Pseudomonas syringae is a pathogen of numerous plant species, including several economically important crops. P. syringae pv. syringae B728a is a resident on leaves of common bean, where it utilizes several well-studied virulence factors, including secreted effectors and toxins, to develop a pathogenic interaction with its host. The B728a genome was recently sequenced, revealing the presence of 1,297 genes with unknown function. This dissertation demonstrates that a 29.9-kb cluster of genes in the B728a genome encodes a novel secretion pathway, the type VI secretion system (T6SS), that functions to deliver at least one protein outside of the bacterial cell. Western blot analyses show that this secretion is dependent on clpV, a gene that likely encodes an AAA+ ATPase, and is repressed by retS, which apparently encodes a hybrid sensor kinase. RetS and a similar protein called LadS are shown to collectively modulate several virulence-related activities in addition to the T6SS. Plate assays demonstrate that RetS negatively controls mucoidy, while LadS negatively regulates swarming motility. A mutation in retS affects B728a population levels on the surface of bean leaves. A model for the LadS and RetS control of B728a virulence activities is proposed, and possible roles for the B728a T6SS are addressed.

F. tularensis is a Gram-negative bacterial pathogen and it is the causative agent of tularemia. It has the ability to replicate to high numbers within a variety of host cells, including macrophages. Little is known of its virulence mechanisms; however, all species of Francisella contain a cluster of virulence genes known as the Francisella Pathogenicity Island (FPI), which is thought to encode a type 6 secretion system. While 14 of the 18 FPI genes encode products required for intracellular growth in macrophages, the functions of most of these proteins remain to be determined. Therefore, further work is required to understand the role played by the FPI in Francisella pathogenesis. In this thesis, the localization of the core FPI proteins IglA, IglB, IglC and IglD, was examined in order to further elucidate of the structure and activities of the FPI-encoded secretion system. Deletion mutagenesis of pdpA was performed to determine how host intracellular signalling might be affected by secretion of the putative FPI effector protein PdpA. In addition, variations in virulence between different biotypes of Francisella were investigated with respect to the role played by the FPI protein PdpD. Considering the highly infectious nature of Francisella and the absence of a quality vaccine, it is clear that this organism represents an excellent model for proof of principle investigations focussing on new vaccine technologies for intracellular pathogens. The second half of this thesis describes the construction and characterization of live attenuated temperature-sensitive vaccines. These vaccines were created in the intracellular pathogen F. novicida through allelic replacement of essential genes with naturally-occurring, cold-adapted, thermolabile homologues isolated from Arctic bacteria. Thus, the objectives of this work were twofold: to provide further characterization of the structural components and effector proteins associated with the FPI-encoded secretion system, and to develop a new and effective vaccine technology for use against intracellular bacteria. / Graduate

No description available.

Nematodes are highly diverse animals capable of interacting with almost every other form of life on Earth from general trophic interactions to intimate and persistent symbiotic associations. Much of their recognition originates from their various parasitic lifestyles. From an agricultural standpoint, plant parasitic nematodes are widely known for the destruction they can cause to crop plants, such as the case of the root-knot nematode Meloidogyne incognita, or livestock animals, like the Trichinella spiralis, which infects pigs and other animals. From a human health perspective, nematodes can cause many debilitating diseases, for example Wuchereria bancrofti, which is a causative agent of lymphatic filariasis or elephantiasis. However, not all parasitic nematodes have bad implications for human health. For instance, the diverse interactions of insect parasitic nematodes can be used to our benefit. Many of these species have been considered as biological control alternatives to different insect pests that wreak havoc on human, animal, and plant health. There still remain many questions surrounding their evolution, ecology, and physiological capabilities. Many of these taxa are hard to cultivate in the lab due to their complex and intimate lifestyles. Entomopathogenic nematodes (EPNs) are of great interest in agriculture because they vector insect pathogenic bacteria, which are capable of causing death to an insect host within 48 hours post-infection. Much of the molecular underpinnings in this system still remain to be discovered, from understanding the basic ability of these two organisms to associate with one another to genetically engineering more robust and host specific pathogens for application in the field. The focus of the research presented herein is on Steinernematidae nematodes and their bacterial symbionts. Specifically, it focused on the relationship between Xenorhabdus bovienii and its Steinernema hosts. Bioassays were designed to investigate insect virulence of X. bovienii alone in two Lepidoptera insect species with known differential susceptibility to Steinernema-Xenorhabdus pairs. A comparative genomic analysis was performed to compare different Xenorhabdus bovienii strains with observed variation in insect virulence. Results from this analysis demonstrated that virulent strains possess a type VI secretion system (T6SS) locus that is completely absent in strains with attenuated virulence. Bacterial competition assays between T6SS+ and T6SS- strains suggest this locus is involved in bacterial competition. Additionally, symbiont preference assays were carried out to investigate whether Steinernema hosts are able to discern between virulent and attenuated X. bovienii strains. Results from these assays revealed that Steinernema nematodes are able to distinguish between cognate and non-cognate X. bovienii symbionts, giving preference to virulent strains over those with attenuated virulence. Altogether these results provide further evidence that supports the notion that symbiont-switching events have occurred over the Steinernema-Xenorhabdus co-evolutionary history. Specifically, the competitive virulence of certain X. bovienii strains may have conferred them the ability to be selected by different Steinernema hosts, therefore contributing to the success of the nematode-bacterium partnership in being pathogenic to diverse insect hosts.

Bacterial pathogenesis relies on a subset of mechanisms including adhesion to various matrices, antibiotic resistance, defence and action against surrounding microorganisms, and secretion of virulence factors. Among the secretion systems, the recently identified Type VI secretion system (T6SS) has been shown to be involved in both virulence against eukaryotic cells and inter-bacterial warfare. T6SS are composed of a minimum of 13 proteins called "core components". It is believe to form a macromolecular system that spans the envelope to assemble an extracellular structure composed of the Hcp protein with a trimer of VgrG located at the tip. This model has been built following in silico and structural analyses demonstrating the link between several T6SS subunits and bacteriophage T4 baseplate and tail elements. Other T6SS subunits include membrane proteins. Using enteroaggregative Escherichia coli as a bacterial model, the aim of my work is to understand how this system assembles in the cell envelope. I recently showed that four of these membrane proteins, SciP, SciS, SciN and SciZ make contact to form a complex [1]. These four subunits are critical components of the T6SS. I then delineated the interaction network, demonstrating that SciZ interacts with SciP, and that SciS interacts with both SciP and SciN. Further characterization of these subunits showed that SciN is a lipoprotein associated with the outer membrane [2, 4], whereas SciP and SciS are inner membrane proteins anchored through a single and three transmembrane segments respectively. SciZ is a polytopic inner membrane protein carrying a peptidoglycan-binding motif within its periplasmic domain. Mutagenesis and peptidoglycan binding experiments demonstrated that SciZ anchors the T6SS to the cell wall [1, 3]. Overall, we have identified and characterized a trans-envelope complex anchored in both membrane and to the peptidoglycan layer. / Bacterial pathogenesis relies on a subset of mechanisms including adhesion to various matrices, antibiotic resistance, defence and action against surrounding microorganisms, and secretion of virulence factors. Among the secretion systems, the recently identified Type VI secretion system (T6SS) has been shown to be involved in both virulence against eukaryotic cells and inter-bacterial warfare. T6SS are composed of a minimum of 13 proteins called "core components". It is believe to form a macromolecular system that spans the envelope to assemble an extracellular structure composed of the Hcp protein with a trimer of VgrG located at the tip. This model has been built following in silico and structural analyses demonstrating the link between several T6SS subunits and bacteriophage T4 baseplate and tail elements. Other T6SS subunits include membrane proteins. Using enteroaggregative Escherichia coli as a bacterial model, the aim of my work is to understand how this system assembles in the cell envelope. I recently showed that four of these membrane proteins, SciP, SciS, SciN and SciZ make contact to form a complex [1]. These four subunits are critical components of the T6SS. I then delineated the interaction network, demonstrating that SciZ interacts with SciP, and that SciS interacts with both SciP and SciN. Further characterization of these subunits showed that SciN is a lipoprotein associated with the outer membrane [2, 4], whereas SciP and SciS are inner membrane proteins anchored through a single and three transmembrane segments respectively. SciZ is a polytopic inner membrane protein carrying a peptidoglycan-binding motif within its periplasmic domain. Mutagenesis and peptidoglycan binding experiments demonstrated that SciZ anchors the T6SS to the cell wall [1, 3]. Overall, we have identified and characterized a trans-envelope complex anchored in both membrane and to the peptidoglycan layer.

Bacteria use quorum sensing, a cell to cell signaling mechanism mediated by small molecules that are produced by specific signal molecule synthases, to regulate gene expression in response to population density. In Vibrio anguillarum, the quorum-sensing phosphorelay channels information from three hybrid sensor kinases VanN, VanQ, CqsS that sense signal molecules produced by the synthases VanM, VanS and CqsA, onto the phosphotransferase VanU, to regulate activity of the response regulator VanO. VanO activates transcription of quorum-sensing regulatory RNAs (Qrr), which work together with the RNA chaperone Hfq to repress expression of the transcriptional regulator VanT. The work presented in this thesis characterizes quorum-sensing independent and quorum-sensing dependent mechanisms that regulate VanT expression. Moreover, an in vivo imaging system was established, as a means to study V. anguillarum infections in the rainbow trout infection model. Two quorum-sensing independent mechanisms regulating VanT expression were identified. First, the sigma factor RpoS indirectly activates VanT expression during transition into stationary growth phase by inhibiting hfq expression. Both, RpoS and VanT are crucial for stress response. Second, a type VI secretion system (T6SS) has a novel function as a signal sensing mechanism to regulate rpoS and vanT expression. Consequently, RpoS, quorum sensing and T6SS form a global network that senses stress and modulates stress response to ensure survival of the bacteria. Further analysis of the quorum-sensing dependent regulation of VanT expression by the phosphorelay system revealed that four qrr genes are expressed continuously during growth. The phosphotransferase VanU is suggested to activate two response regulators, VanO and a predicted second response regulator. Activated VanO induces expression of the Qrr sRNAs, whereas, the predicted response regulator represses expression of the Qrr sRNAs. Thus, VanU has a pivotal role in the regulation of VanT expression. The signal synthase VanM and VanT form a regulatory loop, in which VanM represses VanT by inducing expression of the Qrr sRNAs and VanT directly activates vanM expression to repress its own expression. Moreover, Hfq destabilizes vanM mRNA, repressing vanM expression. VanT forms another regulatory loop with the transcriptional regulator LuxT, in which LuxT activates vanT expression and VanT directly represses luxT expression. V. anguillarum is an opportunistic pathogen that causes vibriosis, a terminal hemorrhagic septicemia. The spatial and temporal progression of the infection was analyzed using the whole animal with an in vivo bioluminescent imaging method. Initial studies showed that colonization of the fish skin requires the siderophore, the RNA chaperone Hfq and the exopolysaccharide transport system, which protects against the innate immunity on the skin. Colonization of the fish skin is crucial for disease.

Intracellular bacteria have developed various mechanisms to enter and persist in host cells and, at the same time, to evade the host immune response. One such pathogen is Francisella tularensis, the etiological agent of tularemia. After phagocytosis, this Gram-negative bacterium quickly escapes from the phagocytic compartment and replicates in the host cell cytosol. For this mode of infection, several components of the Francisella pathogenicity island (FPI) are critical. Interestingly, some FPI proteins share homology to components of Type VI Secretion Systems (T6SSs), but their assembly and functionality remains to be shown in Francisella.The thesis focused on the characterization of several of these FPI components; more specifically, how they contribute to the infection cycle as well as their possible role in the putative T6SS. We identified three unique mutants, ΔiglG, ΔiglI and ΔpdpE, which to various degrees were able to escape the phagosomal compartment, replicate in the host cytosol and cause host cell cytotoxicity. In contrast, ΔiglE as well as mutants within the conserved core components of T6SSs, VgrG and DotU, were defective for all of these processes. In the case of IglE, which is a lipoprotein and localized to the outer membrane of the bacterial cell wall, residues within its N-terminus were identified to be important for IglE function. Consistent with a suggested role as a trimeric membrane puncturing device, VgrG was found to form multimers. DotU stabilized the inner membrane protein IcmF, in agreement with its function as a core T6SS component. The functionality of the secretion system was shown by the translocation of several FPI proteins into the cytosol of infected macrophages, among them IglE, IglC and VgrG, of which IglE was the most prominently secreted protein. At the same time, the secretion was dependent on the core components VgrG, DotU but also on IglG. Although we and others have shown the importance of FPI proteins for the escape of F. tularensis, it has been difficult to assess their role in the subsequent replication, since mutants that fail to escape never reach the growth-permissive cytosol. For this reason, selected FPI mutants were microinjected into the cytosol of different cell types and their growth compared to their replication upon normal uptake. Our data suggest that not only the metabolic adaptation to the cytosolic compartment is important for the replication of intracytosolic bacteria, but also the mechanism of their uptake as well as the permissiveness of the cytosolic compartment per se.

Orientadores: Wanderley Dias da Silveira, Eliana Guedes Stehling / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-17T23:32:04Z (GMT). No. of bitstreams: 1 Pace_Fernandade_D.pdf: 4667016 bytes, checksum: c841e6b899f0efc5569c9c6c6ce46935 (MD5) Previous issue date: 2011 / Resumo: Linhagens de Escherichia coli patogênica para aves (APEC) causam infecções extraintestinais e são responsáveis por significativas perdas econômicas na indústria avícola mundial. Recentemente, foram descritos isolados de APEC geneticamente relacionados a diversas outras E.coli extraintestinais (ExPEC) de origem humana, indicando a possibilidade das mesmas constituírem risco zoonótico para humanos. Alguns dos conhecidos fatores de virulência de APEC incluem adesinas, sistema de aquisição de ferro, citotoxinas, entre outros. Nesse trabalho, demonstramos que a linhagem de APEC SEPT 362, isolada do fígado de uma ave apresentando sinais clínicos de septicemia, expressa o Sistema de Secreção Tipo VI (SST6), causa rearranjo do citoesqueleto de células epiteliais cultivadas in vitro, é capaz de aderir e invadir células HeLa e é viável dentro de macrófagos. Para estudar o envolvimento do SST6 na patogênese da linhagem SEPT362, foram deletados três genes desse sistema: hcp, que codifica para uma proteína estrutural e secretada, clpV, que codifica para uma ATPase e icmF (intracellular multiplication factor), gerando três mutantes, respectivamente. Todos os mutantes demonstraram uma diminuição nos processos de adesão e invasão a células HeLa, formação de biofilme e virulência in vivo. Estudos de transcriptoma mostraram que a expressão da fímbria tipo 1 encontra-se diminuída nesses mutantes, o que poderia ser responsável pela diminuição do processo de adesão e invasão às células epiteliais. Nesse trabalho, demonstramos que o SST6 é importante para o processo de patogenicidade, visto que todos os mutantes tiveram sua virulência atenuada em experimentos realizados in vivo com uma significativa diminuição de características relacionadas à patogenicidade in vitro. Esses resultados demonstram que os genes estudados do SST6 influenciam a expressão da fímbria tipo 1 e contribuem para a patogênese desta linhagem APEC / Abstract: Avian pathogenic Escherichia coli (APEC) strains frequently cause extraintestinal infections and are responsible for significant economic losses in the poultry industry worldwide. APEC isolates are closely related to human extraintestinal pathogenic E. coli (ExPEC) strains and may also act as pathogens for humans. Known APEC virulence factors include adhesins such as type 1 fimbriae and curli, iron acquisition systems, and cytotoxins, among others. Here we demonstrated that APEC strain SEPT362, isolated from a septicemic hen, expresses a type VI secretion system (T6SS), causes cytoskeleton rearrangements, invades epithelial cells, replicates within macrophages, and causes lethal disease in chicks. To assess the contribution of the T6SS to SEPT362 pathogenesis, we generated three mutants, ?hcp (which encodes a protein suggessed to be both secreted and a structural component of the T6SS), ?clpV (encoding the T6SS ATPase) and ?icmF (intracellular multiplication factor). All mutants showed decreased adherence and invasion to HeLa cells and decrease in several other pathogenicity related characteristics. Transcriptome studies showed that the level of expression of type 1 fimbriae was decreased in these mutants, which may account for the diminished adhesion and invasion of epithelial cells. The T6SS seems to be important for the disease process, given that both mutants (?hcp and ?clpV) were attenuated in an infection model in chicks. These results suggest that the T6SS influences the expression of type 1 fimbriae and contributes to the pathogenesis of this APEC strain pathogenesis / Doutorado / Genetica de Microorganismos / Doutor em Genetica e Biologia Molecular

The type VI secretion system (T6SS) is a protein export pathway that mediates competition between Gram-negative bacteria by facilitating the injection of toxic effector proteins from attacking cells into target cells. To function properly, many T6SSs require at least one protein that possesses a proline-alanine-alanine-arginine (PAAR) domain. These PAAR domains are often found within large, multi-domain effectors that possess additional N- and C-terminal extension domains whose function in type VI secretion is not well understood. The work described herein uncovers the function of these accessory domains across multiple PAAR-containing effectors. First, I demonstrated that thousands of PAAR effectors possess N-terminal transmembrane domains (TMDs) and that these effectors require a family of molecular chaperones for stability in the cell prior to their export by the T6SS. Our findings are corroborated by co-crystal structures of chaperones in complex with the TMDs of their cognate effectors, capturing the first high-resolution structural snapshots of T6SS chaperone-effector interactions. Second, I characterize a previously undescribed prePAAR effector named Tas1. My work shows that the C-terminus of Tas1 possesses a toxin domain that pyrophosphorylates ADP and ATP to synthesize the nucleotides adenosine penta- and tetraphosphate (hereafter referred to as (p)ppApp). Delivery of Tas1 into competitor cells drives the rapid accumulation of (p)ppApp, depletion of ADP and ATP, and widespread dysregulation of essential metabolic pathways, resulting in target cell death. These findings reveal a new mechanism of interbacterial antagonism, the first characterization of a (p)ppApp synthetase and the first demonstration of a role for (p)ppApp in bacterial physiology. TMD- and toxin-containing PAAR proteins constitute a large family of over 6,000 T6SS effectors found in Gram-negative bacteria. My work on these proteins has uncovered that different regions found within effectors have distinct roles in trafficking between bacterial cells and in the growth inhibition of the target cell. / Dissertation / Doctor of Philosophy (PhD) / Bacteria constantly compete with their neighbours for resources and space. The type VI secretion system is a protein complex that facilitates competition between Gram-negative bacteria by facilitating the injection of protein toxins, also known as effectors, from attacking cells into target cells. In this work, I characterize several members of a large family of membrane protein effectors. First, I showed that these effectors require a novel family of chaperone proteins for stability and recruitment to the type VI secretion system apparatus. Second, I characterized the growth-inhibitory properties of one of these effectors in-depth and showed that it possesses a toxin domain that depletes the essential nucleotides ATP and ADP in target cells by synthesizing the nucleotides adenosine penta- and tetraphosphate, (p)ppApp. Together, these studies revealed a new mechanism for the intercellular delivery of membrane protein toxins and uncovered the first known physiological role of a (p)ppApp-synthesizing enzyme in bacteria.

Francisella tularensis is a highly virulent, intracellular pathogen that causes the disease tularaemia. Francisella species contain a cluster of genes referred to as the Francisella pathogenicity island (FPI). Several genes contained in the FPI encode proteins needed for the intracellular growth and virulence of Francisella tularensis. Pathogenicity determinant protein A (PdpA), encoded by the pdpA gene, is located within the FPI and has been associated with the virulence of Francisella species. The experiments outlined in this dissertation examine the properties of PdpA protein expression and localization as well as the phenotypes of non-polar F. novicida pdpA mutants. Monoclonal antibody detection of PdpA showed that it is a soluble protein that is upregulated in iron-limiting conditions and undetectable in an mglA or mglB mutant background. Deletion of pdpA resulted in a strain that was highly attenuated for virulence in chicken embryos and mice. The ΔpdpA strain was capable of a small amount of intracellular replication but, unlike wild-type F. novicida, remained associated with the lysosomal marker LAMP-1, suggesting that PdpA is necessary for progression from the early phagosome phase of infection. Infection of macrophages with the ΔpdpA mutant generated a host-cell mRNA profile distinct from that generated by infection with wild type F. novicida. The transcriptional response of the host macrophage indicates that PdpA functions directly or indirectly to suppress macrophage ability to signal via growth factors, cytokines and adhesion ligands. Experiments were designed to mutagenize a putative F-box domain within the amino terminus of PdpA. Deletion of amino acids 112-227 created a strain which was impaired in intracellular replication and exhibited severely reduced virulence. However, alanine mutagenesis of key conserved leucine residues required for the interaction of F-box domains with host proteins had no observed effect on bacterial growth in macrophages and did not affect virulence in chicken embryos or mice. Mono and polyubiquitinated proteins associated with both the wild type F. novicida and ΔpdpA bacterial strains early during the infection of J774A.1 macrophages. After 1 hour of infection the wild type strain developed a more intimate association with mono and polyubiquitinated proteins whereas the ΔpdpA strain did not. Inhibition of the host cell proteasome during infection did not affect the intracellular growth of wild type F. novicida. PdpA research concludes by examining the secretion patterns of F. novicida. PdpA was not detected as a surface exposed protein using biotinylation whereas IglA, IglB and IglC were found to be surface exposed in both wild type and ΔpdpA backgrounds. These observations suggest that PdpA is not involved in the assembly or function of the Francisella secretion system. FLAG tagged PdpA protein could not be detected in the TCA precipitated supernatant of broth grown cultures or in the immunoprecipitated cytosol of infected macrophages suggesting that PdpA is not a secreted protein.