Iron is an essential nutrient that is sequestered by iron-binding proteins in the human host resulting in a hostile environment for microbes. Neisseria gonorrhoeae, however, can utilize numerous iron-binding proteins such as transferrin and lactoferrin to acquire this nutrient. During initial infection, gonococci have access to transferrin and lactoferrin present in semen and vaginal fluids, as well as to hemoglobin present in blood during menses or disseminated infections. Consequently, the gonococcus likely encounters conditions of high iron at some stages in the course of natural infection. Potential contributions of iron to gonococcal invasion have however been largely over looked in the field as most studies investigating invasion represent iron depleted environments. Considering the link between menses in women and ascending gonococcal infections, we hypothesized that high iron concentrations present at this time triggers the induction of membrane proteins that enhance gonococcal pathogenesis. Here, we report the gonococcal iron-induced surface proteome and show evidence of post-translational modification of many of these proteins. We also present evidence of an iron enhanced, Opa-independent invasion mechanism. Finally, we investigated the role of NspA, TdfJ and NGO1063 on Opa-independent iron induced invasion. Our studies underscore the importance of investigating the effect of iron on gonococcal host cell interactions. Given the potential clinical relevancy of this phenomenon, data from our studies represent a solid framework for further investigation of gonococcal pathogenesis.

<p>Originally considered the sole providence of protein coding sequences, evolutionary biology has begun to recognize the importance of non-coding DNA in dictating phenotypic adaptation. Exclusively examined in eukaryotic anatomical development, <em>cis</em>-regulatory modifications have the power to alter the spatial-temporal dynamics of gene expression without the plieotropic consequences of protein modification. Owing to the need to integrate horizontally acquired DNA into existing regulatory networks, <em>cis</em>-regulatory mutations may also significantly contribute to prokaryotic evolution. The horizontal acquisition of <em>Salmonella</em> Pathogenicity Island (SPI)-2 led to the evolutionary divergence of <em>Salmonella enterica</em> from <em>S. bongori</em>. Use of the type 3 secretion system encoded in SPI-2 allowed <em>S. enterica</em> to exploit an intracellular host niche offered by immune cells and allowed for its systemic dissemination. Here we identify ancestrally encoded <em>srfN</em> and <em>dalS</em> and demonstrate that through acquisition of a binding site for the SPI-2 regulator, SsrB, they have contributed to the pathoadaptation of <em>S. enterica</em> to the host environment. We also demonstrate that ancestral regulatory networks contribute to the establishment of an expression hierarchy for SPI-2 <em>in vitro</em> and to transcriptional priming in the host lumen prior to invasion. These findings demonstrate that <em>cis</em>-regulatory modifications have significantly contributed to the evolution of <em>S. enterica</em> as an intracellular pathogen.</p> / Doctor of Philosophy (PhD)

Obligate intracellular bacteria are significant causes of morbidity and mortality with over two hundred and fifty million infections worldwide annually. One such bacterium, Anaplasma phagocytophilum is the etiologic agent of human granulocytic anaplasmosis (HGA), a tick-transmitted febrile illness. Previous studies have shown that A. phagocytophilum lacks genes for cholesterol biosynthesis and solely relies on Niemann Pick protein type C (NPC)1-mediated low density lipoprotein (LDL)-derived cholesterol to complete its infection cycle.Acid sphingomyelinase (ASMase) is a lysosomal enzyme that is essential for diverse cellular processes including liberation of LDL-derived cholesterol from the lysosome. By first studying A. phagocytophilum, we found that functional inhibitors of acid sphinogmyelinase (FIASMAs) arrest the bacterium’s infection cycle in a dose-dependent manner. FIASMAs inhibit vacuole maturation, conversion to the infectious form, and eliminate the production of infectious progeny. NPC1-mediated LDL-derived cholesterol traffic to the ApV is abrogated in the presence of FIASMAs. Similar to the in vitro model, A. phagocytophilum cannot establish a productive infection in both ASMase-/-and FIASMA treated mice. Furthermore, we extended our studies to Coxiella burnetti (Q fever), and Chlamydia spp. (STD, infectious blindness, pneumonia). FIASMA treatment has a rapid bacteriocidal effect on C. burnettiwithin host cells. Additionally, FIASMA treatment inhibits C. trachomatis and C. pneumoniae inclusion expansion and infectious progeny generation, with C. pneumoniae being more severely impacted. These data highlight the critical, yet distinct roles that ASMase plays in these pathogens’ infection cycles. Furthermore, these results signify the therapeutic potential of FIASMAs for treating diseases caused by these pathogens.

Members of the Staphylococcus genus are found as a part of normal microflora in humans and can commonly be found on the skin or in the nasal cavity. However, these microorganisms can cause serious and life-threatening opportunistic infections when there is a break in the physical barrier of skin. These infections have become difficult to treat as resistant strains emerge, particularly Methicillin Resistant Staphylococcus aureus (MRSA). MRSA has become a commonly acquired nosocomial infection which is difficult to treat with conventional antibiotics of the blactam class. Even Vancomycin, a last resort antibiotic, has been ineffective on some infections. Furthermore, S. aureus readily forms biofilms on implanted medical devices which establishes a hardy and difficult to treat infection. These biofilms serve as a point of infection to the bloodstream. Research involving polymicrobial interactions and the inhibitory effects of bacterial-bacterial interactions could be a starting point for the discovery of a new therapeutic treatment for infections. It has been shown in our lab that Alcaligenes faecalishas inhibitory effects on Staphylococcus aureusplanktonic growth. Therefore, in this study, we wanted to examine 1) The mechanism by which A. faecalisinhibitsS. aureus growth and 2) how A. faecalisimpacts the various phases of S. aureusbiofilm growth. It was found that A. faecalislikely inhibits S. aureususing a physical mechanism that requires close contact, rather than using a secreted molecule. However, a Type VI secretion system could also produce similar results. Further research involving the formation of mutants to find the gene allowing A. faecalisto inhibit S. aureuswas started, but no viable mutants were created during the course of this research. A. faecaliswas found to inhibit the formation of S. aureus biofilm growth, but when added to a mature S. aureusbiofilm, the slow growth rate of A. faecaliscould not overtake the quickly replicating S. aureus. Further research in the polymicrobial interactions between S. aureus and A. faecaliscould lead to a finding of a new therapeutic target for antibiotics or the use of A. faecalisin infections.

Toxoplasma gondii is the causative agent of Toxoplasmosis in human and animals. T. gondii isolates are highly diverse. Hundreds of genotypes have been identified, but only three clonal lineages, namely Type I, II and III are prevalent worldwide. In mouse model, T. gondii strains can be divided into three groups based on their virulence, including the virulent (LD100=1), the intermediately virulent (LD50 = 103-104) and the non virulent (LD50 > 105). The clonal Type I, II and III T. gondii strains belong to these three groups, respectively. Epidemiologic studies suggest the difference of virulence in mice may relate to the severity of toxoplasmosis in human infection. Therefore, it is necessary to understand biological differences in genetically different T. gondii strains and their effect on the host responses. To date, the majority of data published on this aspect has been limited to in vitro assays. Here, we used in vivo assays to investigate host responses upon infection of distinct Toxoplasma strains. Our studies examined host response to infection of the three widespread clonal lineages of T. gondii using a mouse model. The following results were revealed: (i) increased tissue burden in mice is the indicator of virulence of T. gondii. Quantification of parasite burden in the spleen of mice showed significantly more parasites for Type I strain than that of Type II and III strains, with the latter two having comparable parasite burdens. Given that the Type II strains are more virulent than the Type III strains in mice; this result suggests that difference in host response is the result of specific parasite-host interaction, which is not simply due to the difference of parasite tissue load. (ii) gene expression in the host is strongly influenced by parasite genetic background. Transcriptional profiles of mice infected with the above three types of T. gondii strains showed that the overall gene expression patterns are similar between Type I and Type II infected mice and both stimulated stronger and more polarized change comparing to Type III strain. These results emphasize the importance of studying T. gondii pathogenesis in the host with the consideration of parasite genetic diversity. Such research could possibly aid in select appropriate regimes to treat toxoplasmosis caused by diverse T. gondii strains.

<p>The <em>Streptococcus</em> Milleri Group (SMG) asymptomatically colonize the gastrointestinal, female urogenital, and upper respiratory tract in the healthy population, and are therefore traditionally considered commensals. The SMG, however, are also pathogens that cause pyogenic and pulmonary infections. The factors that differentiate pathogenic from non-pathogenic isolates have proven difficult to identify, and consequently the determinants of SMG pathogenicity remain unknown. Characterization of the immune response to the SMG is important towards advancing the understanding of SMG pathogenicity, however there are limited studies that have done so.</p> <p>Herein, we sought to investigate the cytokine profiles produced by human peripheral blood mononuclear cells in response to 35 clinical isolates of the SMG. Cytokine profiles varied across isolates resulting in a spectrum of responses that separated into three subgroups including a high, intermediate, and low response group. The responses were consistent across three individuals with the exception of several differences, which are discussed and warrant further studies on host susceptibility to SMG infections. The high and intermediate response groups were enriched with clinical isolates from invasive infections, which were found to induce significantly higher cytokine production than airway isolates. Cytokine induction was independent of TLR2 activation, suggesting that other pattern recognition receptors are involved in the recognition of and response to the SMG. Phenotypic characteristics, which are used in the clinical identification of the SMG, did not correlate with cytokine induction; therefore phenotypic tests are not sufficient to identify immunostimulatory isolates. The host response to the SMG characterized in this study provides foundational knowledge for future studies to investigate the mechanism of recognition as well as the function of downstream effector responses in the control of colonization and infection.</p> / Master of Science (MSc)

<p>Crohn’s Disease is a chronic inflammatory bowel disease characterized by an overzealous immune response to a microbial trigger in genetically susceptible individuals. Although this microbial trigger is unknown, <em>Escherichia coli</em> with adherent and invasive properties (Adherent-Invasive <em>Escherichia coli</em>, AIEC) is preferentially enriched in a proportion of Crohn’s Disease patients. AIEC can adhere to and invade intestinal epithelial cells and replicate intracellularly within epithelial cells and macrophages <em>in vitro</em>. One important colonization factor expressed by AIEC is the type 1 fimbrial adhesin protein FimH. FimH mediates colonization of CEABAC10 transgenic mice and can bind several host cell receptors including the macrophage receptor CD48 <em>in vitro</em> indicating a potential role for FimH in macrophage interaction. However, it was not known whether FimH contributed to phagocytosis of AIEC or colonization of wild-type mice. Here we show that FimH enhances early intracellular AIEC levels <em>in vitro</em> and colonization <em>in vivo</em>. We found that deletion of <em>fimH</em> may reduce intracellular AIEC burden at 2 hours post-infection and that this effect was modulated by bacteria opsonisation. Using a competitive index assay, we show that a Δ<em>fimH</em> mutant is unable to chronically colonize CD-1 mice at the same levels as the parental strain. Our results demonstrate that FimH is an important AIEC colonization factor and may increase interaction with macrophages. Identifying factors such as FimH which contribute to colonization and persistence will further our understanding of AIEC survival strategies within the host. Development of therapeutics targeting FimH may provide a means to reduce harmful bacteria overgrowth particularly after surgical intervention.</p> / Master of Science (MSc)

Abstract ORIENTIA TSUTSUGAMUSHI ANKYRIN REPEAT-PROTEIN FAMILY TARGETING OF THE HOST ENDOPLASMIC RETICULUM By Lauren VieBrock, B.S. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University Virginia Commonwealth University, 2015 Director: Jason A. Carlyon, Ph.D. Professor Microbiology and Immunology Scrub typhus is an understudied, potentially fatal febrile illness, which poses threat to one billion people annually in the Asia-Pacific region. The host-pathogen interactions that facilitate the intracellular survival of the etiologic agent, Orientia tsutsugamushi, are not well understood. The Orientia tsutsugamushi genome encodes a large number of ankyrin repeat-containing proteins (Anks), key virulence factors for other intracellular pathogens, as well as components for Type I (T1SS) and Type 4 secretion systems (T4SS), commonly used to deliver them. We sought to characterize the roles of the Anks in O. tsutsugamushi infection. In this study, we demonstrated that O. tsutsugamushi expressed all 20 anks and the genes for the T1SS, for which they are substrates. Many ectopically expressed Anks displayed a tropism for the host endoplasmic reticulum (ER). These results suggest the importance of the Anks and the ER to Orientia tsutsugamushi pathobiology. We demonstrated that O. tsutsugamushi tightly associated with the ER and induced ER stress and defects in protein secretion of its host cells. Therefore, we hypothesized that the ER-tropic anks expressed during the initial hours of infection are critical for establishing infection and do so by interacting with specific host cell targets to modulate host cell function to benefit intracellular survival. ER-tropic Ank4 was detected as expressed early in infection and was further characterized for its contribution to the alterations of the ER during infection. Bat3 was identified as a target of Ank4, and Ank4 expression correlated with a decrease in Bat3 protein levels, induction of ER stress, and defects in protein secretion. These effects were Ank4 F-box dependent, implicating polyubiquitination and proteosomal degradation of Bat3. As Ank4 colocalized with Bat3, a chaperone component of ER-associated degradation (ERAD) of misfolded proteins, ERAD function was measured in cells expressing Ank4. In an F-box dependent manner, Ank4 expression resulted in decreased degradation of a model substrate and indicated inhibition of the ERAD pathway. Similarly, we demonstrated that in O. tsutsugamushi infection, Bat3 levels were significantly reduced early in infection and ERAD degradation was inhibited. After several days of infection however, Bat3 levels and ERAD degradation had both recovered, suggesting temporal modulation of ERAD in infection. Taken together, these data suggest that O. tsutsugamushi has a large capacity to disrupt the host ER, exemplified by Ank4 mediated ERAD dysfunction by depletion of host Bat3.

Pseudomonas aeruginosa (PA) infections in Cystic Fibrosis (CF) patients are not easily cleared due to the conversion from a nonmucoid to a mucoid phenotype. Alginate is an acetylated exopolysaccharide produced by mucoid PA that is responsible for increased resistance to antibiotics, host phagocytic killing, and propagating biofilm formation. Understanding the interaction between PA and host cells is critical to understanding chronic infection and inflammation in CF. In order to investigate this, we used A549 pulmonary epithelial cells and murine alveolar macrophages (MH-S) to examine host response to nonmucoid versus mucoid PA infection. Adhesion assays in A549 pulmonary epithelial cells revealed that mucoid PA mutants adhere poorly compared to their nonmucoid counterparts. Similarly, phagocytosis assays using MH-S infected with PA revealed that mucoid PA are increasingly resistant to phagocytosis. The alginate acetylation mutant FRD1175 is more susceptible to phagocytic killing than alginate+ FRD1. Adherence and phagocytosis of mucoid FRD1 was increased by increasing the multiplicity of infection (MOI) from 50:1 to 500:1. Furthermore, confocal microscopy revealed that mucoid PA are inherently less inflammatory than nonmucoid strains in both A549 and MH-S. Increasing the MOI of mucoid FRD1 from 50:1 to 500:1 significantly increased caspase-1 activation in MH-S but not in A549, revealing that intensity of inflammatory signaling by epithelial cells is likely independent of increased adherence. FRD1175 infection in both A549 and MH-S revealed that alginate acetylation plays a significant role in reducing inflammasome activation. Western analysis revealed that PA does not actively induce TGF-β secretion by A549 epithelial cells. Similarly, NF-κB expression was reduced in both A549 and MH-S when infected with mucoid FRD strains, but not PA from the PAO background, suggesting FRD strains have accumulated additional mutations facilitating escape of inflammation. MH-S treated with cytochalasin D to block phagocytosis were still able to activate NF-κB signaling, suggesting NF-κB activation is adherence but not phagocytosis dependent. These data increase our understanding of the various mechanisms in which mucoid PA is able to evade host immune defenses and provides insight into potential therapies to treat PA infections.

Pseudomonas aeruginosa is a bacterial pathogen that can infect any human tissue. The lungs of cystic fibrosis patients become chronically infected with Pseudomonas aeruginosa. Virulence factor gene expression is under elaborate regulatory control that remains poorly characterized. Understanding the regulatory hierarchy involved during infection is essential for identifying novel drug targets. RsmA is a post-transcriptional regulatory protein that controls expression of several virulence factors. Previous studies demonstrated alginate regulatory components AlgU and AlgR as regulators of rsmA expression. The aim of this study was to determine how AlgR controls rsmA expression. Western blot analysis of HA-tagged RsmA confirmed lower RsmA levels in an algR mutant. An electrophoretic mobility shift assay using purified AlgR demonstrated direct binding of AlgR to the rsmA promoter. These results indicate AlgR directly controls rsmA expression. We propose a mechanism whereby AlgR and AlgU work together to regulate rsmA.