The morphogenesis of C. albicans is a major aspect of its virulence and is regulated by quorum sensing (QS) molecules they produce, as well as the presence of neighboring microbes.Two mutant transporters, SSU1 and CDR4, were characterized for their ability to form biofilms in the presence of cyclic-di-GMP and 3-oxo-12-homoserine lactone. While homoserine lactone showed a decrease in biofilm density of both mutants compared to the wild-type strain, wild-type and ssu1 biofilm densities increased considerably in the presence of cyclic-di-GMP while testing lower inocula. Additionally, it has been shown that C. albicans mutants lacking the hybrid histidine kinase, Chk1, are refractory to the effects of farnesol, a QS molecule that inhibits morphogenesis.We determined both CDR4 and SSU1 expression is reduced or highly repressed in the chk1, ypd1, and skn7 null strains. Our results suggest these two genes are downstream targets in a pathway regulated by Chk1p.

No description available.

Vibrio salmonicida causes cold-water vibriosis in salmon populations around the world and causes financial damage to fisheries designed to farm these salmon. Very little is known about the physiology of how V. salmonicida causes disease and measures to contain vibriosis are restricted to either vaccinating individual fish against disease or administering antibiotics when an outbreak is detected. These procedures are costly and increase the risk for selection of antibiotic-resistant V. salmonicida strains. A recent reoccurrence of outbreaks in Norwegian fisheries provided incentive to better understand the virulence mechanisms of V. salmonicida. In this thesis, a proteomic approach was used to identify proteins that were differentially expressed when cells were grown in vitro under simulated virulence conditions (i.e. 5˚C and in the presence of exogenously supplied autoinducer 3-oxo-hexanoyl-homoserine lactone). Some examples of proteins with significantly altered expression that stood out at as homologs of potential virulence factors were: an exported serine protease DegQ, a multi-drug transporter HlyD, and an outer membrane protein OmpU. The proteomic approach allowed us to identify large numbers of proteins that are expressed by V. salmonicida, facilitating hypothesis-driven research in order to support possible roles for some of these proteins in virulence

Trypanosoma brucei and related parasites are causative agents of severe diseases that affect global health and economy. T. brucei is responsible for sleeping sickness in humans (African trypanosomiasis) and a wasting disease in livestock. More than 100 years after T. brucei was identified as the etiological agent for sleeping sickness, available treatments remain inadequate, complicated by toxicity, lengthy and expensive administration regiments, and drug-resistance. There is clear need for the development of a new antitrypanosomal drugs. Due to the unique evolutionary position of these early diverging eukaryotes, trypanosomes posses a number of biological properties unparalleled in other organisms, including humans, which could prove valuable for new drug targets. One of the most distinctive properties of trypanosomes is their mitochondrial DNA, called kinetoplast DNA (kDNA). kDNA is composed of over five thousand circular DNA molecules (minicircles and maxicircles) catenated into a topologically complex network. Replication of kDNA requires an elaborate topoisomerase-mediated release and reattachment mechanism for minicircle theta structure replication and at least five DNA polymerases. Three of these (POLIB, POLIC, and POLID) are related to bacterial DNA polymerase I and are required for kDNA maintenance and growth. Each polymerase appears to make a specialized contribution to kDNA replication. The research described in this dissertation is a significant contribution to the field of kDNA replication and the advancement of kDNA replication proteins as putative drug targets for sleeping sickness. Functional characterization of POLIB indicated that it participates in minicircle replication but is likely not the only polymerase contributing to this process. Gene silencing of POLIB partially blocked minicircle replication and led to the production of a previously unidentified free minicircle species, fraction U. Characterization of fraction U confirmed its identity as a population of dimeric minicircles with non-uniform linking numbers. Fraction U was not produced in response to silencing numerous other previously studied kDNA replication proteins but, as we demonstrated here, is also produced in response to POLID silencing. This common phenotype led us to hypothesize that POLIB and POLID both participate in minicircle replication. Simultaneously silencing both polymerases completely blocked minicircle replication, supporting a model of minicircle replication requiring both POLIB and POLID. Finally, we demonstrate that disease-causing trypanosomes require kDNA and the kDNA replication proteins POLIB, POLIC, and POLID. These data provide novel insights into the fascinating mechanism of kDNA replication and support the pursuit of kDNA replication proteins as novel drug targets for combating African trypanosomiasis.

Avian pathogenic Escherichia coli (APEC) is the causative agent of colibacillosis. APEC causes significant economic losses to the poultry industry. In this study, 66 E. coli isolates were collected from broiler flocks across Mississippi and categorized as clinical and non-clinical isolates. Genotypic and phenotypic characterization of virulence factors and antimicrobial resistance profiles of these E. coli were examined. The data disclosed a higher prevalence of virulence genes in clinical isolates than in non-clinical isolates. High differences on the genotypic and phenotypic antimicrobial resistance among clinical and non-clinical isolates were observed. Whole genome sequences of avian E. coli elaborated a diverse range in genetic composition, phylogenic relationship, antimicrobial resistance, and virulence gene profiles. Collectively, the results showed that the virulence factors and phenotypic characteristics of APEC may play a role in the pathogenesis of avian colibacillosis. Therefore, this study provides insight to understand the epidemiological background, microbial behavior, and pathogenesis of APEC.

Acinetobacter baumannii is an increasingly multidrug-resistant pathogen contributing to hospital-acquired infections necessitating the discovery of novel treatments. A bacterial second messenger, cyclic diguanosine monophosphate (cyclic di-GMP), can regulate various persistence factors that are potentially advantageous for survival in hospital environments. Cyclic di-GMP–modulating enzymes and cyclic di-GMP–binding effectors predictively are encoded in the Acinetobacter baumannii genome. I hypothesized that cyclic di-GMP controls motility, biofilm formation, and desiccation tolerance in Acinetobacter baumannii. Disrupting cyclic di-GMP–modulating enzymes or cyclic di-GMP–binding effectors should alter the regulatory effectiveness of these phenotypes. I tested the multidrug-resistant isolate Acinetobacter baumannii strain AB5075 and identified several transposon mutants that altered twitching motility, biofilm formation, and desiccation tolerance; these results suggest that cyclic di-GMP plays a role during these three responses in Acinetobacter baumannii AB5075. Inhibiting these cyclic di-GMP signaling pathways could produce novel mechanisms to combat this pathogen in the hospital environment.

Bacteria have developed various means of secreting proteins that can enter the host cell membrane. In this work I focus on two systems: cholesterol-dependent cytolysins and Type III Secretion. Cholesterol is a molecule that is critical for physiological processes and cell membrane function. Not only can improper regulation lead to disease, but also the role cholesterol plays in cell function indicates it is an important molecule to understand. In response to this need, probes have been developed that detect cholesterol molecules in membranes. However, it has been recently shown that there is a need for probes that only respond to cholesterol that is accessible at the membrane surface. Perfringolysin O (PFO) is a toxin secreted by Clostridium perfringens that has been developed into a probe capable of detecting accessible cholesterol. Recently, researchers have been expanding the capabilities of this probe by substituting residues, modifying residues, truncating the probe, or a combination of the three. However, lack of characterization of these new probes has led to controversial results. To understand the role of a conserved Cys residue, here we perform cholesterol binding assays and measure the pore formation activity of a Cys modified PFO derivative. The Type III Secretion (T3S) system is a syringe-like apparatus used by various pathogens to inject effector proteins into target cells. The apparatus spans both the inner and outer bacterial membrane, extending to make contact with the host cell where it forms a pore known as the translocon. In Pseudomonas aeruginosa, the translocon is made up of two proteins, PopB and PopD. While recent advances have been made on the structure of the needle and injectisome, information on the translocon remains sparse. In this work, the P. aeruginosa T3S translocon is analyzed using both in vivo and in vitro methods.

<p>Recreational beaches are important local resources for attracting tourists. It is critical to keep tracking recreational water quality to prevent public health issues. Waterborne pathogens are one of the main elements that could cause recreational water related diseases. Fecal pollution is the primary source of waterborne pathogens. Therefore, it is important to quantify the amount of fecal pollution indicators that are present in the water, particular the human fecal indicator. The primary objective of this thesis is to develop an integrative microbial quality monitoring system to better understand water quality. The first part of this thesis examined the presence of a general fecal pollution indicator (<em>E. coli</em>) and a human fecal pollution indicator (human-specific <em>Bacteroidales</em>). The correlations between pollution sources and beach water quality were also studied to identify the impact of pollution sources. The results revealed the highly localized correlations at individual beaches depended on the impact from pollution sources. The weak correlations suggested some previous assumed pollution sources may only weakly impacted beach water quality.</p> <p>Because <em>E. coli</em> strains differ enormously in pathogenic potential, it is possible that environmental <em>E. coli</em> have different genetic compositions and differential gene expression in genes such as the global stress regulator <em>rpoD</em> and <em>rpoS</em>. Thus, the second part of this thesis examined genetic composition and gene expression in <em>E. coli</em> environmental strains to study how global gene expression is altered in the natural environment. The results revealed differential RpoSexpression levels in environmental <em>E. coli </em>strains, suggesting that genes regulated by <em>rpoD</em> and <em>rpoS</em> may have differential expression levels in environmental strains, compared to commonly studied laboratory strains.</p> / Master of Science (MSc)

<p>Bacteria demonstrate an extraordinary capacity to survive and adapt to changing environments. In part, this ability to adapt can be attributed to horizontal gene transfer, a phenomenon which introduces novel genetic information that can be appropriated for use in particular niches. Nowhere is this more relevant than in pathogenic bacteria, whose acquisition of virulence genes have provided an arsenal that permits them to thrive within their selected host. Regulatory evolution is necessary for timely regulation of these acquired virulence genes in the host environment. <em>Salmonella enterica</em> serovar Typhimurium is an intracellular pathogen which possesses numerous horizontally-acquired genomic islands encoding pathogenic determinants that facilitate its host lifestyle. One island, <em>Salmonella</em> Pathogenicity Island (SPI)-2, encodes a type-III secretion system (T3SS) which is regulated by the two-component regulatory system SsrA-SsrB. This system coordinates expression of the SPI-2 T3SS as well as an array of virulence effectors encoded in horizontally-acquired regions throughout the <em>Salmonella</em> genome. The studies presented here investigated the mechanisms in which the transcription factor SsrB functions to integrate virulence processes through regulatory adaptation. This work identified the regulatory logic controlling SsrB and defined the associated SsrB regulon. Furthermore, SsrB was found to induce a regulatory cascade responsible for the expression of bacteriophage genes encoded within SPI-12, an island that also contributes to <em>Salmonella</em> virulence. These findings demonstrate the important contribution of regulatory evolution in pathogen adaptation to the host, and show that horizontally-acquired genes, once integrated into appropriate regulatory networks, can contribute to pathogen fitness in specific niche environments.</p> / Doctor of Philosophy (PhD)

Acinetobacter baumannii is an increasingly multidrug-resistant pathogen contributing to hospital-acquired infections, necessitating a greater understanding of how it interacts with its surroundings. Many bacteria utilize different methods of bacterial motility to move about and interact with these surroundings. A bacterial second messenger, cyclic diguanosine monophosphate (c-di-GMP), can regulate various motility factors that are potentially advantageous for survival in and adaptation to their environment. Concentrations of c-di-GMP are regulated by specific synthesizing and degrading enzymes. Controlled levels of c-di-GMP allow interaction between the c-di-GMP and its binding effectors that induce changes in bacterial phenotypes such as biofilm formation and motility. A search of the A. baumannii genome identified two proteins that contain the c-di-GMP-binding PilZ domain. The PilZ protein for which this PilZ domain was named was initially discovered in Pseudomonas aeruginosa where it has been demonstrated to be a part of the type IV pilus machinery. Type IV pili play roles in twitching motility, adhering to surfaces, DNA uptake, protein secretion, and predation. One of the PilZ-containing proteins from A. baumannii resembled this original PilZ protein (PilZ), while the second PilZ-containing protein contained a hydrolase domain with unknown substrate specificity (HydP). I investigated whether these PilZ-containing proteins play a role in motility of A. baumannii by testing two strains: AB5075 that displays twitching motility, and ATCC17978 that displays an uncharacterized form of surface-associated motility. Results suggest PilZ plays a role in twitching motility, while its effect on surface-associated motility phenotypes3 is possibly due to polar effects from mutation. Results also suggest HydP plays a role in surface-associated motility, although its mechanism is not understood. Testing of both proteins’ PilZ domains indicates they may not bind c-di-GMP, implying they may be playing roles in motility regulation through other mechanisms outside of binding c-di-GMP. These findings give us greater insight into the regulatory mechanisms used by A. baumannii to move about its environment.