBRF Borrelia need to adapt to both tick and mammalian environments. The mechanisms of persistent mammalian infection are well understood and driven by antigenic variation. However, the molecular mechanisms enabling vector competence are poorly understood. We hypothesize that TBRF Borrelia adapt to these diverse environments by differentially regulating their gene expression as they complete their tick-mammalian transmission cycle. To test this, our lab developed the Ornithodoros turicata-Borrelia turicatae (vector-pathogen) model to investigate genes involved in TBRF Borrelia vector competence. Through gene expression assays, 18 B. turicatae genes, encoding putative surface lipoproteins, were found to be expressed at significantly higher levels within the vector compared to mammalian infection. To begin characterizing the role of nine of these putative lipoproteins in vector competence, we generated four multi-gene deletion mutants and performed murine needle inoculations and tick transmission studies. All mutants were able to infect mice at levels that allowed us to feed uninfected ticks and assess vector colonization. Since transmission of B. turicatae occurs within seconds of tick attachment, we evaluated the ability of mutants to infect mice by tick transmission. Preliminary studies suggest that a triple (Δbta134-bta136) and a double mutant (Δbta132-bta133) have attenuated transmission phenotypes. These results may aid in identifying targets for a vaccine to prevent the establishment of early mammalian infection after tick transmission.

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DEVELOPMENT OF NOVEL ANTIFUNGALS AGAINST CANDIDA BASED ON AN ANTIFUNGAL PEPTIDE PRODUCED BY E. FAECALIS

Shantanu Guha*, Melissa Cruz, Michael Lorenz, Danielle Garsin (Breakout Room, PD2)

*Postdoctoral Fellow, McGovern Medical School/Microbiology & Molecular Genetics

Fungal resistance to commonly used medicines is a growing public health threat. The most common cause of dangerous, bloodstream, fungal infections is Candida species, and there are emergent strains of Candida resistant to all current antifungals. To increase the probability of successfully treating Candida infections, novel antifungals must be developed. The basis of our project in developing a novel antifungal agent is a secreted bacterial peptide, EntV, which is produced by Enterococcus faecalis and restricts C. albicans to a non-virulent form. By targeting virulence rather than viability, the chances of developing resistance to EntV may be less than traditional antifungals. Our investigation aims to identify the minimal structural features necessary for EntV activity, generate a combinatorial peptide library using the truncated peptide as a template, conduct high- throughput screening to determine gain-of-function peptide variants, and test EntV and its variants in preclinical models to determine its effectiveness and potential usage. We hypothesize that by rationally varying specific residues in combination, we will generate more potent antifungal peptides than the template sequence through synthetic molecular evolution. We will use C. albicans to screen the novel antifungal peptides that we generate, as that is the causative agent behind most Candida infections. We expect that our discoveries will contribute to the development of novel antifungals in the fight against antimicrobial resistant fungi.

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DEVELOPMENT OF BACTERIOPHAGES WITH ANTI-BIOFILM PROPERTIES AS NOVEL TREATMENT FOR CATHETER-ASSOCIATED URINARY TRACT INFECTIONS

Sanchez BC*, Heckman ER, Ramig FR, Kaplan HB, Hines-Munson C, Skelton F, Trautner BW, Maresso AW (Breakout Room, PD3)

*Postdoctoral Fellow, Baylor College of Medicine /Molecular Virology and Microbiology

Escherichia coli is the most common causative agent of Urinary Tract Infections (UTIs). The formation of biofilms in the urinary tract and on urinary catheters, and the increasing rate of antibiotic resistance found in E. coli strains makes treatment of E. coli UTIs extremely challenging. Bacteriophages (phages), are ubiquitous viruses that exclusively infect and kill bacteria irrespective of their antibiotic sensitivity. Phages are active in human urine and can produce enzymes that degrade bacterial extracellular polysaccharides (EPS), such as those in biofilms. The objective of this work is to discover and characterize phages that kill E. coli growing in biofilms using an in vitro model of Catheter-Associated Urinary Tract Infection (CAUTI). Biofilms of E. coli strains isolated from the urine of persons with spinal cord injury were grown for 24 - 48 hours at 37°C in Tryptic Soy Broth (TSB) or human urine. Pre-formed biofilms were treated with 107 PFU/mL of phage for 24 hours and stained with MTT to determine the metabolic output of live cells within the biofilms. Of the 28 phages tested, 8 (28.6%) decreased biofilm viability by >50%, 9 (32.1%) reduced biofilm viability by 25-50%, and 11 phages (39.3%) did not cause a significant reduction in biofilm viability in TSB. Phages HP3, ES17, ES21, ES26, 6915 and 6950 each decreased biofilm viability by >85% when added individually to 48- hour biofilms in human urine of two E. coli strains (DS515 and DS552). Four of these 6 phages with anti-biofilm activity form plaques with halos when plated on bacterial lawns. The presence of halos suggests that enzymatic activity against the bacterial EPS might be associated with biofilm destruction by phage. We have identified six phages (HP3, ES17, ES21, ES26, 6915 and 6950) with anti-biofilm activity against E. coli in human urine. These phages may have therapeutic potential against CAUTIs, and our future studies will test their efficacy in vivo.

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THE PATHOGENESIS OF TUBERCULOSIS–THE KOCH PHENOMENON REINSTATED

Robert Hunter* (Breakout Room, S1)

*Instructor, McGovern Medical School/Pathology & Laboratory Medicine

The paradigm that granulomas are the hallmark of TB is a product of animal research of the late 20th century when human tissues were no longer available to most investigators. Actually, TB in man produces two types of granulomas only one of which is studied in animal models. Primary granulomas occur in many species and are widely studied. Post- primary granulomas, in contrast, develop only in humans to surround retained foci of caseous pneumonia and are seldom studied. Post-primary TB begins as an obstructive lobular pneumonia that spreads via bronchi. It is prolonged subclinical stockpiling of secreted mycobacterial antigens in foamy alveolar macrophages and nearby highly sensitized T cells in preparation for a massive necrotizing hypersensitivity reaction, the Koch Phenomenon. This, in turn, produces caseous pneumonia that is either coughed out to form cavities or is retained to become the focus of post-primary granulomas and fibrocaseous disease. This revised paradigm is supported by nearly 200 years of research and suggests new approaches and animal models to investigate long standing mysteries of human TB and vaccines that inhibit the early lesion to finally end transmission of infection.

24 DIVERSITY AND DISTRIBUTION OF THE TICK-BORNE RELAPSING FEVER SPIROCHETE BORRELIA TURICATAE

Aparna Krishnavajhala*, Brittany A. Armstrong, Alex R. Kneubehl, Sarah M. Gunter, Julie Piccione, Hee J. Kim, Rosa Ramirez, Ivan Castro-Arellano, Walter Roachell, Pete D. Teel, Job E. Lopez (Breakout Room, S2)

*Research Staff, Baylor College of Medicine/Pediatrics

Borrelia turicatae is a causative agent of tick-borne relapsing fever (TBRF) in portions of the United States and Latin America, and there is growing evidence that the pathogen is emerging in densely populated regions of Texas. However, little is known regarding the genetic diversity between B. turicatae isolates and the pathogen’s distribution remains poorly defined. Between 2017 and 2020, we focused efforts to expand the number of B. turicatae isolates. Multilocus sequence typing (MLST) was performed and included previously reported sequences. The four chromosomal loci targeted for MLST were 16S ribosomal RNA (rrs), flagellin B (flaB), DNA gyrase B (gyrB), and the intergenic spacer (IGS). Given the complexity of Borrelia , plasmid diversity was also evaluated. These studies indicate that the IGS locus segregates B. turicatae into genomic groupings and plasmid diversity is extensive between isolates. This work further supports the indication that TBRF spirochetes are distributed in metropolitan settings.

25 EVOLUTIONARY LEAPFROGGING LEADS TO THE FAILURE OF A PROMISING ANTIMICROBIAL STRATEGY Mehta HH*, Ibarra D, Shamoo Y (Breakout Room, S3)

*Research Staff, Rice University/BioSciences

The rise of antibiotic resistant pathogens worldwide threatens to undermine generations of biomedical progress. Using combinations of antibiotics has long been considered a promising strategy, but successes have remained more elusive than anticipated. We investigated the in vitro efficacy of a potent combination of ciprofloxacin and doxycycline against the live vaccine strain of the select agent, Francisella tularensis. We showed that despite the efficacy of the individual drugs and the clear independence of their mechanistic basis of action, the two drugs together did not delay the onset of resistance and, more worryingly, the organism achieved resistance to both drugs on the same timescale as either of the drugs individually. Following the acquisition of an initial generalist that provided a modest increase in resistance to both drugs, the generalized failure of the drug combination was attributed largely to an evolutionary “leapfrogging” cascade that allowed the pathogen to sequentially adapt to each drug as if they were administered separately. We observed a clear pattern where the balance of selection pressure of each drug shifted over time, allowing the organism to leapfrog over each drug independently by creating an asymmetry in the experienced selection environment. These findings suggest that spatial heterogeneities in drug concentration observed in vivo across tissues may provide conditions that favor such an evolutionary leapfrogging and reduce the efficacy of some promising combinatorial antimicrobial therapies.

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Thank you for attending this year. We look forward to seeing you in-person in 2022!

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