Made available in DSpace on 2012-05-07T14:43:59Z (GMT). No. of bitstreams: 2 license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) 000063.pdf: 1337362 bytes, checksum: fdc0d841069eb0184e4ae777bec25b84 (MD5) Previous issue date: 2007 / A Yersinia pestis é o agente causador da peste, doença infecciosa transmitida através da picada de pulgas infectadas. O homem se contamina acidentalmente ao entrar em contato com roedores ou outros animais infectados (raposas, cães e gatos) e suas pulgas. A Y. pestis é uma espécie muito homogênea, fenotipicamente, apresentando um sorotipo, um fagotipo e três biotipos. Diferentes métodos moleculares foram utilizados em estudos epidemiológicos para discriminar cepas bacterianas. No entanto, para Y. pestis isoladas em focos do Nordeste do Brasil, a maioria dos marcadores estudados não conseguiram discriminar as cepas originadas de diferentes hospedeiros, períodos e locais de isolamento. A eletroforese em gel de campo pulsado (PFGE) é caracterizada pela separação de fragmentos de DNA obtidos por digestão dos cromossomos com endonucleases de restrição. Essa técnica é considerada o padrão ouro dos métodos de tipagem molecular, sendo altamente discriminatória e válida para muitos patógenos bacterianos, inclusive Y. pestis de outros focos do mundo. O objetivo deste trabalho foi realizar tipagem molecular de cepas brasileiras de Y. pestis através do PFGE. Das 43 cepas de Y. pestis, 36 foram usadas para padronização da técnica e 22 cepas, obtidas antes e durante um surto de peste ocorrido no Estado da Paraíba, para avaliação do PFGE. De acordo com padrões encontrados com a endonuclease de restrição AscI, 19 perfis foram gerados. Esses genótipos foram enquadrados em oito grupos (A-H) geneticamente relacionados. A técnica do PFGE mostrou se capaz de diferenciar as cepas de Y. pestis obtidas em diferentes municípios, antes e durante o surto de peste. De acordo com a variabilidade dos padrões de restrição e o alto poder discriminatório, a técnica PFGE pode ser utilizada para diferenciação e análise de novos isolados. A padronização do protocolo do PFGE para genotipagem das cepas brasileiras de Y. pestis pode ser útil na compreensão e controle da expansão da peste

Yersinia pestis, a gram-negative bacterium, is the causative agent of plague. Y. pestis is a zoonotic pathogen that occasionally infects humans, and is endemic in the western United States. History gives evidence of three main plague pandemics. The first, originating in Egypt in 541AD, is known as the Justinian plague. The second, perhaps most well-known, is thought to have emerged in 1347AD in China, and is called the Black Death. The third, and current plague pandemic, also emerged in China in 1855. In 1899, Y. pestis was established in California, and the plague in other parts of America evolved from this initial introduction. In order to understand evolutionary patterns, we sequenced and analyzed 22 novel Y. pestis genomes from New Mexico. Performing a multiple genome alignment was the first step of our computational pipeline, after which evolutionary patterns were elucidated. Results from this study include predictions of four genes under negative selection pressure. Three of these genes were located on the Y. pestis chromosome, the fourth on the pCD1 virulence plasmid. This study also revealed 42 sites displaying statistically significant skew in the observed residue distribution when comparing sequences based on the year of isolation, and nine significant sites when comparing sequences based on the host species. Phylogenetic tree reconstruction showed a monophyletic pattern for sequences collected in the United States. Taken together, these analyses shed light on the evolutionary history of this pathogen in the southwestern US over a focused time range.

The pathogenicity of Yersinia pestis, the causative agent of plague, is specified by chromosomal and plasmid encoded genes. At least two plasmids, with sizes of 9.5 and 75 kilobases, are indispensable to the full expression of virulence. Loss of the 75 kb plasmid results in outright avirulence. Strains lacking the 9.5 kb plasmid exhibit LD50s at least six orders of magnitude greater than wild-type following subcutaneous or intraperitoneal infection of mice or guinea-pigs but have LD50s as low as wild-type when injected intravenously. Four biochemical properties are associated with the 9.5 kb plasmid. These include plasminogen activator and coagulase activities in addition to the bacteriocin pesticin, and its immunity determinant. A genetic analysis of this plasmid was undertaken as a first step towards the identification and characterization of its virulence determinant(s). This led to the construction of a physical and genetic map of the plasmid. Four loci were mapped to the plasmid: pst and pim, which encode pesticin and its immunity determinant respectively; pla, which encodes both plasminogen activator and coagulase activities; and ori/inc, the locus containing both the origin of replication and the region responsible for the control of plasmid incompatibility. pst was shown to encode a 45 kD protein but the pim gene product was not identified. pla encodes two outer membrane proteins, α- and β-Pla of 37 and 35 kD, respectively, the latter being derived from the former most probably by a proteolytic processing event. At least one of these proteins is responsible for the highly specific degradation of the YOPs, a set of virulence-associated outer membrane proteins encoded by the 75 kb plasmid. The nucleotide sequence of pla revealed that it possessed significant homology to both prtA (geneE) of Salmonella typhimurium and ompT of Escherichia coli. Subcutaneous infection of mice with isogenic strains of Y. pestis harboring well defined mutations in the genes that reside on the 9.5 kb plasmid revealed that pla is a virulence determinant of Y. pestis, and also that of the genes harbored by the plasmid, pla is both necessary and sufficient to account for the high degree of virulence of Y. pestis for mice from subcutaneous sites of infection. pla encodes an activator of human, rat, and mouse plasminogen but does not induce coagulation of plasma obtained from these species. Treatment of mice with the antifibrinolytic agent, trans-4(aminomethyl)-cyclohexanecarboxylic acid did not affect the outcome of plague infection, indicating that fibrinolysis per se does not play a role in plague pathogenesis.

Yersinia pestis, l'agent de la peste, est une bactérie à Gram-négatif classée comme agent pathogène ré-émergent et potentielle arme de bioterrorisme. De plus, l'apparition d'une souche multi-résistance de cette bactérie souligne la nécessité de mieux comprendre comment cette bactérie hyper-virulente interagit avec son hôte. Afin d'identifier des facteurs génétiques de vulnérabilité à la peste, notre laboratoire travaille sur la réponse de souris résistantes versus sensibles à Y. pestis. Notre stratégie pour identifier les facteurs génétiques impliqués dans la résistance/sensibilité à la peste combine une approche de cartographie de QTL (Quantitative Trait Locus) et d'analyse d'expression génique. Nous avons précédemment décrit la lignée SEG/Pas, issue de Mus spretus, comme la première résistante à une souche virulente de Y. pestis, alors que la plupart des lignées murines de laboratoire, telle que la lignée C57BL/6J, sont extrêmement sensible à la bactérie. Des croisements entre SEG/Pas et C57BL/6J nous ont permis d'identifier trois QTL impliqués dans la résistance à Y. pestis, localisés sur les chromosomes 3, 4 et 6. Deux des QTL (situés sur les chromosomes 4 et 6) ont pu être confirmés par l'analyse de lignées congéniques. Plus de 40 % des femelles bi-congéniques hétérozygotes pour ces deux QTL ont survécu à l'infection, alors que tous les témoins C57BL/6J ont succombé. La dissection de ces deux QTL par l'analyse de lignées sous-congéniques, nous a permis d'affiner l'architecture génétique de la résistance à la peste chez SEG/Pas. Nous avons conclu qu'un minimum de quatre facteurs génétiques, au sein de ces deux QTL, sont nécessaires pour augmenter la résistance à Y. pestis chez la Souris. Cependant, la production de plusieurs lignées congéniques portant le QTL situé sur le chromosome 3, dont une lignée triple congénique, ne nous a pas permis de confirmer l'existence de ce QTL. En parallèle de l'analyse génétique, nous avons déterminé que les macrophages de SEG/Pas et de C57BL/6J présentaient des caractéristiques différentes après exposition à Y. pestis. Une analyse différentielle du profil transcriptionnel des macrophages de ces deux lignées a été réalisée à l'aide de puces à ADN. Nos résultats montrent une forte activation de la production cytokinique dans les macrophages de SEG/Pas en réponse à la bactérie, activation qui n'est pas observée dans la lignée C57BL/6J. Ces résultats suggèrent que les souris SEG/Pas sont capables de mettre en place une réponse immune innée plus forte ou peut-être plus précoce que C57BL/6J. Nous avons ensuite étudié par qRT-PCR l'expression en cinétique de 44 gènes dans des macrophages de SEG/Pas, C57BL/6J et des bi-congéniques portant les QTL sur les chromosomes 4 et 6. Cette étude nous a permis de confirmer que les souris SEG/Pas sont capables se mettre en place une forte réponse inflammatoire lors de l'infection. Cependant, aucune différence significative n'a été observée entre la lignée bicongénique et la lignée parentale C57BL/6J. D'autres expériences seront nécessaires afin de mieux comprendre les mécanismes biologiques impliqués dans la résistance intermédiaire de cette lignée. La dissection génétique associée à l'analyse de l'expression génique de ces lignées résistante et sensible permet d'augmenter notre compréhension de la réponse de l'hôte à Y. pestis.

Host survival during plague, caused by the Gram-negative bacterium Yersinia pestis, is favored by a robust early innate immune response initiated by IL-1β and IL-18. Precursors of these cytokines are expressed downstream of TLR signaling and are then enzymatically processed into mature bioactive forms, typically by caspase-1 which is activated through a process dependent on multi-molecular structures called inflammasomes. Y. pestis evades immune detection in part by using a Type three secretion system (T3SS) to inject effector proteins (Yops) into host cells and suppress IL-1β and IL-18 production. We investigated the cooperation between two effectors, YopM and YopJ, in regulating inflammasome activation, and found that Y. pestis lacking both YopM and YopJ triggers robust caspase-1 activation and IL-1Β/IL-18 production in vitro. Furthermore, this strain is attenuated in a manner dependent upon caspase-1, IL-1β and IL-18 in vivo, yet neither effector appears essential for full virulence. We then demonstrate that YopM fails to inhibit NLRP3/NLRC4 mediated caspase-1 activation and is not a general caspase-1 inhibitor. Instead, YopM specifically prevents the activation of a Pyrin-dependent inflammasome by the Rho-GTPase inhibiting effector YopE. Mutations rendering Pyrin hyperactive are implicated in the autoinflammatory disease Familial Mediterranean Fever (FMF) in humans, and we discuss the potential significance of this disease in relation to plague. Altogether, the Y. pestis T3SS activates and inhibits several inflammasome pathways, and the fact that so many T3SS components are involved in manipulating IL-1β/IL-18 underscores the importance of these mechanisms in plague.

Yersinia pestis, the causative agent of plague, is estimated to have claimed the lives of 30-50% of the European population in five years. Although it can now be controlled through antibiotics, there are still lurking dangers of outbreaks from biowarfare and bioterrorism; therefore, ongoing research to further our understanding of its strong virulence factors is necessary for development of new vaccines. Many Gram-negative bacteria, including Y. pseudotuberculosis, the evolutionary ancestor of Y. pestis, produce a hexa-acylated lipid A/LPS which can strongly trigger innate immune responses via activation of Toll-like receptor 4 (TLR4)-MD2. In contrast, Y. pestis grown at 37ºC generates a tetra-acylated lipid A/LPS that poorly induces TLR4-mediated immune activation. We have reported that expression of E. coli lpxL in Y. pestis, which lacks a homologue of this gene, forces the biosynthesis of a hexa-acylated LPS, and that this single modification dramatically reduces virulence in wild type mice, but not in mice lacking a functional TLR4. This emphasizes that avoiding activation of innate immunity is important for Y. pestis virulence. It also provides a model in which survival is strongly dependent on innate immune defenses, presenting a unique opportunity for evaluating the relative importance of innate immunity in protection against bacterial infection. TLR signaling is critical for the sensing of pathogens, and one implication of TLR4 engagement is the induction of the pro-forms of the potent inflammatory cytokines IL-1β and IL-18. Therefore Y. pestis is able to suppress production of these which are generated through caspase-1-activating nucleotide-binding domain and leucine-rich repeat (NLR)-containing inflammasomes. For my thesis, I sought to elucidate the role of NLRs and IL-18/IL-1β during bubonic and pneumonic plague infection. Mice lacking IL-18 signaling led to increased susceptibility to wild type Y. pestis, and an attenuated strain producing a Y. pseudotuberculosis-like hexa-acylated lipid A. I found that the NLRP12, NLRP3 and NLRC4 inflammasomes were important protein complexes in maturing IL-18 and IL-1β during Y. pestis infection, and mice deficient in each of these NLRs were more susceptible to bacterial challenge. NLRC4 and NLRP12 also directed interferongamma production via induction of IL-18 against plague, and minimizing inflammasome activation may have been a central factor in evolution of the high virulence of Y. pestis. This is also the first study that elucidated a pro-inflammatory role for NLRP12 during bacterial infection.

<p>The pathogenic bacteria Yersinia pestis and Y. pseudotuberculosis are related to the degree where the former is considered a subspecies of the latter, and still they cause disease of little resemblance in humans. Y. pestis is the causative agent of lethal bubonic and pneumonic plague, while Y. pseudotuberculosis manifests itself as mild gastroenteritis. An important virulence determinant for these species is their ability to secrete and inject toxins (Yop effectors) into immune cells of the infected host, in a bacterium-cell contact dependent manner. This ability depends on the extensively studied type III secretion system, a highly complex multicomponent structure resembling a needle. The induction of Yop secretion is a strictly controlled event. The two structural type III secretion components YscU and YscP are here shown to play a crucial role in this process, which is suggested to require an YscP mediated conformational change of the C-terminus of YscU. Proteolytic cleavage of YscU within this domain is further revealed to be a prerequisite for functional Yop secretion. The needle subcomponent itself, YscF, is recognised as a regulatory element that controls the induction of Yop effectors and their polarised delivery into target cells. Potentially, the needle might act as a sensor that transmits the inducing signal (i.e. target cell contact) to activate the type III secretion system. Secondly a, for Yersinia, previously unexplored system, the Twin arginine translocation (Tat) pathway, is shown to be functional and absolutely required for virulence of Y. pseudotuberculosis. A range of putative Yersinia Tat substrates were predicted in silico, which together with the Tat system itself may be interesting targets for future development of antimicrobial treatments.</p>

The pathogenic bacteria Yersinia pestis and Y. pseudotuberculosis are related to the degree where the former is considered a subspecies of the latter, and still they cause disease of little resemblance in humans. Y. pestis is the causative agent of lethal bubonic and pneumonic plague, while Y. pseudotuberculosis manifests itself as mild gastroenteritis. An important virulence determinant for these species is their ability to secrete and inject toxins (Yop effectors) into immune cells of the infected host, in a bacterium-cell contact dependent manner. This ability depends on the extensively studied type III secretion system, a highly complex multicomponent structure resembling a needle. The induction of Yop secretion is a strictly controlled event. The two structural type III secretion components YscU and YscP are here shown to play a crucial role in this process, which is suggested to require an YscP mediated conformational change of the C-terminus of YscU. Proteolytic cleavage of YscU within this domain is further revealed to be a prerequisite for functional Yop secretion. The needle subcomponent itself, YscF, is recognised as a regulatory element that controls the induction of Yop effectors and their polarised delivery into target cells. Potentially, the needle might act as a sensor that transmits the inducing signal (i.e. target cell contact) to activate the type III secretion system. Secondly a, for Yersinia, previously unexplored system, the Twin arginine translocation (Tat) pathway, is shown to be functional and absolutely required for virulence of Y. pseudotuberculosis. A range of putative Yersinia Tat substrates were predicted in silico, which together with the Tat system itself may be interesting targets for future development of antimicrobial treatments.

Innate immunity is the first line of defense against invading pathogens. Vertebrate innate immunity provides both initial protection, and activates adaptive immune responses, including memory. As a result, the study of innate immune signaling is crucial for understanding the interactions between host and pathogen. Unlike mammals, the insect Drosophila melanogasterlack classical adaptive immunity, relying on innate immune signaling via the Toll and IMD pathways to detect and respond to invading pathogens. Once activated these pathways lead to the rapid and robust production of a variety of antimicrobial peptides. These peptides are secreted directly into the hemolymph and assist in clearance of the infection. The genetic and molecular tools available in the Drosophila system make it an excellent model system for studying immunity. Furthermore, the innate immune signaling pathways used by Drosophilashow strong homology to those of vertebrates making them ideal for the study of activation, regulation and mechanism. Currently a number of questions remain regarding the activation and regulation of both vertebrate and insect innate immune signaling. Over the past years many proteins have been implicated in mammalian and insect innate immune signaling pathways, however the mechanisms by which these proteins function remain largely undetermined. My work has focused on understanding the molecular mechanisms of innate immune activation in Drosophila. In these studies I have identified a number of novel protein/protein interactions which are vital for the activation and regulation of innate immune induction. This work shows that upon stimulation the Drosophila protein IMD is cleaved by the caspase-8 homologue DREDD. Cleaved IMD then binds the E3 ligase DIAP2 and promotes the K63-polyubiquitination of IMD and activation of downstream signaling. Furthermore the Yersinia pestis effector protein YopJ is able to inhibit the critical IMD pathway MAP3 kinase TAK1 by serine/threonine-acetylation of its activation loop. Lastly TAK1 signaling to the downstream Relish/NF-κB and JNK signaling pathways can be regulated by two isoforms of the TAB2 protein. This work elucidates the molecular mechanism of the IMD signaling pathway and suggests possible mechanisms of homologous mammalian systems, of which the molecular details remain unclear.

Yersinia pestis, the gram-negative causative agent of plague, is a master of immune evasion. The bacterium possesses a type three secretion system which translocates Yop effector proteins into host immune cells to inhibit a number of immune and cell signaling cascades. Interestingly, this apparatus is not expressed at low temperatures such as those found within the flea vector and is therefore neither in place nor functional when the bacteria are first transmitted into a mammalian host. However, the bacterium is still able to avoid activating the immune system, even very early during infection. When grown at 37°C (human body temperature) Y. pestis produces a tetra-acyl lipid A molecule, which is antagonistic to the human Toll like receptor 4/MD2, the major lipopolysaccharide recognition receptor. Although tetra-acyl lipid A binds this receptor complex, it does not induce signaling, and in fact inhibits the receptors interaction with other stimulatory forms of lipid A. The work undertaken in this thesis seeks to determine if the production of tetra-acyl lipid A by Y. pestis is a key virulence determinant and was a critical factor in the evolution of Y. pestis from its ancestral parent Yersinia pseudotuberculosis. By examining the enzymes involved in the lipid A biosynthesis pathway, it has been determined that Y. pestis lacks LpxL, a key enzyme that adds a secondary acyl chain on to the tetra acyl lipid A molecule. In the absence of this enzyme, Y. pestis cannot produce a TLR4 stimulating form of lipid A, whereas Y. pseudotuberculosis does contain the gene for LpxL and produces a stimulatory hexa acyl lipid A. To determine if the absence of LpxL in Y. pestis is important for virulence, LpxL from E. coli and Y. pseudotuberculosis were introduced into Y. pestis. In both cases the addition of LpxL led to bacterium which produced a hexa-acylated lipid A molecule and TLR4/MD2 stimulatory LPS. To verify the LpxL phenotype, lpxL was deleted from Y. pseudotuberculosis, resulting in bacteria which produce tetra-acylated lipid A and nonstimulatory LPS. Mice challenged with LpxL expressing Y. pestis were found to be completely resistant to infection. This profound attenuation in virulence is TLR4 dependent, as mice deficient for this receptor rapidly succumb to disease. These altered strains of the bacterium also act as vaccines, as mice infected with Y. pestis expressing LpxL then challenged with wild type Y. pestis do not become ill. These data demonstrate that the production of tetra-acyl lipid A is a critical virulence determinant for Y. pestis, and that the loss of LpxL formed a major step in the evolution of Y. pestis from Y. pseudotuberculosis. These bacterial strains were also used as tools to determine the contributions of different innate immune receptors and adaptor molecules to the host response during Y. pestis infection. The use of LpxL expressing Y. pestis allowed identification of the innate immune pathways critical for protection during Y. pestis infection. This model also established that CD14 recognition of rough LPS is critical for protection from Y. pestisexpressing LpxL, and activation of the IL-1 receptor and the induction of IL-1β plays a major role in this infection as well. The lipid A acylation profile of gram negative bacteria can have a direct and profound effect on the pathogenesis of the organism. This work illustrates a previously unknown and critical aspect of Y. pestis pathogenesis, which can be extended to other gram-negative pathogens. The greater detail of the contributions which different host adaptor and receptor molecules make to the overall innate immune signaling pathway will allow a better insight into how gram negative infections progress and how they are counteracted by the immune system. Alterations of the lipid A profile of Y. pestis have important implications for the production of vaccines to Y. pestis and other gram negative pathogens. Taken together, this work describes a novel mechanism for immune evasion by gram negative bacteria with consequences for understanding the immune response and the creation of more effective vaccines, both of which will decrease the danger posed by this virulent pathogen.