Shiga toxin-producing <i>E. coli</i> (STEC) is an important food- and water-borne pathogen of humans, causing Hemorrhagic Colitis and Haemolytic Uremic Syndrome. Colonization of both cattle and human hosts is mediated through the action of effector molecules secreted via a type III secretion system (T3SS), which forms attaching and effacing lesions (A/E). The necessary effectors which form A/E by manipulation of host signalling and actin nucleation are present on a pathogenicity island called the Locus of Enterocyte Effacement (LEE).<p> It has been reported that vaccination of cattle with Type III-secreted proteins (T3SPs) from STEC O157 resulted in decreased shedding. In order to extend this to non-O157 STEC serotypes, we examined the serological cross-reactivity of T3SPs of serotypes O26:H11, O103:H2, O111:NM and O157:H7. Groups of cattle were vaccinated with T3SPs produced from each of the serotypes and the magnitude and specificity of the responses were measured resulting in limited cross reactivity. Overall, results suggest that vaccination of cattle with T3SPs as a means of reducing the risk of STEC transmission to humans will induce protection that is serotype specific.<p> To pursue the possibility of a cross-protective vaccine, we investigated the protective properties of a chimeric Tir protein against STEC serotypes. Several studies have reported that Tir is highly immunogenic and capable of producing high antibody titers. Potter and colleagues also demonstrated that the vaccination of cattle with ∆tir STEC O157 strain did not protect as well as the wildtype strain. We constructed thirty-mer peptides to the entire STEC O157 Tir protein, as well as to the intimin binding domain of the Tir protein from STEC serotype O26, O103 and O111. Using sera raised against STEC O157 and non-O157 T3SPs, we identified a number of immunogenic peptides containing epitopes unique to a particular serotype. Two different chimeric Tir proteins were constructed containing the STEC O157 Tir protein fused with six STEC non-O157 peptides with or without the Leukotoxin produced by <i>Mannheimia haemolytica</i>. However, the vaccination of mice with the chimeric protein did not protect against challenge with STEC O157 or STEC O111. These results suggest that to achieve cross protection against STEC serotypes using a recombinant protein vaccine, other immunogenic and protective antigens must also be included.<p> In order to identify other immunogenic and cross-protective antigens we cloned and expressed the genes coding for 66 effectors and purified each as histidine-tagged proteins. These included 37 LEE-encoded proteins and 29 non-LEE effectors. The serological response against each protein was measured by Western blot analysis and an enzyme-linked immunosorbent assay (ELISA) using sera from rabbits immunized with T3SPs from four STEC serotypes, experimentally infected cattle and human sera from 6 HUS patients. A total of 20 proteins were recognized by at least one of the STEC T3SP- vaccinated rabbits using Western blots. Sera from experimentally infected cattle and HUS patients were tested using an ELISA against each of the proteins. Tir, EspB, EspD, EspA and NleA were recognized by the majority of the samples tested. Overall, proteins such as Tir, EspB, EspD, NleA and EspA were highly immunogenic for both vaccinated and naturally infected subjects.<p> Based on the above results, two different mixtures of secreted proteins (5 proteins and 9 proteins) were used to vaccinate mice and test the level of shedding following challenge with STEC O157. Overall, the cocktail vaccine containing 9 immunogenic effectors including Tir, EspB, EspD, NleA and EspA was capable of reducing shedding as effectively as the current STEC T3SPs vaccine, Econiche®.

Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) has been identified as a significant cause of salmonellosis in humans. Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) each encode a specialized type III secretion system (T3SS) that enables Salmonella to manipulate host cells at various stages of the invasion/infection process. The SPI-2 T3SS has been identified as vital for survival and replication of S. Typhimurium and S. Enteritidis in mouse macrophages, as well as full virulence in mice. In order to test the ability of SE SPI-2 mutants to survive in vivo we used a chicken isolate of SE (Sal18). In one study, we orally co-challenged 35-day-old specific pathogen free (SPF) chickens with two bacterial strains per group. The control group received two versions of the wild-type (WT) strain Sal18: Sal18 attTn7::tet and Sal18 attTn7::cat, while the other two groups received the WT strain (Sal18 attTn7::tet) and one of two mutant strains (Sal18 attTn7::cat ÄspaSÄssaU or Sal18 ÄSPI-1ÄSPI-2::cat). From this study we conclude that S. Enteritidis deficient in the SPI-1 and SPI-2 systems are out-competed by the WT strain. In a second study, groups of SPF chickens were challenged at 1 week of age with four different strains; a WT strain and three other strains missing either one or both of the SPI-1 and SPI-2 regions. On days 1 and 2 post-challenge (PC) we observed a reduced systemic spread of the SPI-2 mutants, but by day 3 the mutants systemic distribution levels matched that of the WT strain. Based on these two studies, we conclude that the SPI-2 T3SS facilitates invasion and systemic spread of S. Enteritidis in chickens, but alternative mechanisms for these processes appear to exist. Several structural components of the T3SSs encoded by SPI-1 and SPI-2 are exposed to the hosts immune system prior to/during the infection/invasion process, making them potential vaccine candidates. Several of these candidates genes were cloned, the proteins overproduced, purified, and formulated as vaccines for use in further studies. SPI-2 T3SS proteins used for vaccine studies included the secretin, SsaC, the needle, SsaG, the filament, SseB, and a part of the translocon, SseD, as well as a number of effectors, SseI, SseL, SifA, and SifB. The first vaccine study involved vaccination of SPF chickens with SseB and SseD, followed by challenge with the WT S. Enteritidis strain Sal18. Additional studies evaluated whether hens vaccinated with SPI-2 T3SS structural or effector components could mount a significant humoral immune response (as measured by serum immunoglobulin Y [IgY] titres), whether these antibodies could be transferred to progeny (as measured by egg yolk IgY titres), and whether vaccinates and progeny of vaccinates could be protected against challenge with the WT S. Enteritidis strain Sal8. The results of our studies show that vaccinated chickens do produce high levels of SPI-2 T3SS specific serum IgY that they are able to transfer to their progeny. It was demonstrated that vaccinates and progeny of vaccinates had lower overall countable recovered SE per bird in most situations. In order to better identify the role of the SPI-2 T3SS in chickens, we used the well-known gentamicin protection assay with activated HD11 cells. HD11 cells are a macrophage-like chicken cell line that can be stimulated with phorbol 12-myristate 13-acetate (PMA) to exhibit more macrophage-like morphology and greater production of reactive oxygen species (ROS). Activated HD11 cells were infected with a WT S. Typhimurium strain, a SPI-2 mutant S. Typhimurium strain, a WT S. Enteritidis strain, a SPI-2 mutant S. Enteritidis strain, or a non-pathogenic Escherichia coli (E. coli) strain. SPI-2 mutant strains were found to survive as well as their parent strain at all time points post-infection (PI) up to 24 h PI, while the E. coli strain was no longer recoverable by 3 h PI. We can conclude from these observations that the SPI-2 T3SS is not important for survival of Salmonella in the activated macrophage-like HD11 cell line, and that Salmonella must employ other mechanisms for survival in this environment as E. coli is effectively eliminated.

Pseudomonas aeruginosa causes various infections in humans, animals and plants. Type III secretion system (T3SS) is one of the essential virulence factors used by P. aeruginosa. In this study, a previously uncharacterized gene PA0466 and its role in T3SS regulation have been examined. The results indicate that PA0466 is a novel T3SS regulator. It regulates T3SS directly through an unknown pathway and has a minor effect on the GacA-RsmA pathway. Besides the role in the interaction between the pathogen and the host, T3SS may also play a role in the interspecies interaction. A real-time PCR based Competitive Index (CI) assay was used to compare the wild type and T3SS mutant with and without the presence of Staphylococcus spp.. The results indicate that PAO1 was more competitive than exsA mutant and the difference was even bigger in the presence of Staphylococcus, suggesting T3SS may play a significant role in bacterium-bacterium interaction.

Doctor of Philosophy / Department of Plant Pathology / Xiaoyan Tang / P. syringae is a group of bacterial phytopathogens that can infect a wide variety of plants. These bacteria rely on the type III secretion system (TTSS) to deliver effectors into plant cells for infection. The TTSS genes, that encode the TTSS apparatus and the effectors, are repressed when bacteria grow in nutrient rich media but are strongly induced in the plants and in minimal medium (MM). Plant cutin monomers appear to negatively regulate the P. syringae TTSS genes. It is poorly understood how bacteria sense the environmental signals to regulate the TTSS genes. By genetic screen, four sets of transposon insertion mutants displaying aberrant TTSS gene expression were isolated: KB and fin mutants derepress the TTSS genes in rich medium KB and in the presence of a cutin monomer precursor in MM, respectively; min and pin mutants are defective in induction of TTSS genes in MM and in plants, respectively. A putative two-component sensor histidine kinase, RohS, is identified to be required for the induction of avrPto-LUC in MM and in plants. The rohS gene is in an operon containing a two-component response regulator gene rohR. Mutation of rohS in P. s. phaseolicola and P. s. tomato reduced the bacterial pathogenicity on hosts and HR-inducing activity on non-hosts. Our results suggested that RohS acts upstream of HrpR/HrpS. The phosphorylated RohR represses TTSS genes. It is likely that RohS acts as phosphatase of RohR in the TTSS-inducing conditions, and subsequently derepresses TTSS genes. Simple sugars such as glucose, sucrose and fructose are known to be inducers of the TTSS genes. Isolation of four min mutants defective in fructose-uptake enabled us to study if sugars serve as extracellular signals or as essential nutrients. Our results suggest that fructose acts as an essential nutrient for the activation of type III genes. These mutants slightly compromised induction of avrPto promoter in Arabidopsis and pathogenicity on the host bean plant, but displayed normal HR elicitation on non-host plant tobacco. The reduced pathogenicity suggested that exploitation of fructose from the host tissue is an important means for pathogenesis of P. s. phaseolicola.

Orientador: Rodrigo Tavanelli Hernandes / Resumo: O principal mecanismo de virulência de Escherichia coli enteropatogênica (EPEC) é a capacidade de causar uma lesão histopatológica na mucosa intestinal denominada attaching and effacing (AE), caracterizada pela aderência íntima das bactérias, destruição das microvilosidades e formação de estruturas semelhantes a pedestais, ricos em F-actina, nos enterócitos infectados. Genes do locus of enterocyte effacement (região LEE) codificam todas as proteínas necessárias para a formação da lesão AE. As EPEC são divididas em típicas (tEPEC) e atípicas (aEPEC), com base na presença do EPEC adherence factor plasmid no primeiro grupo. A partir de um conjunto de sete amostras de aEPEC pertencentes ao sorotipo O2:H16, obtidos de surtos e casos esporádicos de diarreia, mostramos que cinco deles produziram uma adesão híbrida localizada/difusa (AL/AD) em células HeLa. Neste estudo, uma amostra de aEPEC deste sorotipo (282/14), que produziu o padrão AL/AD, foi selecionada para investigar as estruturas bacterianas envolvidas em seu fenótipo adesivo. Para este propósito, a amostra de aEPEC 282/14 foi mutagenizada usando o kit EZ::TN <R6Kyori/KAN-2> Tnp transposome kit, gerando uma biblioteca de inserções Tn5. Esses mutantes de inserção Tn5 foram testados quanto a perda ou redução da capacidade aderente, em ensaios realizados em 6 h de incubação com células HeLa. Dentre 320 clones pesquisados, nove foram considerados deficientes em sua capacidade de interagir com células epiteliais e quatro deles a... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The main virulence mechanism of enteropathogenic Escherichia coli (EPEC) is the capacity to cause a histopathological lesion on the intestinal mucosa, termed Attaching and Effacing (AE); characterized by intimate bacterial adherence, microvillus destruction and formation of F-actin rich pedestal-like structures, in infected enterocytes. Genes of the locus of enterocyte effacement (LEE region) encode all proteins necessary for AE lesion formation. EPEC are divided in typical (tEPEC) and atypical (aEPEC), based on the presence of the EPEC adherence factor plasmid in the former group. From a collection of seven aEPEC O2:H16, obtained from outbreak and sporadic cases of diarrhea, we showed that five of them produced a hybrid localized/diffuse adherence (LA/DA) in HeLa cells. In this study, we selected one aEPEC isolate of this serotype (282/14) that produced the LA/DA pattern, to investigate the bacterial structures involved in its adhesive phenotype. For this purpose, aEPEC 282/14 was mutagenized using the EZ::TN < R6Kyori/KAN-2 > Tnp transposome kit, generating a library of Tn5 insertions. These Tn5 insertion mutants were screened for non-adherent or less adherent mutants, in assays performed in 6 h of incubation with HeLa cells. Among the 320 clones screened, nine were considered deficient in their ability to interact with epithelial cells, and four of them presented the Tn5 insertion in genes within the LEE region, such as tir, escV, and grlR. In order to confirm the role of ... (Complete abstract click electronic access below) / Doutor

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a Gram-negative facultative anaerobe that induces severe inflammation resulting in gastroenteritis. In the case of S. Typhimurium infection, induction of an inflammatory response has been linked to its primary virulence mechanism, the type III secretion system (T3SS). The T3SS secretes protein effectors that exploit the host’s cell biology to facilitate bacterial entry and intracellular survival, and to modulate the host immune response. One such effector, SifA, is a bi-functional T3SS effector protein that plays an important role in Salmonella virulence. The N-terminal domain of SifA binds SifA-Kinesin-Interacting-Protein (SKIP), and via an interaction with kinesin, forms tubular membrane extensions called Sif filaments (Sifs) that emanate from the Salmonella Containing Vacuole (SCV). The C-terminal domain of SifA harbors a WxxxE motif that functions to mimic active host cell GTPases. Taken together, SifA functions in inducing endosomal tubulation in order to maintain the integrity of the SCV and promote bacterial dissemination. Since SifA performs multiple, unrelated functions, the objective of this study was to determine how each functional domain of SifA becomes processed. In the present study, we demonstrate that a linker region containing a caspase-3 cleavage motif separates the two functional domains of SifA. To test the hypothesis that processing of SifA by caspase-3 at this particular site is required for function and proper localization of the effector protein domains, we developed two tracking methods to analyze the intracellular localization of SifA. We first adapted a fluorescent tag called phiLOV that allowed for T3SS mediated delivery of SifA and observation of its intracellular colocalization with caspase-3. Additionally, we created a dual-tagging strategy that permitted tracking of each of the SifA functional domains following caspase-3 cleavage to different subcellular locations. The results of this study reveal that caspase-3 cleavage of SifA is required for the proper localization of functional domains and bacterial dissemination. Considering the importance of these events in Salmonella pathogenesis, we conclude that caspase-3 cleavage of effector proteins is a more broadly applicable effector processing mechanism utilized by Salmonella to invade and persist during infection.

Indiana University-Purdue University Indianapolis (IUPUI) / Cystic fibrosis (CF) is a fatal, incurable genetic disease that affects over 30,000 people in the United States alone. People with this disease have a homozygous mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) which causes defects in chloride transport and leads to build up of mucus in the lungs and disruption of function in various organs. CF patients often suffer from chronic bacterial infections within the lungs, wherein the bacteria persist as a biofilm, leading to poor prognosis. Two of these pathogens, Stenotrophomonas maltophilia and Pseudomonas aeruginosa, are often found in the lungs of patients with CF and are an increasing medical concerns due to their intrinsic antimicrobial resistance. Both species can readily form biofilms on biotic and abiotic surfaces such as intravascular devices, glass, plastic, and host tissue. Biofilm formation starts with bacterial attachment to a surface and/or adjacent cells, initiating the acute infection stage. Chronic, long-term infection involves subsequent or concurrent altered genetic regulation, including a downregulation of virulence factors, resulting in the bacteria committing to a sessile lifestyle, markedly different from the planktonic one. Many of these genetic switches from an acute to chronic lifestyle are due to pressures from the host immune system and lead to permanently mutated strains, most likely an adaptive strategy to evade host immune responses. Biofilms are extremely problematic in a clinical setting because they lead to nosocomial infections and persist inside the host causing long-term chronic infections due to their heightened tolerance to almost all antibiotics. Understanding the genetic networks governing biofilm initiation and maintenance would greatly reduce consequences for CF and other biofilm-related infections and could lead to the development of treatments and cures for affected patients. This study showed that in S. maltophilia, isogenic deletion of phosphoglycerate mutase (gpmA) and two chaperone-usher pilin subunits, S. maltophilia fimbrae-1 (smf-1) and cblA, lead to defects in attachment on abiotic surfaces and cystic fibrosis derived bronchial epithelial cells (CFBE). Furthermore, Δsmf-1 and ΔcblA showed defects in long-term biofilm formation, mimicking that of a chronic infection lifestyle, on abiotic surfaces and CFBE as well as stimulating less of an immune response through TNF-α production. This study also showed that in P. aeruginosa, the Type III secretion system (T3SS), an important virulence factor activated during the acute stage of infection, is downregulated when polB, a stress-induced alternate DNA polymerase, is overexpressed. This downregulation is due to post-transcriptional inhibition of the master regulatory protein, ExsA. Taken together, this project highlights important genes involved in the acute and chronic infection lifestyle and biofilm formation in S. maltophilia and genetic switches during the acute infection lifestyle in P. aeruginosa.

Salmonella can reside in healthy animals without the manifestation of any adverse effects on the carrier. If raw products of animal origin are not handled properly during processing or cooked to a proper temperature during preparation, salmonellosis can occur. In this research, microbial aspects related to Salmonella as a food pathogen are investigated. A bioluminescent reporting system was developed for Salmonella to monitor the attachment and growth of the pathogen on food products. Twelve and eleven Salmonella strains from the broiler production continuum were tagged with bioluminescence by plasmid and integration of the lux operon into the chromosome, respectively. To assess the usefulness of bioluminescent Salmonella strains in food safety studies, an attachment model using chicken skin was developed. Variables including washing and temperature were tested in the attachment model to determine the effects on attachment of Salmonella strains to chicken skin, a characteristic that enhances persistence during processing. Additionally, the invasion process for two serovars of Salmonella with differing host tropism was examined with emphasis on the initial establishment of the bacterium in the host. The major facilitator for invasion, type III secretion system, was inactivated through deletion mutation to evaluate invasion of human epithelial cell line by additional means. The difference in host tropism between the two subspecies of Salmonella was also taken into account when evaluating invasion. Results showed that invasion of human epithelial cells can be initiated despite inactivation of the type III secretion system. A serovar of Salmonella that is not typically associated with human illness was also shown to initiate invasion of human epithelial cells, a result that carries public health implication as this serovar has recently been shown to be multi-drug resistant.

No description available.

Problem: Shigella is a gram-negative enteropathogen that, when passed through fecal particles from one host to the oral cavity of another host, causes an infectious disease known as shigellosis. One of the distinctive features of the infection by Shigella is its ability to bypass its host’s autophagic defenses. It does this through the use of a Type III secretion system, found in gram-negative pathogens like Shigella, which injects virulent proteins into the host cell. One of these proteins is IcsB; however, its exact function is not well understood. This study aims to better understand the role of this protein in the infection. Methods: A yeast two-hybrid screening test is used in this case to examine the interactions between variations of the protein IcsB, and a library of host proteins. Given IcsB’s high yeast toxicity and that resulted in the total absence of yeast colony formation, the first aim was to identify IcsB variants which expression would not prevent yeast growth. The second aim was to use the mutant with reduced cytotoxicity to perform a Y2H screen that will allow for the identification of candidate host proteins interacting with IcsB. Results: Two mutations of the IcsB protein grew in the Y2HG yeast strain, indicating a significant reduction in the protein’s toxicity. Of the cultures that reacted, high stringency and strong interaction was observed between four genes and IcsB proteins. Among the four identified clones that grew, three corresponded to the gene RNF2, while the last one corresponds to a non-coding sequence. Key control experiments revealed that the interaction of IcsB with RNF2 is likely false-positive. Thus, when screened full-length IcsB using new epithelial cells cDNAVI libraries, strong interaction was observed between three genes and our IcsB proteins. All the three genes DDX3X, FANCL, and SGT1 passed the false-positive interaction tests. It is interesting to notice that DDX3X and SGT1 interacted with catalytically active and inactive IcsB, suggesting that the interactions established between IcsB and prey proteins does not require the catalytic - C306A mutation and that IcsB most likely does not function as a protease against these two proteins. By contrast, FANCL bound catalytically inactive, but not catalytically active IcsB, suggesting it could be a substrate of IcsB. The literature provides some support for the putative role of DDX3X, FANCL, and SGT1 in regulating the vacuole escape of Shigella through IcsB action. Conclusion: The aim of this study was to determine the functional of IcsB in the vacuole escape of Shigella. This study successfully identified three candidates interacting partner proteins for IcsB. Key control experiments confirmed the interaction of IcsB with DDX3X, FANCL and SGT1. This study provides a basis for further research, with further study aimed at confirming these results during Shigella infection