No description available.

Thesis (doctoral)--Katholieke Universiteit te Nijmegen.

No description available.

Chlamydia is the most prevalent sexually transmitted bacterial infection in many developed countries, including Canada. Untreated infections in women can lead to a number of complications including pelvic inflammatory disease, tubal factor infertility, and ectopic pregnancy. Public health programs, including screening for at-risk individuals, partner identification, and antibiotic treatment, have had limited success in controlling the rising incidence of chlamydial infections over the past two decades. A chlamydia vaccine that prevents infection and its pathological sequelae is the next essential step to control this persistent public health problem. Chlamydia spp. utilize the highly conserved type III secretion (T3S) system as an essential virulence factor for infection and intracellular replication. Here, we evaluated a novel chimeric antigen (BD584) consisting of three T3S proteins from C. trachomatis (CopB, CopD, and CT584) as a potential chlamydia vaccine candidate. Intranasal immunization with BD584 elicited strong humoral responses that neutralized infection in vitro. Following intravaginal challenge with C. muridarum, immunized mice had a 95% reduction in chlamydial shedding and a 87.5% reduction in incidence of upper genital tract pathology compared to control mice. BD584 immunization generated strong cell-mediated and mucosal antibody responses in mice with different genetic backgrounds, and conferred protection against an intravaginal C. trachomatis infection in two out of three strains of mice. BD584 formulated with NE01, a mucosal adjuvant known to be safe and effective in humans, was shown to be highly immunogenic and efficacious against C. trachomatis infection in mice. These results suggest that BD584 may represent a promising antigen for use in a chlamydia vaccine. / Thesis / Doctor of Philosophy (PhD) / Chlamydia is the most common sexually transmitted bacterial infection in the world. The goal of this thesis is to evaluate a novel chlamydia vaccine in a mouse model of genital chlamydia infection. We engineered a fusion protein, BD584, made up of three highly conserved type III secretion (T3S) proteins CopB, CopD, and CT584. We show that vaccination with BD584 generated strong immune responses and protected mice from chlamydia infection and the associated reproductive tract disease. Interestingly, the level of protection afforded by BD584 vaccination is dependent upon the genetic background of the animal. Furthermore, we have identified particular antibody subtypes directed against BD584 as markers of BD584-mediated protective immunity. Lastly, we show that vaccination with BD584 formulated with a clinically safe and effective mucosal adjuvant generates robust immune responses and confers protection against chlamydia in mice. Together, these results provide support for the use of T3S proteins in a chlamydia vaccine.

Bacteria are under constant threat by their viral parasites, the bacteriophages (phages) and have evolved a range of anti-phage systems to defend themselves. One of these systems is termed abortive infection (Abi) where, upon phage infection, an Abi system may be activated which initiate a bacteriostatic or bactericidal response. While the infected bacteria do not obviously benefit from the activation of these systems, the cessation of bacterial growth or premature cellular suicide prevents the release of phage progeny. Thus Abi can be viewed as an altruistic process as only the remaining clonal bacterial population benefits. The Type III toxin-antitoxin systems have previously been shown to be involved in Abi, however the mechanisms through which these systems are activated are still poorly understood. A common approach to reveal the phage product involved in triggering these systems is to first determine the mutations that a previously sensitive phage evolves to escape after exposure to an Abi system. Analysis of viral "escape" mutants has been used in this study to try to elucidate the activation mechanism(s) of two Type III systems (ToxIN$_P$$_a$ and TenpIN$_P$$_l$) of several environmental phages. Several new phage products were identified in escape mutants as candidate factors involved in circumventing Abi - and possible roles in phage metabolism predicted. Furthermore, the genomes of several phages that could not evolve escapes, or were insensitive to Abi, are sequenced and these data exposed interesting curiosities regarding Abi (as well as the discovery of several novel and rare phages). Previously, no coliphage was identified that was capable of escape of the ToxIN$_P$$_a$ or TenpIN$_P$$_l$ systems. However, this study defined and characterised the first ToxIN$_P$$_a$ and TenpIN$_P$$_l$ coliphage escapes as well as a new method for isolating host-dependent coliphage escapes. Finally, multiple phages that infect the insect pathogen $\textit{Photorhabdus luminescens}$ TT01 (the bacterial strain from which the TenpIN$_P$$_l$ system originated) were isolated, genomically sequenced and characterised in terms of host range. The results revealed a large superfamily of flagellum-dependent phages that exhibit remarkable host promiscuity, possibly defining the most promiscuous phages thus far identified.

A plasmid-encoded Type III Secretion System (T3SS) is employed by human pathogenic yersiniae to inject effector proteins, termed Yops, directly into host cells. The secretion of Yops is tightly regulated, and occurs only upon contact with a eukaryotic cell in vivo or in media devoid of calcium in vitro. A complex containing the secreted protein YopN, its heterodimeric chaperone SycN/YscB, and TyeA is required to prevent secretion of effector Yops until the appropriate secretion-triggering signals are encountered. The mechanism by which these proteins regulate the T3S process is unknown. A mutational analysis of YopN and TyeA was performed to identify regions and residues of these proteins that are required to regulate Yop secretion. Amino-acid residues of TyeA were identified that were specifically required for the interaction of TyeA with YopN, confirming that the YopN/TyeA interaction is essential for the regulation of Yop secretion. Furthermore, analysis of TyeA mutants identified a surface-exposed region that was critical for the regulation of Yop secretion, but not required for interaction with YopN. YopN residues critical for the regulation of secretion clustered within the N- and C-terminal regions of YopN that were required to interact with the SycN/YscB chaperone and TyeA, respectively. No residues critical for the regulation of secretion were identified in the central region of YopN, suggesting that this region acts primarily to maintain proper positioning of the functional N- and C-terminal regions of this complex. A novel role for the chaperone binding domain (CBD) of YopN in the regulation of Yop secretion was identified. This role was separate from its role in binding the SycN/YscB chaperone and targeting YopN for secretion. Finally, it was demonstrated that the SycN/YscB chaperone is dispensable for the regulation of secretion if the expression of both YopN and TyeA is increased, indicating that these chaperones have no direct role in the regulation of Yop secretion. These results indicate that the YopN secretion signal and SycN/YscB chaperone function to efficiently target the YopN/TyeA complex to the T3S apparatus, whereas the YopN CBD and C-terminal region of YopN complexed with TyeA mediate the block in Yop secretion.

<i>Salmonella enterica</i> serovar Enteritidis (<i>S.</i> Enteritidis) is a major cause of gastrointestinal disease in humans worldwide that is mainly associated with the consumption of contaminated poultry meat and eggs. During the course of infection, <i>S.</i> Enteritidis uses two Type 3 Secretion Systems (T3SS), one of which is encoded by <i>Salmonella</i> Pathogenicity Island-1 (SPI-1). SPI-1 plays a major role in the invasion process.<p> In order to study the role of SPI-1 in the colonization of chickens, we constructed deletion mutants affecting either the complete SPI-1 region (40 kb) or <i>invG</i>, a single gene located on this pathogenicity island. The mutants were impaired in the secretion of effector proteins and were less invasive compared to the wild type strain in polarized Caco-2 cells. Similarly, when chicken cecal and small intestinal explants were co-infected with the wild type and ÄSPI-1 mutant strains we found that the ÄSPI-1 mutant strain was less invasive relative to the wild type strain. Oral challenge of 1-week-old chickens with the wild type or ÄSPI-1 strains demonstrated that there was no difference in chicken cecal colonization. However, systemic infection, measured as levels of <i>Salmonella</i> in the liver and spleen, was delayed in birds that were challenged with the ÄSPI-1 strain. This demonstrates that SPI-1 facilitates systemic infection but is not essential for invasion and systemic spread of S. Enteritidis in chickens.<p> Based on the above results, we examined the effect of sera against SPI-1 T3SS components to <i>S.</i> Enteritidis invasion. Anti-SipD serum protected Caco-2 cells against entry of wild type <i>S.</i>Enteritidis, but not against invasion of a mutant strain lacking sipD. On the other hand, sera against InvG, PrgI, SipA, SipC, SopB, SopE and SopE2 did not affect S. Enteritidis entry. To illustrate the specificity of anti-SipD mediated inhibition, SipD specific antibodies were depleted from the serum. Depleted serum restored the invasion of S. Enteritidis, demonstrating that the SipD protein may be an important target in blocking SPI-1 mediated virulence.<p> To determine if SPI-1 T3SS proteins were protective against <i>S.</i> Enteritidis oral challenge, chickens were vaccinated subcutaneously twice at 14 and 28 days of age with PrgI and SipD. The results indicate that these proteins induce strong IgG antibody responses and confer significant protection against infection of the livers in vaccinated birds. In another study, we vaccinated hens with selected SPI-1 T3SS proteins to determine if their progeny could be protected from <i>S.</i> Enteritidis oral challenge. The proteins induced strong antibody responses but did not affect the levels of the challenge strain in the ceca or internal organs of the vaccinates. Taken together, our results establish that <i>S.</i> Enteritidis SPI-1 is an important virulence factor in chickens and that the proteins associated with this T3SS may form components of a subunit vaccine used for protection against colonization by <i>S.</i> Enteritidis in poultry.

<i>Salmonella enterica</i> serovar Enteritidis (<i>S.</i> Enteritidis) is a major cause of gastrointestinal disease in humans worldwide that is mainly associated with the consumption of contaminated poultry meat and eggs. During the course of infection, <i>S.</i> Enteritidis uses two Type 3 Secretion Systems (T3SS), one of which is encoded by <i>Salmonella</i> Pathogenicity Island-1 (SPI-1). SPI-1 plays a major role in the invasion process.<p> In order to study the role of SPI-1 in the colonization of chickens, we constructed deletion mutants affecting either the complete SPI-1 region (40 kb) or <i>invG</i>, a single gene located on this pathogenicity island. The mutants were impaired in the secretion of effector proteins and were less invasive compared to the wild type strain in polarized Caco-2 cells. Similarly, when chicken cecal and small intestinal explants were co-infected with the wild type and ÄSPI-1 mutant strains we found that the ÄSPI-1 mutant strain was less invasive relative to the wild type strain. Oral challenge of 1-week-old chickens with the wild type or ÄSPI-1 strains demonstrated that there was no difference in chicken cecal colonization. However, systemic infection, measured as levels of <i>Salmonella</i> in the liver and spleen, was delayed in birds that were challenged with the ÄSPI-1 strain. This demonstrates that SPI-1 facilitates systemic infection but is not essential for invasion and systemic spread of S. Enteritidis in chickens.<p> Based on the above results, we examined the effect of sera against SPI-1 T3SS components to <i>S.</i> Enteritidis invasion. Anti-SipD serum protected Caco-2 cells against entry of wild type <i>S.</i>Enteritidis, but not against invasion of a mutant strain lacking sipD. On the other hand, sera against InvG, PrgI, SipA, SipC, SopB, SopE and SopE2 did not affect S. Enteritidis entry. To illustrate the specificity of anti-SipD mediated inhibition, SipD specific antibodies were depleted from the serum. Depleted serum restored the invasion of S. Enteritidis, demonstrating that the SipD protein may be an important target in blocking SPI-1 mediated virulence.<p> To determine if SPI-1 T3SS proteins were protective against <i>S.</i> Enteritidis oral challenge, chickens were vaccinated subcutaneously twice at 14 and 28 days of age with PrgI and SipD. The results indicate that these proteins induce strong IgG antibody responses and confer significant protection against infection of the livers in vaccinated birds. In another study, we vaccinated hens with selected SPI-1 T3SS proteins to determine if their progeny could be protected from <i>S.</i> Enteritidis oral challenge. The proteins induced strong antibody responses but did not affect the levels of the challenge strain in the ceca or internal organs of the vaccinates. Taken together, our results establish that <i>S.</i> Enteritidis SPI-1 is an important virulence factor in chickens and that the proteins associated with this T3SS may form components of a subunit vaccine used for protection against colonization by <i>S.</i> Enteritidis in poultry.

Blood vessel growth is mediated by angiogenesis, which is defined as the formation of new blood vessel out of existing vessels, as well as vasculogenesis, a process that circulating progenitor cells contributes to adult neovascularization. Cilostazol, a commercially available drug holding antiplatelet and vasodilating effects, increases intracellular cyclic adenosine monophosphate (cAMP) levels through inhibiting the activity of phosphodiesterase 3. Interestingly, this chemical compound has a lot of cellular effects. In current work, we demonstrated that cilostazol promoted proliferation and migration of human umbilical cord vein endothelial cells (HUVECs), enhanced in-vitro vascular tube formation, and increased releasing of cAMP and NO from them. Furthermore, cilostazol activated eNOS and PI3-K/Akt signaling pathways. We also examined the angiogenic and vasculogenic effects of cilostazol in a murine hindlimb ischemia model. Our data showed that cilostazol enhanced angiogenesis and vasculogenesis with resultant flow recovery after murine hindlimb ischemia partly mediated by promoting mobilization of bone marrow-derived stem cells into circulation and increasing in situ expression of some proteins involved in angiogenesis. In addition, cilostazol significant increased colony forming unit of human endothelial progenitor cells. These results are unique and clinically significant with potential in translational therapy. According to our report, further preclinical and clinical studies of cilostazol on the other ischemic situations such as myocardial infarction will be justified.

Successful bacterial pathogens manipulate crucial intracellular host processes as a virulence strategy. One particular potent mechanism utilized by bacterial phytopathogens is to inject virulence factors (effectors) directly into the host cell. While many effectors have been identified and shown to suppress plant immune responses, very few have well-characterized enzymatic activities or host targets. To overcome the challenges of functional analysis of effectors, I designed two heterologous screens to characterize effector proteins of the bacterial phytopathogen Pseudomonas syringae. Specifically, my objective was to identify those P. syringae effectors that target evolutionarily conserved host proteins or processes and to subsequently elucidate the molecular mechanisms of these effectors. The first heterologous screen that I performed was to utilize tandem-affinity-purification (TAP)-tagged effectors in human cells to identify potential interacting host proteins. The second heterologous screen iii utilized a high-throughput genomics approach in yeast, known as the pathogenic genetic array (PGA), to characterize P. syringae effectors. Using the first heterologous approach, I have identified HopZ1a as the first bacterial phytopathogen effector that binds tubulin. I have shown that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor, phytic acid. In vitro, activated HopZ1a acetylates itself and tubulin. In Arabidopsis thaliana, activated HopZ1a causes microtubule destruction, disrupts the secretory pathway and suppresses cell wall-mediated defense. The acetyltransferase activity of HopZ1a is dependent on the conserved catalytic cysteine residue (C216) and a conserved lysine residue (K289). Using the second heterologous screen in yeast, I have shown that HopZ1a may target the mitogen-activated protein kinase (MAPK) signaling cascades. Together, my work has identified novel eukaryotic targets and elucidated the virulence functions of HopZ1a.