2022 MOLECULAR BASIS OF INFECTIOUS DISEASES (MBID) - MCGOVERN MEDICAL SCHOOL
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2022 Molecular Basis of
Infectious Diseases (MBID)
Poster Abstracts
Sponsors
The Department of Microbiology & Molecular Genetics, UTHealth McGovern Medical School, UTHealth Houston
The Molecular Basis of Infectious Diseases (MBID) Training Program
Organizing Committee
Theresa M. Koehler, Ph.D., Professor & Chair, Microbiology & Molecular Genetics, UTHealth McGovern Medical School
Michael Lorenz, Ph.D., Professor, Microbiology & Molecular Genetics, UTHealth McGovern Medical School
Heidi B. Kaplan, Ph.D., Associate Professor, Microbiology & Molecular Genetics, UTHealth McGovern Medical School
Shelby Simar, MBID Fellow, UTHealth School of Public Health/Epidemiology
Jacob Rutherford, MBID Fellow, Texas A&M Institute for Biosciences and Technology
0
John Taylor, MBID Fellow, Texas A&M Institute for Biosciences and Technology
Hannah Wilson, MBID Fellow, MD Anderson UTHealth Graduate School of Biomedical SciencesPRESENTER POSTER TITLE
(1) Benjamin, Aaron Efficacy of Cathelicidin Antimicrobial Peptoids against Staphylococcus aureus
(2) Buda De Cesare, Characterization of the Mechanism of Action of the Enterococcus faecalis
Giuseppe Bacteriocin EntV on Candida albicans
Functional Genomics of Enteroaggregative E. coli and Diverse
(3) Carter, Hannah Gastrointestinal E. coli Isolates from Patients with Hematological
Malignancies
Structural Analyses of Hypothetical Proteins from Bacteriophage Proteome
(4) Chang, James
Using AlphaFold2
(5) Clark, Justin Genomic Analytic for Therapeutic Bacteriophages
(6) Clinton, Jennifer Generating an Immunogenic Elephant Endotheliotropic Herpesvirus (EEHV)
Vaccine
Virulence and Biofilm Formation in Candida albicans are Inhibited by Short
(7) Cristy, Shane
Peptide Subunits of EntV
Characterizing the Roles of Putative Lipoproteins during Tick-Borne
(8) Curtis, Michael
Relapsing Fever Borrelia Vector Competence
Characterization Of Virulence Mechanisms In Staphylococcus aureus
(9) Duran Ramirez, Jesus
Isolates Colonizing Urinary Catheters
(10) Dutta, Soumita BrnQ-type Branched-chain Amino Acid Transporters Influence Bacillus
anthracis Growth and Virulence
CarSR Controlling Fructose Metabolism Promotes Synergistic and
(11) G C, Bibek Competitive Interactions between Fusobacterium nucleatum and the
Other Oral Microbes
Investigating Calcium Dysregulation and Enteric Virus Virulence using
(12) Gebert, J. Thomas
Forward and Reverse Genetics
The TAILΦR 12: A Report on the Use of Tailored Phage Cocktails
(13) Green, Sabrina
Against Multidrug Resistant Infections in 12 Patients
3PRESENTER POSTER TITLE
Lighting the Viral Dark Matter: Revealing Earth's Cryptic Genosphere through
(14) Gu Liu, Carmen
Environmental Sampling
Development of Novel Antifungals Against Candida based on an Antifungal
(15) Guha, Shantanu
Peptide Produced by E. faecalis
(16) Kang, Alex An in vitro Cell Culture Model for Pyoverdine-Mediated Virulence
Non-specific Effects of BCG Vaccination Enhancing Efficacy of COVID-19 and
(17) Koster, Kent
Influenza Vaccines
(18) Krishnavajhala, Comparative Genome Analysis of Clonal Populations of in vitro Cultivated
Aparna Tick-Borne Relapsing Fever Spirochete Borrelia turicatae
(19) Kumar, Santosh Dissecting the Molecular Function of BamE in the Essential Bam complex
Stabilization of Tuberculosis Reporter Enzyme Fluorescence (REFtb)
(20) Lebedev, Maxim
Diagnostic System for Use at the Point-of-Care
Expression of Hyphal-associated Virulence Factors is Necessary for Candida
(21) Mauk, Kelsey
albicans Persistence in the Murine Gastrointestinal Tract
Human Milk Oligosaccharides Reduce Murine Group B Streptococcus Vaginal
(22) Mejia, Marlyd
Colonization with Minimal Impact on the Vaginal microbiota
(23) Miramon, Pedro Development of an Invertebrate Virulence Model for the Emerging Fungal
Pathogen Candida auris
Bacteriophage-containing Biodegradable Microsphere Technology to
(24) Narro, Analisa
Treat Osteomyelitis
(25) Ottinger, Samantha An In vivo Model of Urinary Tract Infection-Associated Preterm Birth
(26) Portilla, Sabrina Antibiotic Sensitivities of Mixed Bacterial Cultures
Study of Fusobacterium nucleatum FAS-II Using Molecular and
(27) Rutherford, Jacob
Chemical Genetics
(28) Scribano, Francesca Calcium Signaling in Enteric Infection and Diarrheal Disease
Emergence of Tn1549-mediated vanB Vancomycin Resistance in
(29) Simar, Shelby
Multidrug-resistant Enterococcus faecalis ST6 in the United States
4PRESENTER POSTER TITLE
In situ Architecture of Porphyromonas gingivalis Type IX Secretion
(30) Song, Liqiang
System
The TAILΦR Initiative at Year One: Personalized Medicine for
(31) Terwilliger, Austin
Dynamic Infections
Determining the Contributions of C. albicans Metabolism and
(32) Wilson, Hannah
Morphogenesis to Phagosomal Alkalinization
Immunization with the Invasin-Like Autotransporter Protein, SinH,
(33) Xing, Yikun Protects Against ExPEC Caused Sepsis and Cystitis in the Murine
Models
Initial Characterization of Small Cytotoxic Membrane Vesicles
(34) Xu, Qi
Produced by Pseudomonas aeruginosa
The Master Regulator for Entry into Sporulation in Bacillus subtilis
(35) Zarazua-Osorio,
Becomes the Mother Cell Specific Transcription Factor Controlling
Brenda
Forespore Engulfment
Uropathogenic E. coli Resistance to Phage ES17 Decreases Bacterial
(36) Zulk, Jacob
Fitness
Streptococcus gallolyticus subspecies gallolyticus stimulates host cell
Taylor, John proliferation by directly activating the Epidermal Growth Factor
Receptor
5EFFICACY OF CATHELICIDIN ANTIMICROBIAL PEPTOIDS AGAINST
STAPHYLOCOCCUS AUREUS
Aaron B. Benjamin*, Maruti K. Didwania, Madeline Moule, Jonathan Hardy, Panatda
Saenkham, Preeti Sule, Yoriel Marcano, Borries Demeler, Jennifer S. Lin, Christopher H.
Contag, Annelise E. Barron, and Jeffrey D. Cirillo
(1)
*Postdoctoral Fellow, Texas A&M Health Science Center - Microbial Pathogenesis and
Immunology
Staphylococcus aureus is one of the most common pathogens associated with infection
in wounds. The current standard of care uses a combination of disinfection and drainage
followed by conventional antibiotics such as methicillin. Methicillin and vancomycin
resistance has rendered these treatments ineffective, often causing the reemergence of
infection. This study examines the use of novel antimicrobial peptoids (sequence-specific
poly-N-substituted glycines) that mimic naturally occurring host defense peptides as an
alternative to conventional antibiotics. These peptoids provide efficient killing of
methicillin- susceptible S. aureus (MSSA) and MRSA at low micromolar concentrations
without exhibiting major cytotoxic side-effects. Additionally, these novel peptoids show
excellent efficacy against biofilm formation and detachment for both MSSA and MRSA.
By comparison, conventional antibiotics were unable to detach or prevent formation of
biofilms. In comparison, Peptoid 1 shows great promise, as it could prevent formation of
or detach biofilms at concentrations as low as 1.6μM. Characterization of aqueous
Peptoid 1 solutions by Analytical Ultracentrifugation shows multimeric structures,
possibly contributing to its high antimicrobial activity. The use of a bioluminescent S.
aureus murine incision wound model demonstrated clearance of infection in peptoid-
treated mice within 8 days, showing another advantage these peptoids have over
conventional antibiotics.These results provide clear evidence of the potential for
antimicrobial peptoids for treatment of S. aureus wound infections.
6CHARACTERIZATION OF THE MECHANISM OF ACTION OF THE
ENTEROCOCCUS FAECALIS BACTERIOCIN EntV ON CANDIDA ALBICANS
G. Buda De Cesare*, A. Zito, S. Guha, SA. Christy, MR. Cruz, DA. Garsin, and MC.
Lorenz
(2)
*Postdoctoral Fellow, UTHealth McGovern Medical School - Microbiology & Molecular
Genetics
Candida albicans, an opportunistic fungal pathogen, causes systemic and superficial
infections, especially in immunocompromised patients. Treatment of fungal infections is
complicated by limited antifungal options and the development of drug resistance. As a
result, these infections are associated with high mortality. Previous work from our group
demonstrated that the Gram-positive bacterium Enterococcus faecalis, antagonizes
hyphal morphogenesis, biofilm formation, and virulence in C. albicans through the
production of EntV, a bacteriocin and antimicrobial peptide. The main aim of this work is
to unveil the molecular mechanism behind the activity of EntV on C. albicans. Using
fluorescence and electron microscopy, we showed differences in peptide binding
patterns relative to cellular morphology, with a greater binding of the peptide to hyphae
compared to yeast cells, indicative of a higher amount of the peptide target in hyphae.
Moreover, the binding of the peptide to extracellular vesicles (EVs) of another
opportunistic fungal pathogen, Cryptococcus neoformans, suggests a connection with
the antivirulence activity of EntV. Finally, we performed RNA sequencing to detect
transcriptomic changes associated to the activity of EntV on C. albicans. The transcript
of genes involved in arginine pathway as well as transcriptional variations of enzymes
involved in cell wall biogenesis and maintenance were detected in presence of EntV
compared to the control, indicating possible metabolic and cell wall remodelling
mechanisms associated to peptide exposure. Identifying the molecular target of EntV in
regard to the anti-virulence mechanisms of C. albicans is an important step in its further
development as a therapeutic addition to the classical antifungal agents and in tackling
the problem of drug resistance.
7FUNCTIONAL GENOMICS OF ENTEROAGGREGATIVE E. COLI AND DIVERSE
GASTROINTESTINAL E. COLI ISOLATES FROM PATIENTS WITH
HEMATOLOGICAL MALIGNANCIES
Hannah Carter*, Justin Clark, Lily G. Carlin, Anubama Rajan, Adilene Olvera, Mary K.
Estes, Pablo C. Okhuysen, and Anthony Maresso
(3)
*Graduate Student, Baylor College of Medicine - Molecular Virology and Microbiology
Infections are the leading cause of death in cancer patients. Among patients with
hematological malignancies, bacterial infections are responsible for 43% of deaths and
polymicrobial infections are responsible for an additional 11% of deaths. E. coli is a
diversee species which can cause infections at multiple body sites. Chemotherapy
treatments, and the antibiotics often prescribed to cancer patients, can increase
vulnerability to E. coli infections in the gastrointestinal tract and facilitate the
translocation of some types of E. coli into the blood. Enteroaggregative E. coli (EAEC)
has been identified in cancer patients as a cause of abdominal pain and diarrhea. In
healthy populations, EAEC infections can cause symptomatic disease, mainly watery
diarrhea with or without blood and mucus, abdominal pain, nausea, vomiting, and fever.
Interestingly, EAEC can also cause infections which are asymptomatic or are
undetectable but cause subclinical inflammation. Maintenance of an EAEC reservoir as
asymptotic infections in healthy people risks transmission to more vulnerable
populations. From twenty-nine patients with hematological malignancies, ongoing
diarrhea, and stool samples testing positive for AggR, we could recover a typical EAEC
strain from only six patients. From those which we could not recover a typical EAEC,
we isolated E. coli and Citrobacter possessing various degrees of adherence and
cytotoxic potential including several novel strains with intriguing properties. Of these
diverse GI E. coli, many are most likely commensal E. coli harboring little to no virulence
factors while several are likely currently inert, but harbor virulence genes – including
two strains of ExPEC bacteria – a group of bacteria which translocate across the
intestinal epithelium and cause infections at other body sites. Several of our isolates
showed the ability to adhere to human intestinal colonoids in equal number as prototype
strains of EIEC, ETEC, EHEC, and EPEC and to disturb HIE monolayer attachment.
These results suggest existence of E. coli that lie outside of the classical pathotype
paradigm that may be responsible for diarrhea in immunocompromised patients (or
enteric pathogens of the intestinal tract).
8STRUCTURAL ANALYSES OF HYPOTHETICAL PROTEINS FROM
BACTERIOPHAGE PROTEOME USING ALPHAFOLD2
James Chang*, Carmen Gu Liu, Justin Clark, and Anthony Maresso
(4)
*Postdoctoral Fellow, Baylor College of Medicine - Molecular Virology and Microbiology
Bacteriophages have received renewed attention in recent years to combat bacterial
pathogens. Ubiquitous in nature, these lytic viruses infect and replicate within bacteria
for propagation, which leads to bacterial death. This parasitic lifecycle for killing bacteria
stands in contrast to conventional antibiotics' biochemical modes of action, and phages
thus promise a new way to combat antibiotic resistant bacterial pathogens. However,
much of phages' protein machineries that exploit bacterial biology remain poorly
characterized, especially given difficulties in both identifying and culturing phages. In
2021, Google Deepmind's AlphaFold 2 won critical acclaim by winning protein fold
prediction competition CASP14 with protein structural predictions that were on par with
experimentally solved structures. In this study, we used AlphaFold 2 to predict
structures of these hypothetical proteins from genes identified through our
metagenomic analysis. We demonstrate the capability of these predictions to identify
functions of these previously unknown proteins, and highlight unique structural
adaptations in these proteins that may aid phage in overwhelming bacteria. With these
analyses, we show how AlphaFold 2 can be used to form experimental hypothesis and
guide investigations of these unknown phage proteins.
9GENOMIC ANALYTIC FOR THERAPEUTIC BACTERIOPHAGES
Justin Clark*, Austen Terwilliger, Sabrina Green, Keiko Salazar, and Anthony Maresso
(5)
*Research Staff, Baylor College of Medicine - Molecular Virology and Microbiology
The number of deaths that result from antibiotic resistance is rising so rapidly that some
predict it will surpass the deaths that result from cancer in less than 30 years. One of the
major problems with combating this growing threat is the fact that bacteria can acquire
resistance to new antibiotics at a rate orders of magnitude higher than humans develop
them. Simply put, new, non-antibiotic, strategies are needed. By far the most advanced
and promising alternative to antibiotics is bacteriophage—or phage—therapy. Phage
have been used as a therapeutic for nearly a century, even in the US. However, the low
cost and availability of antibiotic sapped interesting for this therapy. The rise of antibiotic
resistance has instead sparked changed that, especially in dire cases when antibiotic
have failed completely. Against this grim backdrop, we founded of Tailored
Antimicrobials and Innovative Laboratories for Phage (Φ) Research (TAILΦR) at Baylor
College of Medicine, a phage service center dedicated to advancing the understanding
of phage biology and produce therapeutic-grade phage cocktails. An important part of
realizing this mission is the analysis of phage genomes. Therapeutic phages are
expected to be devoid of any virulence factors, antibiotic resistance genes, or a lysogenic
life cycle. Here we will describe the bioinformatic processes TAILΦR uses to help ensure
phage are safe for therapeutic use. This includes the assembly and analysis of the phage
gnome through short-read sequencing and tools used to screen these assemblies for
problematic genes that would be unsuitable for use in patients.
10GENERATING AN IMMUNOGENIC ELEPHANT ENDOTHELIOTROPIC
HERPESVIRUS (EEHV) VACCINE
Jennifer L. Spencer Clinton*, Taylor Pursell, Tabitha Hoornweg, Jie Tan, Rongsheng
Peng, Willem Schaftenaar, Victor P. Rutten, Cornelius A. M. de Haan, and Paul D. Ling
(6)
*Postdoctoral Fellow, Baylor College of Medicine - Molecular Virology and Microbiology
Asian elephants are an endangered species facing many threats, including severe
hemorrhagic disease (HD) caused by the elephant endotheliotropic herpesvirus (EEHV).
EEHV-HD is the leading cause of death in captive juvenile Asian elephants in North
America and Europe, and affects elephants in their natural range countries. EEHV can
also cause lethal disease in captive African elephants, with at least 11 cases and four
fatalities in North American zoos since 2019. Due to the rapid severe onset of EEHV,
detection and treatment options are limited. Thus, our goal is to develop a vaccine
eliciting strong antibody and cell-mediated immunity (CMI) against EEHV to prevent
lethal disease. Previous studies with EEHV and human herpesviruses indicate that
glycoproteins B, H, and L (gB, gH, gL) are likely to induce protective humoral immunity
and CMI. Therefore, our vaccine approach will include two strategies: (1) protein subunit,
and (2) a recombinant virus vector using Modified Vaccinia Ankara (MVA). We have
successfully generated an MVA recombinant expressing the EEHV gB glycoprotein and
purified recombinant gB protein from mammalian cells. In preclinical studies, we have
shown that MVA-gB or gB subunit vaccinated mice induce robust gB-specific antibodies
and polyfunctional CD4+ and CD8+ T cell responses after homologous prime-boosts.
We also observed that a single priming vaccine and one boost are sufficient to induce
immune responses and are not significantly different than two subsequent vaccine
boosts. Future studies will incorporate multiantigenic MVA recombinants expressing
EEHV gH/gL antigens in addition to gB, as well as compare immunogenicity of
heterologous prime-boost vaccines with MVA and purified antigen subunits. Completion
of these studies will provide support for the possible use of these vaccines in elephants.
11VIRULENCE AND BIOFILM FORMATION IN CANDIDA ALBICANS ARE INHIBITED
BY SHORT PEPTIDE SUBUNITS OF EntV
Shane A. Cristy*, Michael C. Lorenz
(7)
*Graduate Student, UTHealth McGovern Medical School/Microbiology & Molecular
Genetics, MD Anderson UTHealth Graduate School of Biomedical Sciences
Candida albicans, the most clinically significant fungal pathogen, commonly causes
topical mucosal infections such as oral cavity and urogenital tract infections. It also less
frequently causes severe invasive and bloodstream infections. Invasive infections are
most prevalent amongst patients with compromised innate immune responses, such
as those receiving chemotherapy or recovering from surgery. C. albicans can also
form biofilms on implanted medical devices. Fungal infections are difficult to treat due
to the paucity of therapeutic options, and this problem is compounded by the
resistance properties of biofilm infections. C. albicans exists as a member of the
commensal flora of the mucosa where many complex polymicrobial interactions occur
with genera such as Pseudomonas and Streptococcus. Some of these interactions
potentiate or inhibit virulence. One such interaction is with Enterococcus faecalis, a
bacterial gastrointestinal commensal species. E. faecalis produces a small peptide,
EntV, that modulates C. albicans virulence. The mature 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 is 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 or improved efficacy in inhibiting C.
albicans virulence and biofilm formation. These smaller peptides inhibit initial
adhesion to abiotic surfaces, reduce final biofilm biomass, and reduce the size of
mature biofilms as 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.
12CHARACTERIZING 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, and
Job E Lopez
(8)
*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
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 transcriptional analysis, 18 B. turicatae genes, encoding putative surface
lipoproteins, were found to be expressed at significantly higher levels during vector
colonization 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. Preliminary studies suggest that a triple (Δbta134-
bta136) and a double mutant (Δbta132-bta133) have attenuated murine infection
following tick transmission. Furthermore, studies have shown that both mutants have a
growth defect during cultivation at 22°C. Together these results suggest that these five
inactivated genes may have important roles in vector adaptation.
13CHARACTERIZATION OF VIRULENCE MECHANISMS IN STAPHYLOCOCCUS
AUREUS ISOLATES COLONIZING URINARY CATHETERS
Jesus M. Duran Ramirez*, Chloe L. Pinker, Scott J. Hultgren, Jennifer N. Walker
(9)
*Graduate Student, UTHealth School of Public Health; Research Staff, UTHealth
McGovern Medical School – Microbiology & Molecular Genetics
Catheter-associated urinary tract infections (CAUTIs) are the most common
hospital- associated infections in the US and can results in severe morbidity and
mortality. Importantly, CAUTIs are caused by a broader range of pathogens than
non-catheter related UTIs. Many “atypical uropathogens” that cause CAUTI remain
unstudied, despite being responsible for thousands of infections annually. Of these
pathogens, Staphylococcus aureus is particularly problematic as recent reports
indicate S. aureus strains causing CAUTI are primarily methicillin resistant S.
aureus (MRSA), making them difficult to treat. Furthermore, S. aureus encodes the
enzyme urease, which promotes catheter encrustations that facilitate biofilm
formation, further increasing recalcitrance to antibiotics, and contributing to device
failure. Investigating how S. aureus urease contributes to CAUTI may provide
insights into the development of better treatment strategies. To study the role of
urease in S. aureus CAUTI, we adapted the Christensen Agar assay to test for
urease activity in several S. aureus strains isolated from urinary catheters. Notably,
urease activity in serially isolated strains was increased compared to the initial
isolate. Thus, using this assay, we began dissecting the pathways regulating urease
expression. We found that the regulators sigB, agr, and codY controlled urease
activity. The assay demonstrated sigB and agr were activators, while codY was a
repressor. Additionally, selected isolates were assessed for their ability to cause
CAUTI in a mouse model. These results supported our previous work
demonstrating S. aureus requires a catheter to cause UTI. Together these data
suggest that S. aureus isolates colonizing urinary catheters encode the urease
operon and that urease may be an important virulence factor for promoting long-
term urinary tract colonization. Thus, therapies that reduce or prevent urease
activity may be developed as an antibiotic-sparing therapy.
14BRNQ-TYPE BRANCHED-CHAIN AMINO ACID TRANSPORTERS INFLUENCE
BACILLUS ANTHRACIS GROWTH AND VIRULENCE
Soumita Dutta*, Ileana D. Corsi, Naomi Bier and Theresa M. Koehler
(10)
*Postdoctoral Fellow, UTHealth McGovern Medical School - Microbiology & Molecular
Genetics
Bacillus anthracis, the anthrax agent, exhibits robust proliferation in diverse niches of
mammalian hosts. Metabolic attributes of B. anthracis that permit rapid growth in multiple
mammalian tissues have not been established. We posit that branched-chain amino acid
(BCAA: Isoleucine, leucine and valine) metabolism is key to B. anthracis pathogenesis.
Increasing evidence indicates relationships between B. anthracis virulence and
expression of BCAA-related genes. Expression of some BCAA-related genes is altered
during culture in bovine blood in vitro and the bacterium exhibits valine auxotrophy in a
blood serum mimic medium. Transcriptome analyses have revealed that the virulence
regulator AtxA, that positively affects expression of the anthrax toxin and capsule genes,
negatively regulates genes predicted to be associated with BCAA biosynthesis and
transport. Here, we show that B. anthracis growth in defined media is severely restricted
in the absence of exogenous BCAAs, indicating that BCAA transport is required for
optimal growth in vitro. We demonstrate functional redundancy among multiple BrnQ-
type BCAA transporters. Three transporters are associated with isoleucine and valine
transport, and deletion of one, BrnQ3, attenuates virulence in a murine model for
anthrax. Interestingly, an ilvD-null mutant lacking dihydroxy-acid dehydratase, an
enzyme essential for BCAAs synthesis, exhibits unperturbed growth when cultured in
media containing BCAAs, but is highly attenuated in the murine model. Finally, our data
show that BCAAs enhance AtxA activity in a dose-dependent manner, suggesting a
model in which BCAAs serve as a signal for virulence gene expression.
15CarSR CONTROLLING FRUCTOSE METABOLISM PROMOTES SYNERGISTIC AND
COMPETITIVE INTERACTIONS BETWEEN FUSOBACTERIUM NUCLEATUM AND
THE OTHER ORAL MICROBES
Bibek G C*, Chenggang Wu
(11)
*Postdoctoral Fellow, UTHealth McGovern Medical School - Microbiology & Molecular
Genetics
Fusobacterium nucleatum (F. nucleatum) is a common opportunistic pathogen, shown
to have important role in oral diseases such as periodontitis, halitosis, dental pulp
infection as well as extra oral diseases such as pericarditis, brain abscess,
osteomyelitis, chorioamnionitis, colorectal cancer etc. F. nucleatum has the most
powerful ability of coaggregation with many oral microbes suggesting its critical role in
regulating dental biofilm formation as well as spreading extraoral sites, such as the
placenta, and gastrointestinal tract. Our previous studies have shown that a two-
component signal transduction system (TCS), named CarSR, regulates RadD
mediated fusobacterial coaggregation, and methionine-derived AI-2 is shown to be
signal molecule for sensor kinase, CarS. Our working hypothesis is that AI-2 can be
sensed by CarS, which undergoes autophosphorylation and transfers its phosphate
group to response regulator CarR to regulate fusobacterial virulence. Deletion of carR
results in significant increase in radD expression as well as downregulation of fruRAB
in F. nucleatum suggesting it as transcriptional repressor and activator of radB and
fruRAB respectively. In addition, RadD mediated coaggregation is shown to decrease
frucAB expression in F. nucleatum, while increase the expression of frucAB genes in its
binding partners including Streptococcus gordonii, Streptococcus oralis and
Actinomyces oris. In fact, coaggregation with other oral bacteria itself is shown to
promote RadD expression in F. nucleatum. These results thus show intricate regulation
of CarSR in controlling fructose metabolism and coaggregation between F. nucleatum
and the other oral microbes. We further seek to explore CarR regulon and identify
genome-wide CarR-binding regions in F. nucleatum.
16INVESTIGATING CALCIUM DYSREGULATION AND ENTERIC VIRUS VIRULENCE
USING FORWARD AND REVERSE GENETICS
J. Thomas Gebert*, Kristen Engevik, Francesca Scribano, J. Lance Perry, Joseph
Hyser
(12)
*Graduate Student, Baylor College of Medicine - Molecular Virology & Microbiology
Acute gastroenteritis (AGE) remains the 2nd leading cause of death among children
under the age of 5 worldwide. AGE becomes fatal when pathogen-associated
upregulation of secretory activity in the intestine causes severe volume depletion and
multi-system failure. While enteric viruses remain the most common cause of fatal AGE
in kids, the drivers of their virulence (i.e. secretory activity) remain poorly understood.
We recently found that cells infected with rotavirus, the most prevalent enteric virus in
kids, release adenosine diphosphate (ADP) to coordinate signals known as “intercellular
Ca2+ waves,” which spread through uninfected neighboring cells. This dysregulates
Ca2+ signaling pathways, enhancing fluid secretion and thereby contributing to volume
depletion. Pharmacological blockade of Ca2+ waves in rotavirus-infected mouse pups
decreases disease severity, suggesting they are integral to rotavirus virulence.
Understanding how rotavirus triggers intercellular Ca2+ waves may allow us to design
safer, more effective vaccines and therapeutics, but we still lack a mechanistic
understanding of this process. Here, we report data showing that recombinant
expression of a single rotavirus protein, non-structural protein 4 (NSP4), is sufficient to
induce Ca2+ waves. Using the rotavirus reverse genetics system and human intestinal
organoids, we show that both the diminished Ca2+ wave phenotype and the diminished
secretory stimulation associated with an attenuated strain of rotavirus segregate with
NSP4. Furthermore, we show that this attenuation is attributable to a single amino acid
polymorphism at the c-terminal end of NSP4. This implicates the c-terminal end and a
putative PDZ-binding motif therein as key upstream mediators of intercellular Ca2+
waves. These findings deepen our understanding of pathogenesis, offer novel targets
for anti-secretory therapeutics, and expand foundational knowledge to support the
development of improved live-attenuated vaccines.
17THE TAILΦR 12: A REPORT ON THE USE OF TAILORED PHAGE COCKTAILS
AGAINST MULTIDRUG RESISTANT INFECTIONS IN 12 PATIENTS
Sabrina I. Green*, Austen L. Terwilliger, Justin R. Clark, Kyle E. Weesner, Haroldo H.
Santos, and Anthony W. Maresso
(13)
*Director of Research and Development, Baylor College of Medicine - Molecular Virology
and Microbiology
Antibiotic resistant bacteria are a major threat to the lives of human beings worldwide.
New treatments are needed now to fight against multidrug resistant bacterial infections.
Previously we outlined the major goals of TAILΦR (Tailored Antimicrobials and
Innovative Phage Research) an initiative at Baylor College of Medicine which aims to
provide tailored phage cocktails for treatment of antibiotic resistant infections. Within our
phage program thus far 12 patients have been treated with one patient receiving
retreatment with phages originally isolated from raw sewage and other environmental
sources or sourced from other phage labs across the world. These personalized phage
cocktails were extensively characterized—lytic assays, stability testing, endotoxin and
other toxin screening, sterility testing, sequencing analysis, and antibiotic synergy
assays—prior to treatment for eventual submission and approval by the FDA for
expanded access clinical use. Patient infections ranged from urinary tract infections
(UTI) to infections from devices or prosthesis (prosthetic joint infection or PJI; left
ventricular assist device infection or LVAD), surgical would infections (sternotomy) and
bacteremia. And causative organisms spanning from the ESKAPE pathogens—E.
cloacae, K. aerogenes, K. pneumonia, S. aureus, P. aeruginosa and E. coli. Here we
report a summary of data on these cases to call for a personalized or tailored approach
for phage therapy against drug resistant infections. Phage centers like TAILΦR are a
valuable resource to address an unmet clinical gap to help patients suffering from
antibiotic failure.
18LIGHTING THE VIRAL DARK MATTER: REVEALING EARTH'S CRYPTIC
GENOSPHERE THROUGH ENVIRONMENTAL SAMPLING
Gu Liu C*, Chang J, Thompson BE, Min L, Weesner KE, Heckmann, ER, Clark JR,
Green SI, Terwilliger AL, Kaplan HB, Maresso AW
(14)
*Graduate Student, Baylor College of Medicine - Molecular Virology and Microbiology
Bacteriophages (or phages) are abundant (estimated 1031 particles), diverse (in terms
of their genome, morphology, and host), and ubiquitous. However, only a small number
of phage genomes have been sequenced and around 60-80% of their sequences do not
share homology with previously classified genes. Hence, phages represent the largest
repository of undiscovered new biology – what we term as the cryptic genosphere. The
main goal of this study is to identify novel phage genes, and their corresponding protein
function and structure that supplant existing bacterial cell processes, specifically, genes
that may overcome oxidative stress and/or repair damaged DNA. To do this, freshwater,
seawater, and wastewater samples (~360 liters) were collected around Austin and
Houston, TX. Sludge, solids, and contaminants were removed via centrifugation. The
viral fraction was recovered and enriched via mixed cellulose esters filter and chemical
flocculation, detected with plaque assay and visualized with transmission electron
microscopy. Viral DNA was extracted, purified, and sequenced from all samples. Raw
reads were analyzed via both the iVirus pipeline and MG-RAST, and several DNA
libraries were constructed for functional screening. These DNA libraries were
transformed into deletion mutants, ΔrecA and Δcatalases, and either hydrogen peroxide
or mitomycin C was added as the stressor for the functional screening. Genes that
overcame the stressor and complemented the mutants were analyzed via gel
electrophoresis, sequenced, and re-transformed for confirmation. Overall, we
successfully isolated and enriched the viral fraction from all water samples. Freshwater
yielded more viral diversity compared to the other two samples, both freshwater and
seawater had more diverse functional potential, and ~70 positive hits were obtained from
the functional assays. Some of these hits aligned to genes that are involved in DNA
repair, potential iron sequestration, and many more. Additionally, we obtained ~100 high
quality and complete metagenome-assembled- genomes (MAGs) and a large proportion
of these MAGs are novel viruses that have not been previously identified.
19DEVELOPMENT OF NOVEL ANTIFUNGALS AGAINST CANDIDA BASED ON AN
ANTIFUNGAL PEPTIDE PRODUCED BY E. FAECALIS
Shantanu Guha*, Melissa R. Cruz, Giuseppe Buda De Cesare, Shane Christy, Michael
C. Lorenz, Danielle A. Garsin
(15)
*Postdoctoral Fellow, UTHealth 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.
20AN IN VITRO CELL CULTURE MODEL FOR PYOVERDINE-MEDIATED VIRULENCE
Donghoon (Alex) Kang*, Natalia V. Kirienko
(16)
*Graduate Student, Rice University - BioSciences (Biochemistry & Cell Biology Program)
Pseudomonas aeruginosa is a Gram-negative multidrug-resistant pathogen that causes
life-threatening infections in immunocompromised patients. One key virulence factor in
this pathogen is the siderophore pyoverdine, which not only provides the bacterium with
iron, but also regulates the production of several secreted toxins. We recently
demonstrated that pyoverdine can also directly exert virulence against Caenorhabditis
elegans by translocating into host tissue and disrupting iron and mitochondrial
homeostasis. Due to a combination of these functions, pyoverdine production is
necessary for P. aeruginosa virulence during murine lung infection. To investigate the
consequences of pyoverdine intoxication on a cellular level, we developed an in vitro cell
culture model using murine macrophages. We demonstrated that pyoverdine-rich
filtrates from P. aeruginosa exhibit substantial cytotoxicity, and that the inhibition of
pyoverdine production (genetic or chemical) is sufficient to mitigate virulence.
Furthermore, consistent with previous observations made in C. elegans, pyoverdine
translocates into cells and disrupts host mitochondrial homeostasis (as indicated by the
fragmentation of mitochondrial networks). Interestingly, pyoverdine seems to utilize both
endocytic and nonendocytic pathways for translocation. While pyoverdine is primarily
localized to endosomal compartments in both phagocytic (macrophages) and non-
phagocytic cells (epithelial cells), the siderophore is also able to diffuse across the
cellular membrane in giant plasma membrane vesicles derived from lung epithelial cells.
Most importantly, using our macrophage pathogenesis model, we observed a strong
correlation between pyoverdine production and virulence in P. aeruginosa clinical
isolates, confirming pyoverdine’s value as a promising target for therapeutic intervention.
We expect this in vitro cell culture model to provide further mechanistic insight into the
consequences of pyoverdine-mediated damage.
21NON-SPECIFIC EFFECTS OF BCG VACCINATION ENHANCING EFFICACY OF
COVID-19 AND INFLUENZA VACCINES
Kent Koster*, Zoe Spieler, Gabriel Neal, Madeline Moule, Aaron Benjamin, Maxim
Lebedev, Sathish Kumar, Seth Lerner, Robert Hutchison, Theresa Ofili, Pablo Avalos,
George Udeani, Ashish Kamat, Moshe Arditi, Andrew DiNardo, Jeffrey D. Cirillo
(17)
*Postdoctoral Fellow, Texas A&M Health Science Center - Microbial Pathogenesis and
Immunology
Evidence suggests that protection provided by COVID-19 specific vaccines declines
with time following vaccination. It is possible that vaccination with the live attenuated
Mycobacterium bovis strain Bacillus Calmette-Guérin (BCG) can enhance the
effectiveness of disease-specific vaccines through immune priming mechanisms. Small
sample sizes and short periods of time between BCG vaccination and treatment with
the disease-specific vaccine can make it difficult to evaluate existing data. The BCG As
a Defense Against SARS-CoV-2 (BADAS) clinical trial was begun to investigate the
trained immunity effects of BCG against severe COVID-19 disease. BADAS has
provided a large population of subjects, including healthcare practitioners, first
responders, and persons in high-risk groups, that can be examined to explore BCG’s
ability to enhance responses to specific vaccines that have been given to subjects
throughout the study. We investigated the effects of BCG vaccination on the strength
and duration of immune responses generated by COVID-19 and influenza vaccination
through serology and induction of ex vivo cytokine responses. We present a portion of
these data regarding the ability of BCG to enhance effectiveness of COVID-19 and
influenza vaccines, and propose a strategy for investigating ex vivo cytokine responses
as indicators of the duration of vaccine-mediated protection. Overall, these data are
suggestive of a beneficial response with BCG vaccination and point toward the need for
further investigation of the potential use of BCG to improve health.
22COMPARATIVE GENOME ANALYSIS OF CLONAL POPULATIONS OF IN VITRO
CULTIVATED TICK-BORNE RELAPSING FEVER SPIROCHETE BORRELIA
TURICATAE
Aparna Krishnavajhala*, Alex Kneubehl and Job Lopez
(18)
*Research Staff, Baylor College of Medicine - Pediatrics
Tick-borne relapsing fever (TBRF) is an infectious disease caused by Borrelia turicatae
and transmitted by Ornithodoros turicatae. Prior work indicated that B. turicatae
remained infectious by needle inoculation during serial cultivation. Further, the effect of
prolonged in vitro cultivation of B. turicatae by tick bite indicated that infection
frequencies in the mice that were fed on by infected ticks diminished after 120
generations (g120) and infectivity was lost after 300 generations (g300). At g120, B.
turicatae is a clear mixture of infectious and non-infectious bacteria. Our current study
focused on whole genome sequencing of clonal populations of B. turicatae propagated
for 60, 120 and 300 generations to identify the genes that are lost or disrupted during
prolonged in vitro propagation, genes essential for tick colonization and to establish
mammalian infection. At least two clones per generation of B. turicatae were used for
whole genome sequencing. The assembly of the genomes indicated on an average B.
turicatae g60, g120 and g300 have 16, 14 and 15 contigs; 1.72MB, 1.64 MB, and
1.62MB total genome; 41.8, 35.9 and 41.2 assembly consensus quality value (QV); and
99.57, 98.55 and 99.77 completeness of the genome, each respectively. Average
nucleotide identity (ANI) and pangenome analysis will be performed to understand
genetic similarities and differences at nucleotide level.
23DISSECTING THE MOLECULAR FUNCTION OF BamE IN THE ESSENTIAL Bam
COMPLEX
Santosh Kumar*, Muralidhar Tata, Anna Konovalova
(19)
*Postdoctoral Fellow, UTHealth McGovern Medical School - Microbiology & Molecular
Genetics
A defining feature of Gram-negative bacteria is the outer membrane (OM). The OM is
an essential selective permeability barrier and a major factor of intrinsic antibiotic
resistance. Outer membrane β-barrel proteins (OMPs) play crucial roles in nutrient
transport, signaling, and many serve as adhesion or virulence factors. OMPs are
inserted into the OM by the β-barrel assembly machinery (Bam). Because the Bam
complex is highly conserved and essential in gram-negative bacteria, it emerged as a
major target of antibiotic development. Therefore, much effort to understand the
mechanism of the Bam complex function is put in to facilitate antibiotic discovery. The
Bam complex consists of BamA, an OMP itself, and four associated lipoproteins, BamB,
C, D, and E. BamA and BamD are the only essential and best-studied core
components. BamB,C,E are individually not essential, but collectively play an essential
function in regulating BamAD core. The specific function of each of these components
remains elusive. Our laboratory discovered that BamE plays a specific role in the Bam
complex facilitating assembly of the OMP- dependent surface exposed lipoproteins,
such as RcsF (a regulator of capsule synthesis protein F). We use the RcsF/OMP
complex as a model substrate to study the molecular function of BamE in the Bam
complex. We showed that BamE coordinates BamA and BamD to ensure that BamA
adopts a competent conformation to promote RcsF/OMP assembly. The results of my
biochemical experiments demonstrate that BamE interacts directly with both BamA and
BamD, stabilizing the BamAD complex. Because BamE interaction with both BamA and
BamD is required for RcsF/OMP assembly, we propose that BamE directly regulate
BamA conformations to ensure functional coordination with BamD.
24STABILIZATION OF TUBERCULOSIS REPORTER ENZYME FLUORESCENCE
(REFtb) DIAGNOSTIC SYSTEM FOR USE AT THE POINT-OF-CARE
Maxim Lebedev*, Aaron B. Benjamin, Kent J. Koster, Sathish Kumar, Sugeily Ramos,
Joe Jilka, Jeffrey D. Cirillo
(20)
*Postdoctoral Fellow, Texas A&M Health Science Center - Microbial Pathogenesis and
Immunology
Tuberculosis is currently one of the top causes of death in humans worldwide. One of the
primary reasons tuberculosis remains a public health threat is that diagnosis can take
weeks to months, often is not sensitive enough to detect all infections and cannot be
easily accomplished in the remote environments where many of the cases occur. A
rapid, sensitive and inexpensive point-of-care (POC) diagnostic, even if used only as a
triage test to identify cases for followup would have a major impact on tuberculosis
eradication efforts. The new tuberculosis diagnostic system REFtb is based on specific
detection of the constitutively expressed β-lactamase (BlaC) in Mycobacterium
tuberculosis (Mtb) using a custom fluorogenic substrate, designated CDG-3. REFtb has
great potential as a diagnostic for tuberculosis that could be used at the POC and provide
diagnosis within 10 minutes, but components are currently in liquid form, leaving them
susceptible to degradation and difficult to transport. As a first step toward improving
REFtb, we tested the effects of component lyophilization on performance of REFtb. We
examined mannitol, lactose and raffinose as lyophilization excipients. We found that
lyophilization of REFtb components produces an easily reconstituted powder that
displays similar performance to the liquid system and raffinose represents one of the
most promising excipients for further study. These studies provide the foundation for
production of a stable POC REFtb system that could be easily distributed worldwide with
minimal refrigeration requirements.
25EXPRESSION OF HYPHAL-ASSOCIATED VIRULENCE FACTORS IS NECESSARY
FOR CANDIDA ALBICANS PERSISTENCE IN THE MURINE GASTROINTESTINAL
TRACT
Kelsey Mauk*, Lynn Bimler, J. Morgan Knight, David B. Corry
(21)
*Graduate Student, Baylor College of Medicine - Immunology and Microbiology Program
Candida albicans is a dimorphic fungus that infects mucosal surfaces of humans.
Although often considered a commensal organism, C. albicans has the potential to cause
severe disease in immunocompromised individuals and has been associated with
multiple chronic diseases. Despite its pathogenic potential, and the fact that C. albicans
can be effectively cleared from other mucosal sites, C. albicans colonizes and persists
within the gastrointestinal (GI) tract of a large portion of the human population. However,
the mechanisms by which C. albicans avoids elimination from the gut are unclear. To
investigate these mechanisms, we established a chronic murine model of C. albicans
intestinal infection. We have observed that mice infected with C. albicans remain
colonized for at least two months and experience significant growth retardation,
suggesting a more pathogenic relationship to the host. However, these mice did not
develop a robust inflammatory response in the intestine upon exposure to C. albicans,
suggesting the murine host is unable to effectively respond to the infection. To determine
if C. albicans virulence factors play a role in intestinal persistence, we infected mice with
strains lacking known virulence factors, including candidalysin, Als3, and Sap2. We
found that no virulence factor-deficient strains colonize the GI tract as well as the wild-
type strain, suggesting these factors are crucial for intestinal colonization and
persistence. Our additional data demonstrate that C. albicans enters intestinal epithelial
cells during infection, which may be an important mechanism of immune evasion and
intestinal persistence. Thus, we hypothesize that persistent infection with C. albicans is
accomplished through the expression of multiple virulence factors required for gut
epithelial invasion. Understanding how C. albicans persists within the GI tract will
elucidate the widespread presence of this fungus in humans and its participation in
multiple diseases.
26HUMAN MILK OLIGOSACCHARIDES REDUCE MURINE GROUP B
STREPTOCOCCUS VAGINAL COLONIZATION WITH MINIMAL IMPACT ON THE
VAGINAL MICROBIOTA
Marlyd E. Mejia*, Samantha Ottinger, Alison Vrbanac, Priyanka Babu, Jacob Zulk,
David Moorshead, Lars Bode, Victor Nizet, and Kathryn A. Patras
(22)
*Graduate Student, Baylor College of Medicine - Molecular Virology and Microbiology
Group B Streptococcus (GBS) colonizes the vaginal mucosa of a significant percentage
of healthy women and is a leading cause of neonatal bacterial infections. Currently,
pregnant women are screened in the last month of pregnancy and GBS-positive women
are given antibiotics during parturition to prevent bacterial transmission to the neonate.
Recently, human milk oligosaccharides (HMOs) isolated from breastmilk were found to
inhibit GBS growth and biofilm formation in vitro, and women that make certain HMOs
are less likely to be vaginally colonized with GBS. Using in vitro human vaginal epithelial
cells and a murine vaginal colonization model, we tested the impact of HMO treatment
on GBS burdens and the composition of the endogenous microbiota by 16S rRNA
amplicon sequencing. HMO treatment reduced GBS vaginal burdens in vivo with
minimal alterations to the vaginal microbiota. HMOs displayed potent inhibitory activity
against GBS in vitro, but HMO pretreatment did not alter adherence of GBS or the
probiotic Lactobacillus rhamnosus to human vaginal epithelial cells. Additionally,
disruption of a putative GBS glycosyltransferase (Δsan_0913) rendered the bacterium
largely resistant to HMO inhibition in vitro and in vivo but did not compromise its
adherence, colonization, or biofilm formation in the absence of HMOs. We conclude
that HMOs are a promising therapeutic bioactive to limit GBS vaginal colonization with
minimal impacts on the vaginal microenvironment.
27DEVELOPMENT OF AN INVERTEBRATE VIRULENCE MODEL FOR THE
EMERGING FUNGAL PATHOGEN CANDIDA AURIS
Pedro Miramon*, Melissa Marti-nez, Andrew Pountain, Melissa Cruz, Danielle Garsin,
Michael C. Lorenz
(23)
*Research Staff, UTHealth McGovern Medical School - Microbiology & Molecular
Genetics
Candida auris is an emerging fungal pathogen, with reported cases all over the world.
Susceptible individuals infected with this yeast have poor outcomes, stressing the
importance of understanding the pathogenicity attributes of this pathogen. Murine
models for the study of C. auris virulence are inadequate since mice are naturally
resistant. Therefore, we investigated whether the invertebrate worm (Caenorhabditis
elegans) model is suitable for virulence studies. We challenged the worms with strains
from multiple genetic clades of C. auris, showing that most, but not all, strains rapidly
killed the worms. As proof of principle, we used a CRISPR/Cas9-based approach to
create a deletion in ADE2, encoding an enzyme involved in purine biosynthesis that is
easy to monitor as mutants in this gene accumulated a red intermediate and ade2∆
mutants are highly attenuated in other fungal pathogens. Worms infected with the
ade2∆ deletion mutants exhibited increased survival, indicating that ADE2 is required
for full virulence in this model. Thus, the worm model can detect avirulent mutants. We
next examined the interaction with innate immune cells, which represent one of the first
lines of defense during infection. We investigated the transcriptional response during
coincubation with murine macrophages. Like other Candida spp., C. auris
downregulates glycolysis and protein biosynthesis while inducing stress responses and
alternative carbon metabolism. However, a remarkable response in C. auris is the
upregulation of multiple transport systems such as oligopeptide transporters and drug
resistance genes (ABC and MFS transporters). This suggests that C. auris is effectively
ready to utilize alternative carbon sources that may be present in host tissues and
upregulated antifungal resistance determinants to cope with such compounds.
28BACTERIOPHAGE-CONTAINING BIODEGRADABLE MICROSPHERE
TECHNOLOGY TO TREAT OSTEOMYELITIS
Analisa J. Narro*, Erin Brown, Heidi B. Kaplan, Catherine G. Ambrose
(24)
*Medical Student, UTHealth McGovern Medical School - Microbiology & Molecular
Genetics
Background: The rise in antimicrobial resistant infections is a growing concern with few
antibiotics in the pipeline. Thus, bacteriophage could provide a unique biological
approach. Several concerns around delivery of active phage using degradable drug
delivery systems have been raised. In manufacturing poly (lactic-co-glycolic acid)
(PLGA) microspheres, the steps that present possibility for phage inactivation are contact
with dichloromethane (DCM) and lyophilization. As lyophilization is less likely to result in
significant loss of phage activity, in this study we investigated two protocols for
microsphere manufacture with focus on exposure to DCM.
Methods: Two phage, K and SA4, were tested for their ability to lyse a Staphylococcus
aureus isolate obtained from an osteomyelitis infection. The PLGA microspheres were
manufactured according to a water-oil-water protocol. In method 1, a phage solution was
added directly to a PLGA-DCM mixture. Contact time between the phage and the solvent
was reduced with 2-propanol to precipitate the microspheres. In Method 2, a phage
solution was added to polyvinyl alcohol before adding this to a PLGA-DCM solution. Both
were evaluated with an elution assay with eluent collections at 1, 3 and 7 days. Each
eluent was spotted against S. aureus and plaque forming units were counted. Total
phage eluted over the 7 days and average entrapment efficiency was calculated. SPSS
software and a t-test were completed for data analysis.
Results: Method 1 and 2 had a total of 2.0 x 107 phage and 5.1 x 105 phage eluted after
7 days, yielding a 0.6% and 0.02% entrapment efficiency, respectively (p=0.012).
Conclusion: Method 1 eluted a statistically greater number of active phage and had a
greater entrapment efficiency. These results indicate that further studies should be
performed to determine the effectiveness of our microspheres against S. aureus in in
vitro biofilms and in in vivo animal models.
29AN IN VIVO MODEL OF URINARY TRACT INFECTION-ASSOCIATED PRETERM
BIRTH
Samantha Ottinger*, Vicki Mercado-Evans, Jacob J. Zulk, Marlyd E. Mejia, Mallory B.
Ballard, Kathryn A. Patras
(25)
*Graduate Student, Baylor College of Medicine - Immunology and Microbiology
Preterm birth is the leading cause of neonatal mortalities and morbidities, resulting in
over 1 million neonatal deaths each year and countless physical, neurodevelopmental,
and socioeconomic sequelae. Maternal infections are major causes of spontaneous
preterm labor, including urinary tract infection (UTI). Clinical studies have repeatedly
shown increased risk of preterm birth in patients who are diagnosed with an UTI any
point during pregnancy; however, the mechanism by which UTI causes preterm birth
has not been explored. To study this phenomenon, we have developed an in vivo model
of UTI- associated preterm birth with uropathogenic E. coli (UPEC), the causative agent
of over 75% of UTIs. In this model, mice were mated over 3 days, infected
transurethrally at embryonic day 13.5, then monitored twice daily for signs of preterm
labor. We found that 52% of pregnant mice experienced preterm labor, with preterm
mice experiencing significantly higher adverse pup outcomes including death.
Interestingly, preterm pregnancies had significantly lower bladder and placental
bacterial burdens compared to term pregnancies. Additionally, dams given an UTI with
UV-inactivated bacteria still experienced preterm birth at the same rate as dams treated
with live bacteria. These findings suggest that maternal inflammation during UTI may
be contributing to preterm birth independently of bacterial replication or uterine
ascension. In addition to lower bacterial burdens, there were lower maternal bladder
concentrations of IL1β and CXCL2 (MIP2), suggesting bladder dysfunction in response
to bacterial components may drive uterine inflammation and initiation of labor. Together,
these data support a murine model of UTI-associated preterm birth that replicates
adverse outcomes seen in humans. This model enables us to further study the
relationship between maternal urinary tract infections and reproductive outcomes,
enabling future work to identify therapies to prevent or delay preterm birth.
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