Evaluating The Role Of Gras And Fadxdeba In Promoting Staphylococcus Aureus Adaptation To Host-Derived Fatty Acids Encountered At Sites Of Colonization And Infection, Robert C. Kuiack

Electronic Thesis and Dissertation Repository

Staphylococcus aureus is an opportunistic pathogen that asymptomatically colonizes 30% of humans, where it is well adapted to survive on the skin in the presence of innate defense mechanisms such as antimicrobial free fatty acids (FFA). While antimicrobial FFA function to inhibit the growth of S. aureus, they also provide a valuable source of lipids for membrane synthesis and energy production. We hypothesized that S. aureus possesses a novel antimicrobial FFA resistance pathway that is activated under conditions found on human skin, and that under these conditions, S. aureus can metabolize exogenous fatty acids to fuel growth and virulence …

Towards More Complete Metagenomic Analyses Through Circularized Genomes And Conjugative Elements, Benjamin R. Joris

Electronic Thesis and Dissertation Repository

Advancements in sequencing technologies have revolutionized biological sciences and led to the emergence of a number of fields of research. One such field of research is metagenomics, which is the study of the genomic content of complex communities of bacteria. The goal of this thesis was to contribute computational methodology that can maximize the data generated in these studies and to apply these protocols human and environmental metagenomic samples.

Standard metagenomic analyses include a step for binning of assembled contigs, which has previously been shown to exclude mobile genetic elements, and I demonstrated that this phenomenon extends to all conjugative …

Exploring The Use Of Covellite As A Proxy For Corrosion Of Native Copper By Sulphur Reducing Bacteria, Manan K. Joshi

Undergraduate Student Research Internships Conference

We are analyzing the effect of sulphate reducing bacteria on native copper, and using that evidence to further support the initiative of having a deep geological repository to store nuclear material. Sulphate reducing bacteria are a concern for the deep geological repository as they cause the corrosion of regular copper. However native copper has gone billions of years without corrosion, which could either mean that it had not been exposed to sulphate reducing bacteria over the billions of years, or native copper is able to withstand corrosion despite the contact of sulphate reducing bacteria. We can find out by trying …

Tetr Family Regulator Farr Variation Controls Antimicrobial Fatty Acid Efflux In Staphylococcus Aureus, Camryn M. Bonn

Electronic Thesis and Dissertation Repository

To colonize human skin and survive within abscesses, Staphylococcus aureus has evolved mechanisms to evade host innate defenses. USA300 has become the predominate community-acquired methicillin-resistant S. aureus (CA-MRSA) clone, which can be in part attributed to detoxification of unsaturated free fatty acids (uFFA) found in sebum and the nares. Our lab has previously identified the TetR family regulator FarR responsible for induction of the resistance-nodulation-division (RND) superfamily efflux pump FarE to promote efflux of toxic uFFA. However, RND family efflux pumps remain poorly characterized in Gram-positive species and the mechanism by which FarR regulates FarE has yet to be determined. …

Screening For Purine Transporters In S. Aureus, Tothong Sonpaveerawong

Electronic Thesis and Dissertation Repository

Staphylococcus aureus is an important human pathogen and crucial part of its pathogenesis depends on its ability to acquire purines to cause disease. In this study, I screened a library of individual mutants under purine import-dependent conditions by inhibiting the purine biosynthesis using the pharmacological agents methotrexate and 6-mercaptopurine and supplementing inosine monophosphate and/or guanine as an exogenous purine source. I identified an ATP-Binding Cassette transporter mutant that failed to grow under the selective purine transport conditions. Further growth characterization of the mutant revealed that the growth defect was not due to an inability to transport purines but rather to …

The Role Of Coagulase-Negative Staphylococcal Secreted Products On Staphylococcus Aureus And Staphylococcus Lugdunensis Infections, Denny Chin

Electronic Thesis and Dissertation Repository

The Staphylococcus genus is comprised of over 40 bacterial species. The most well-studied species in this genus is the notorious human pathogen Staphylococcus aureus, a bacterium that produces coagulase among many other virulence factors. Since S. aureus is a major health burden and causes a plethora of diseases in humans, it has received significant attention and much research has been done to understand its biology to treat diseases caused by this pathogen. However, the coagulase-negative staphylococci (CoNS) make up most of the staphylococcal species and have received less attention since they are thought to have a lesser impact on …

Fishing For The Right Probiotic: Investigating The Mechanism Of Mucosal-Bacterial Interactions At The Interface Of Health And Productivity In Salmonid Aquaculture, Luana Langlois

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Aquaculture is vital for the global food supply, but the high incidence of infectious diseases threatens the industry’s productivity. The intestinal mucosa is a key port of entry for pathogens and provides an extensive interface for host-microbe interactions. Tight junctions are at the core of gut barrier function and the mucosal health of finfish. Disruption of these complexes gives rise to sepsis, which leads to systemic inflammation and death. The present study employs a combinatorial approach that integrates in vitro and in vivo analyses to gain actionable insights into the mechanism of microbial-mediated modulation of host health. The experiments outlined …

Assessing Selective Plasmids For Bradyrhizobium Sp. Doa9 And Mesorhizobium Loti, Aiden J. Wilcox, Rebecca Meaney, Bogumil Karas

Undergraduate Student Research Internships Conference

No abstract provided.

Staphylococcus Aureus Small Colony Variants Use Heme And Staphyloferrin B For Iron Acquisition, Izabela Z. Batko

Electronic Thesis and Dissertation Repository

Iron is an essential nutrient for the bacterium Staphylococcus aureus. Wild-type S. aureus utilizes various iron acquisition systems to support growth in iron deplete conditions. S. aureus small colony variants (SCVs) are associated with chronic infections, yet the mechanisms by which these variants acquire iron are unknown. Mutation of hemB, involved in heme biosynthesis, generated a stable SCV that was auxotrophic for hemin and formed small colonies on solid media. To support growth under iron deplete conditions, my data revealed that S. aureus hemB synthesizes the siderophore staphyloferrin B, but not staphyloferrin A, although both siderophores could be …

Investigation Of The Growth And Survival Of Staphylococcus Aureus In Cftr-Deficient Macrophages, Bita Azad

Electronic Thesis and Dissertation Repository

Staphylococcus aureus and its small colony variants (SCVs) are commonly isolated from the lungs of cystic fibrosis (CF) patients. Although studies have suggested that cystic fibrosis transmembrane regulator (CFTR)-deficient macrophages are diminished in their ability to kill intracellular pathogens, whether this is true for CFTR-deficient macrophages infected with S. aureus or its SCVs is unknown. I employed gentamicin protection and eFluorÔ-670-based proliferation assays to assess the intracellular replication of S. aureus in CFTR inhibitor treated THP-1 and primary human macrophages, and in primary macrophages derived from CF patient blood. My work shows that the susceptibility of CFTR-deficient macrophages to the …

Investigating The Regulation Of Fatty Acid Efflux Pump Fare By Tetr Family Regulator Farr In Staphylococcus Aureus, Katherine A. Ferguson

Electronic Thesis and Dissertation Repository

The success of the USA300 strain of methicillin resistant Staphylococcus aureus can be attributed in part to its enhanced ability to overcome innate defenses of the skin including sebum, which provides a source of antimicrobial unsaturated free fatty acids (uFFA). We have previously identified farE and farR genes that confer S. aureus resistance to uFFA, respectively encoding a uFFA efflux pump and a TetR family regulator required for farE expression. However, the exact regulatory mechanism of FarR remains to be elucidated. Here, we show the importance of a conserved TAGWTTA motif in FarR operator sites, such that the loss of …

Metabolomic And Genomic Analysis Of Lactobacillus Crispatus Vaginal Isolates, Emiley Watson

Electronic Thesis and Dissertation Repository

Lactobacillus crispatus is the dominant species in the vagina of many women. To add to our knowledge of its properties, the metabolic readout of twenty strains was analyzed using liquid chromatography-mass spectrometry (LC-MS). This led to a separation of the strains into two groups, Groups A and B. Notably, five Group B strains produced tyramine, known to raise vaginal pH, and make the environment more favourable to vaginal pathogens. The origin of Group B strains did not correlate with their host having bacterial vaginosis (BV), a condition associated with elevated pH. Five bacteriocins were detected in the genomes. Therefore, differences …

The Group A Streptococci Bacteriocins Facilitate A Competitive Advantage During Nasopharyngeal Infection, Lana Estafanos

Electronic Thesis and Dissertation Repository

Pathogenic streptococci have evolved specific systems to eliminate bacterial competitors within their biological niche. In microbial environments, niche competition is often driven by the production of short antimicrobial peptides called bacteriocins; this provides a mechanism by which Streptococcus pyogenes may compete for ecological stability and establish infection. Recent findings from our laboratory have identified two novel Class IIb bacteriocin systems – Streptococcus pyogenes bacteriocin (Spb) JK and MN in the M18 serotype S. pyogenes strain MGAS8232 – that may contribute to nasopharyngeal infection. Here, we show that galactose and CO₂ are distinct regulatory cues which induce antimicrobial activity. Under …

Identification And Characterization Of Farr And Fare As A Regulator And Effector Of Fatty Acid Resistance In Staphylococcus Aureus, Heba Alnaseri

Electronic Thesis and Dissertation Repository

Although Staphylococcus aureus is exposed to antimicrobial fatty acids on the skin, in nasal secretions and in abscesses, specific mechanisms for regulating gene expression and intrinsic resistance in response to these fatty acids have not been reported. Through in vitro selection for increased resistance of S. aureus to linoleic acid, I identified fatty acid resistant clone FAR7, where a single nucleotide polymorphism caused a His₁₂₁Tyr substitution in an uncharacterized member of the TetR family of transcriptional regulators, which is divergently transcribed from a gene encoding a member of the resistance-nodulation-division superfamily of multi-drug efflux pumps. I named these …

Iron Acquisition Strategies Employed By Staphylococcus Lugdunensis, Jeremy R. Brozyna

Electronic Thesis and Dissertation Repository

Iron is crucial for many cellular processes including DNA synthesis and respiration. The majority of iron in mammals is in heme within hemoproteins, inside cells, or transported through circulation by the glycoprotein transferrin, which constitutes the greatest iron source in serum. Limiting iron availability is an important facet of nutritional immunity to help prevent infection.

Staphylococcus lugdunensis is a human skin commensal and opportunistic pathogen capable of causing a variety of infections, including particularly aggressive endocarditis. It is an emerging pathogen with elevated virulence compared to other species of coagulase-negative staphylococci. The versatility of S. lugdunensis to infect multiple niches …

Recombinase-Based In Vivo Expression Technology Identifies A Streptococcus Pyogenes Bacteriocin Important For Niche Adaptation In The Nasopharynx, Brent D. Armstrong

Electronic Thesis and Dissertation Repository

Streptococcus pyogenes is a Gram-positive, human-specific bacterial pathogen with the ability to cause a wide range of diseases from strep throat to necrotizing fasciitis and toxic shock syndrome. In addition, S. pyogenes may also induce post-streptococcal sequelae including rheumatic fever, acute glomerulonephritis, and reactive arthritis. Although primarily recognized as a pathogen, S. pyogenes also colonizes the skin and throat often without causing disease, and while numerous surface adhesions are important to attach to these surfaces, additional factors important for colonization and persistence by S. pyogenes are poorly understood. In addition to host defence mechanisms, the upper respiratory tract also contains …

Characterization Of Fatty Acid Resistant Staphylococcus Aureus Mutants Containing Snps In Cvfa, Melissa N. Loyzer

Electronic Thesis and Dissertation Repository

Upon colonization, Staphylococcus aureus must withstand the actions of many host defense mechanisms, including the unsaturated free fatty acids (uFFAs) secreted in sebum. Linoleic acid, a representative uFFA, is toxic to S. aureus at concentrations ≥50 µM; however, we selected for mutants capable of growing at elevated concentrations of linoleic acid. Five mutants had single nucleotide polymorphisms (SNPs) in the cvfA gene encoding an RNase that is a component of the RNA degradosome, and expression of the SNP-containing cvfA genes from a plasmid engendered S. aureus with high level resistance to linoleic acid. The SNPs were located within a region …

Granulocyte Colony-Stimulating Factor: Its Role In Gut-Homing Macrophage Generation And Colitis, And Production By Probiotics, Shahab Meshkibaf

Electronic Thesis and Dissertation Repository

The pleiotropic cytokine granulocyte-colony stimulatory factor (G-CSF) is mainly required for the generation of neutrophils, but its role in macrophage generation has also been reported. In addition, G-CSF is effective for the down-regulation of inflammatory cytokines and ameliorating gut disorders, such as colitis. However, the G-CSF function in macrophage generation and gut immunity remains unclear. The first focus of this thesis was to assess the role of G-CSF in macrophage generation and its contribution to gut immunity. G-CSF was found to promote the generation of Gr-1high/F4/80+ macrophages in macrophage (M)-CSF-treated bone marrow cells, most likely through suppressing cell death. Gr-1high …

The Role Of Streptococcus Salivarius As A Modulator Of Homeostasis In The Oral Cavity, Kyle W. Macdonald

Electronic Thesis and Dissertation Repository

The oral cavity contains many different surfaces all colonized by prokaryotes, of which over 700 have been identified. While almost all people have some degree of plaque formation, the more concerning diseases of caries, candidiasis and periodontal disease afflict many patients and represent a major public health concern. As these are all diseases which have a component attributable to parts of the microbiota, efforts to manipulate the microbes has until recently involved use of antimicrobial agents. However, due to side effects, resistance and failure to restore homeostasis, this approach is limited. As an alternative, the administration of beneficial microbes (probiotics) …

Role Of Peptidoglycan Modifications In Predation By Bdellovibrio Bacteriovorus 109j, Diane C. Szmiett

Electronic Thesis and Dissertation Repository

Bdellovibrio bacteriovorus is a prey dependent bacterium that traverses the outer membrane and peptidoglycan, and enters the periplasmic space of gram negative bacteria, from where it utilizes the cytoplasmic contents for growth. Soluble lytic transglycosylases could be responsible for creating the entry pore during predation. Bacteria can modify their peptidoglycan by the addition of acetate to the C-6 hydroxyl group of the N-acetylmuramic acid residues, which renders the peptidoglycan insensitive to cleavage by lytic transglycosylases. It was hypothesized that the degree of peptidoglycan O-acetylation of the prey cell affects predation efficiency. In this study it was shown that; …

Hcpe, A Potential Immuno-Modulatory Protein From Helicobacter Pylori That Is Dependent On The Disulfide Bond Protein Dsbhp, Jeff Lester

Electronic Thesis and Dissertation Repository

H. pylori is a human gastric pathogen that colonizes ~ 50% of the world’s population. It can cause gastritis, gastric or duodenal ulcers and also gastric cancer. H. pylori produces Helicobacter cysteine rich protein HcpE, a secreted protein which may play a role in virulence. In this study we show that HcpE is secreted in the culture supernatant both as a soluble protein and in association with outer membrane vesicles, and may play a role in the modulation of H. pylori inflammatory responses. We identified that Dsb_HP is necessary for HcpE production and secretion in H. pylori, and …

The Role Of Superantigens During Staphylococcus Aureus Nasal Colonization And Infection, Stacey Xu

Electronic Thesis and Dissertation Repository

Superantigens (SAgs) are potent toxins produced by bacteria such as Staphylococcus aureus that function to overactivate T cells resulting in massive cytokine production and immune activation. Despite decades of research on the structure and function of these proteins, as well as their role in severe diseases such as toxic shock syndrome, the question as to why strains of S. aureus produce SAgs and the role that they play in the life cycle of these bacteria remains unanswered. The contribution of SAgs towards pathogenicity and bacterial survival in vivo were assessed using isogenic SAg deletion knockouts in conjunction with SAg-sensitive humanized …

Chemical Communication Of Antibiotic Resistance By Highly Resistant Bacteria., Omar M. El-Halfawy

Electronic Thesis and Dissertation Repository

The overall antibiotic resistance of a bacterial population results from the combination of a wide range of susceptibilities displayed by subsets of bacterial cells. Bacterial heteroresistance to antibiotics has been documented for several opportunistic Gram-negative bacteria, but the mechanism of heteroresistance is unclear. I use Burkholderia cenocepacia as a model opportunistic bacterium to investigate the implications of heterogeneity in the response to the antimicrobial peptide polymyxin B (PmB) and also other bactericidal antibiotics. Here, I report that B. cenocepacia is heteroresistant to PmB. Population analysis profiling identified B. cenocepacia subpopulations arising from a seemingly homogenous culture that are resistant to …

Probiotic Therapy For Heart Failure: Investigating The Potential Anti-Hypertrophic Properties Of Probiotics, Grace L. Ettinger

Electronic Thesis and Dissertation Repository

Heart failure patients face a five-year 50% mortality rate, due to impaired cardiac function and hypertrophy of the heart. Probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host. Considering the established cardiovascular benefits of some probiotics, including reduction of cholesterol and hypertension, it was hypothesized that probiotics can improve outcomes of heart failure. Probiotics or controls were administered orally to an animal model for heart failure. Those receiving probiotics showed a significant improvement in cardiac hypertrophy and an attenuation of heart failure compared to control. No changes in gut microbial composition occurred. …

Sub-Inhibitory Antibiotics Enhance Virulence, Persistence, And Pathogenesis Of Uropathogens, Lee W. Goneau

Electronic Thesis and Dissertation Repository

In addition to their bactericidal effects, antibiotics are potent signal mediators at sub-inhibitory levels in the environment. The ability to modulate community structure in this niche raises concerns over their capacity to influence pathogenesis in patients during antibiotic therapy. This concept forms the basis of this thesis, and is explored using models of prophylactic therapy for recurrent urinary tract infection (UTI) management.

Sub-inhibitory ciprofloxacin, ampicillin, and gentamicin were found to augment virulence in vitro, increasing adherence and urothelial cell invasion in uropathogenic Escherichia coli (UPEC) and Staphylococcus saprophyticus. In addition, biofilm formation was increased, and swarming motility decreased. …

Pegylation As A Novel Tool To Investigate The Topology Of Escherichia Coli Weca, A Membrane Enzyme Involved In Lipopolysaccharide O Antigen Initiation, Stéphanie L. Lamothe

Electronic Thesis and Dissertation Repository

O-antigen, the most surface exposed moiety of bacterial lipopolysaccharide (LPS), plays several roles in pathogenicity. The biosynthesis of O-antigen starts by the formation of a phosphoanhydride bond linking a sugar phosphate with a membrane isoprenoid lipid phosphate. Two distinct families of integral membrane proteins catalyze this reaction. The protein WecA is the prototypic member of one of these families, termed the polyisoprenyl-phosphate N-acetylaminosugar-1-phosphate transferase (PNPT) family. Because the donor nucleotide sugar is only available in the cytosol, cytosolic exposed regions of WecA are expected to be critical for the catalytic activity of the enzyme. Therefore, elucidating an accurate topological …

Characterization Of Staphylococcus Aureus Lipase, Vithooshan Vijayakumaran

Electronic Thesis and Dissertation Repository

USA300, a strain of community-associated methicillin resistant Staphylococcus aureus (CA-MRSA), has become prevalent in the community. Colonization of human skin requires mechanisms that allow this bacterium to overcome the innate immune defenses on the skin, including secretion of antimicrobial lipids. Antimicrobial lipids inhibit S. aureus growth and induce the staphylococcal proteolytic cascade, producing aureolysin (Aur) which processes the lipase glycerol ester hydrolase (Geh). Nearly all S. aureus strains secrete Geh, yet little information exists concerning its function. Using purified Aur and Geh we confirm that aureolysin processes proGeh to Geh. We then confirmed that geh was required for lipase activity …

Staphylococcus Aureus Response To Long Chain Antimicrobial Fatty Acids, Benjamin Arsic

Electronic Thesis and Dissertation Repository

Staphylococcus aureus is a common pathogen that has the ability to infect virtually every tissue and organ system of the body. Despite its propensity to cause invasive infection, S. aureus is also a commensal organism, asymptomatically colonizing ~25% of the population. Much research has gone into resolving this paradox, focusing on both human and bacterial factors that may contribute to colonization. Antimicrobial fatty acids present on the skin and in the nasal mucosa are important components of the innate immune system, and thus we undertook to further understand how S. aureus responds to these fatty acids, and how this response …

Sequesteration Of Lead, Cadmium And Arsenic By Lactobacillus Species And Detoxication Potential, Marc A. Monachese

Electronic Thesis and Dissertation Repository

Toxic metals are a class of elements with no biological role but with extreme toxicity. On average only 50% of ingested toxins are absorbed into the human body, for reasons still unknown. It was hypothesized that the gut microbiota plays a role in reducing toxin absorbance. The aim of this study was to determine if constituents of the gut, namely Lactobacillus species, are able to sequester arsenic, lead and cadmium from the environment. Lactobacilli were incubated with the metals, both in vitro and with a Caco-2 cell line. Analysis of metal concentrations was conducted to determine if these were reduced …

Colonization Of Zea Mays By The Nitrogen Fixing Bacterium Gluconacetobacter Diazotrophicus, Nikita Eskin

Electronic Thesis and Dissertation Repository

To achieve high yields, corn fields are supplemented with nitrogen fertilizers. Nitrogen fertilizers account for a significant portion of production costs, and are harmful to the environment. Gluconacetobacter diazotrophicus, an endophytic nitrogen-fixing bacterium supplies its natural host sugarcane with a significant amount of nitrogen. This study investigated the colonization of G. diazotrophicus in seven different corn genotypes consisting of sweet and grain corn via three different methods of inoculation: soil drench, root dip, and aseptic inoculation. Sucrose content of the corn genotypes and nitrogenase activity were also analysed. Colonization was confirmed by PCR analysis. G. diazotrophicus colonization was detected using …