Caulobacters are gram-negative bacteria that have a biphasic life cycle consisting of a swarmer and a stalked stage. As a result they have elicited interest as a simple developmental model. Less attention has focussed on their role in the environment, although they have been found in almost every aquatic environment as well as in many soils. Caulobacters are often described as oligotrophic bacteria because of their prevalence in pristine waters but have now been isolated from the relatively nutrient-rich wastewater environment. In order to learn more about this population some basic characterization was carried out and an assay system to determine their prevalence in sewage plants was designed. Most of the organisms isolated from sewage treatment facilities had similar gross morphological features, but differed in holdfast composition, total protein profile, antibiotic resistance and restriction fragment length polymorphism, thereby indicating a greater diversity than originally assumed. Most of the organisms hybridized with flagellin and surface array genes that had previously been cloned, and only one of 155 non-Caulobacter sewage isolates hybridized with the flagellin gene probe; consequently these were used in a DNA-based enumeration strategy. DNA was isolated directly from sewage and probed with the flagellin and the surface array gene probes. The signals obtained were compared to standards made up of pooled Caulobacter DNA from the sewage isolates and non-Caulobacter DNA from organisms also present in sewage. Using this assay Caulobacters could only be detected above the 1% level, which was higher than their proportion in the wastewater environment. It appears that this approach will not be useful in monitoring Caulobacters in treatment plants unless a more highly conserved or higher copy number probe is found. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

A leptospirose é uma zoonose causada por bactérias do gênero Leptospira. A patogênese da doença em humanos é observada principalmente no pulmão, fígado e rins. Neste trabalho, foi avaliado o papel da resposta imune inata na proteção contra a leptospirose usando camundongos como modelo experimental. Os animais foram infectados com L. interrogans e o desenvolvimento da doença foi acompanhado, observando-se a morte de animais C3H/HeJ, enquanto C3H/HePas apresentou icterícia e BALB/c não apresentou sintomas. O perfil de mRNA foi medido por qPCR nas amostras de rim, fígado e pulmão e as concentrações de proteinas TNF-<font face=\"Symbol\">&#945;, TGF-<font face=\"Symbol\">b, MCP-1, MIP-1<font face=\"Symbol\">&#945;, MIP-2 e IL-8 foram analisadas por ELISA em extratos dos tecidos e no soro. Os resultados demonstraram que L. interrogans estimula a expressão prematura de TNF-<font face=\"Symbol\">&#945;, TGF-<font face=\"Symbol\">b, MCP-1, MIP-1<font face=\"Symbol\">&#945;, MIP-2 e IL-8 na linhagem BALB/c resistente à infecção. A análise histológica indica que estes mediadores podem estar relacionados com o influxo de diferentes células do sistema imune desempenhando importantes funções na proteção contra leptospirose. / Leptospirosis is a worldwide zoonosis caused by Leptospira. The pathogenesis in humans is mainly observed in lungs, livers and kidneys. In this work the role of innate immune response in protection against leptospirosis is being studied using different mice models. The animals were infected intraperitoneally with virulent cells of L. interrogans serovar Copenhageni and the development of the disease was followed, being observed mortality of C3H/HeJ mice, whereas C3H/HePas presented jaundice and BALB/c mice remained asymptomatic. Samples of liver, kidney, lungs and sera were analyzed following the profiles of mRNA and protein of the cytokines TNF-<font face=\"Symbol\">&#945; and TGF-<font face=\"Symbol\">b and chemokine MCP-1, MIP-1<font face=\"Symbol\">&#945;, MIP-2 and CXCL1/IL-8. We showed that Leptospira infection stimulates early expression of cytokine TNF-<font face=\"Symbol\">&#945; and TGF-<font face=\"Symbol\">b and chemokine MCP-1, MIP-1<font face=\"Symbol\">&#945;, MIP-2 and IL-8 in the resistant mice strain BALB/c. Histological analysis indicates that the expression of those molecules can be related to the influx of distinct immune cells, which play a role in the naturally acquired protective immunity.

Thesis (D.Sc.)--Boston University, Henry M. Goldman School of Dental Medicine, 2004 (Oral Biology). / Includes bibliography (leaves 107-151). / Lipopolysaccharide (LPS), a glycolipid found in the outer membrane of Gram negative bacteria, induces the secretion of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-a) and interleukin (IL )-1, by monocytes/macrophages. Excessive and uncontrolled secretion of these compounds leads to multiple pathological conditions, such as septic shock. LPS receptors have been shown to be CD14, TLR4 and MD-2. LPS interaction with these receptors mediates many monocyte/macrophage functions. Even though only CD14 was demonstrated to bind to LPS, and TLR4/MD-2 were capable of transducing signals, data only show that LPS and CD 14 were in close proximity to TLR4 and no direct binding was reported. Quite recently, moesin, a member of the ERM family of proteins, has been also found to function as a receptor for LPS. We have shown that anti-moesin antibody inhibited the release of TNFa by LPS stimulated monocytes. Moesin was also found to be necessary for the detection of LPS, where homozygous knockout mice exhibited 3-fold reduction in neutrophil infiltrates in LPS injected sites when compared to their wild type controls. When moesin gene expression was completely suppressed with antisense oligonucleotides, there was a significant reduction of LPS-induced TNF-a secretion. LPS stimulation of mononuclear phagocytes activates several intracellular signaling pathways including the phosphorylation of IKBa, mitogen-activated protein kinase (MAPK) pathways: extracellular signal-regulated kinases (ERK) 1 / 2 (P44/42), p38. These signaling pathways in tum activate a variety of transcription factors including NF-KB, which coordinates the induction of several genes encoding inflammatory mediators. [TRUNCATED]

No description available.

The Type VII secretion system (T7SS) is linked to virulence and long-term pathogenesis in a broad range of Gram-positive bacteria, including the human commensal and pathogen Staphylococcus aureus. The Type VIIb secretion system (T7SSb) is responsible for the export of small toxic proteins, which induce antibacterial immune responses and mediate bacterial persistence in the host. In addition, it is also involved in bacterial competition. The T7SSb requires several proteins to build up the secretion machinery. This work focuses on the structural and functional investigation of the motor ATPase EssC and the putative pore forming, multi-pass membrane component EsaA. Both proteins are indispensable for substrate secretion. EssC belongs to the FtsK/SpoIIIE ATPase family and is conserved among the T7SSs. It contains three C-terminal, cytosolic ATPase domains, designated as EssC- D1, -D2 and -D3, whereby EssC-D3 is the most distal one. In this thesis, I am presenting the crystal structure of the EssC-D3 at 1.7 Å resolution. As the deletion of EssC-D3 abrogates substrate export, I have demonstrated that this domain comprises a hydrophobic, surface-exposed pocket, which is required for substrate secretion. More specifically, I have identified two amino acids involved in the secretion process. In addition, my results indicate that not only EssC-D3 is important for substrate interaction but also EssC-D2 and/or EssC-D1. Unlike in the related Yuk T7SSb of Bacillus subtilis, the ATPase activity of D3 domain contributes to substrate secretion. Mutation of the modified Walker B motif in EssC-D3 diminishes substrate secretion completely. The membrane protein EsaA encompasses an extracellular segment spanning through the cell wall of S. aureus. I was able to reveal that this part folds into a stable domain, which was crystallized and diffracted up to 4 Å. The first attempts to dissolve the structure failed due to a lack of homologues structures. Therefore, crystals for single-wavelength anomalous dispersion, containing selenomethionyl-substitutes, were produced and the structure solution is still in progress. Preliminary experiments addressing the function of the extracellular domain indicate an important role in substrate secretion and bacterial competition. / Das Typ VII Sekretionssystem (T7SS) ist wichtig für Virulenz und Langzeit- Pathogenität von Gram-positiven Bakterien. Zu diesen gehört auch Staphylococcus aureus, bekannt als Kommensal und Pathogen im Menschen. Das Typ VIIb Sekretionssystem (T7SSb) exportiert kleine, toxische Proteine, die antibakterielle Immunantworten auslösen und für bakterielle Persistenz verantwortlich sind. Außerdem ist es an dem Konkurrenzkampf zwischen Bakterien beteiligt. Das System benötigt verschiedene Komponenten, um eine Sekretion zu ermöglichen. Diese Doktorarbeit konzentriert sich auf zwei dieser Proteine, die ATPase EssC und das Membranprotein EsaA. Beide Komponenten sind unentbehrlich für eine vollständige Funktionalität. EssC gehört zu der Familie der FtsK/SpoIIIE ATPasen und ist evolutionär in allen T7SSs erhalten. EssC besitzt drei C-terminale, zytosolische ATPase Domänen, bezeichnet als EssC-D1, -D2 und D3, wobei EssC-D3 C-terminal gelegen ist. In dieser Arbeit präsentiere ich die Kristallstruktur der ATPase Domäne EssC-D3, aufgelöst bis zu 1.7 Å. Die Domäne ist unabdingbar für die Sekretion. Durch die Strukturauflösung wurde eine hydrophobe, Oberflächen-exponierte Substrat- Bindetasche bestimmt, die eine essenzielle Rolle für den Export der toxischen Substrate einnimmt. Durch dieses Projekt konnten zwei Aminosäuren in dieser Tasche bestimmt werden, die für den Prozess der Substratsekretion wichtig sind. Weiterhin wurde bewiesen, dass nicht nur EssC-D3, sondern auch die ATPase Domäne EssC-D2 und/oder EssC-D1 mit den Substraten interagieren kann. Im Gegensatz zu dem verwandten T7SSb in Bacillus subtilis, verfügt EssC-D3 über ATPase Aktivität und ermöglicht dadurch den Substratexport. Das Membranprotein EsaA besitzt einen extrazellulären Abschnitt, der sich durch die Zellwand von S. aureus erstreckt. Dieser extrazelluläre Part besteht aus einer stabilen Domäne, welche kristallisiert werden konnte und bis zu 4 Å diffraktiert. Aufgrund von fehlenden homologen Strukturen konnte die Struktur der Domäne noch nicht bestimmt werden. Für die Phasenbestimmung, die wichtig für die Strukturauflösung ist, wurden Kristalle mit Selenomethionyl-Substituten hergestellt. Die Strukturauflösung ist noch nicht beendet. Erste Experimente bezüglich der extrazellulären Domäne zeigen, dass diese ebenfalls wichtig für die Substratsekretion und zusätzlich am Konkurrenzkampf zwischen Bakterien beteiligt ist.

The biosynthetic enzymes involved in wall teichoic acid biogenesis in Grampositive bacteria have been the subject of renewed investigation in recent years with the benefit of modem tools of biochemistry and genetics. Nevertheless, there have been only limited investigations into the enzymes that glycosylate wall teichoic acid. Decades-old experiments in the model Gram-positive bacterium, Bacillus subtilis 168, using phage resistant mutants implicated tagE (also called gtaA and rodD) as the gene coding for the wall teichoic acid glycosyltransferase. This study and others have provided only indirect evidence to support a role for TagE in wall teichoic acid glycosylation. In this work, we showed that deletion of tagE results in the loss of a-glucose at the C-2 position of glycerol in the poly(glycerol phosphate) polymer backbone. We also report the first kinetic characterization of pure, recombinant wall teichoic acid glycosyltransferase using clean synthetic substrates. We investigated the substrate specificity ofTagE using a wide variety of acceptor substrates and showed that this enzyme has a strong kinetic preference for the transfer of glucose from UDP-glucose to glycerol phosphate in polymeric form. Further, we showed that the enzyme recognizes its polymeric (and repetitive) substrate with a sequential kinetic mechanism. This work provides direct evidence that TagE is the wall teichoic acid glycosyltransferase in B. subtilis 168 and provides a strong basis for further studies on the mechanism of wall teichoic acid glycosylation, a largely uncharted aspect of wall teichoic acid biogenesis. / Thesis / Doctor of Philosophy (PhD)

No description available.

No description available.

A multirresistência aos antibióticos observada em bacilos gram-negativos é um grave problema de saúde pública devido a alta morbidade e mortalidade apresentada, especialmente em instituições assistenciais de saúde. Como consequência do intenso uso de antibióticos, a multirresistência a esses fármacos é principalmente mediada por enzimas hidrolisantes, onde destaca-se as enzimas ?-lactamases, principal mecanismo de resistência aos ?-lactâmicos verificado em bacilos gram-negativos. Os esgotos de origem hospitalar e de estações de tratamento de esgoto (ETE) são considerados como reservatórios de bactérias multirresistentes pela presença de antibióticos que as selecionam e por favorecem a transmissão de determinantes de resistência. Nesse sentido, o presente estudo objetivou avaliar a multirresistência a antibióticos e a produção de enzimas ?-lactamases em bacilos gram-negativos isolados de efluente hospitalar e da estação de tratamento de esgoto, na cidade de Ribeirão Preto, SP. No hospital terciário, amostras de esgotos foram coletadas dos ambulatórios, das enfermarias e da junção do esgoto hospitalar. Na ETE, amostras foram coletadas na caixa de entrada do esgoto bruto e após ao tratamento. Dez microlitros foram semeados em ágar MacConkey, SalmonellaShigella, Cetrimide e TCBS e a identificação dos bacilos gram-negativos foi realizada pelo kit Bactray®. O teste de susceptibilidade aos antibióticos foi realizado pelo método de discodifusão em ágar. A detecção fenotípica de bacilos produtores de ESBL foi realizada pelos testes de sinergia de disco-duplo e disco combinado com ácido clavulânico, e para detecção de isolados produtores de carbapenemases foi utilizado os testes de disco combinado com ácido fenilborônico e EDTA e o teste Blue Carba. A PCR foi utilizada para amplificação dos genes codificadores de ESBL e carbapenemases. No total, 45 bacilos gram-negativos foram isolados, sendo as espécies Klebsiella pneumoniae e Pseudomonas aeruginosa as de maiores prevalências. Ampla resistência foi verificada aos antibióticos ?-lactâmicos, sendo a resistência ao aztreonam, a cefepime e a cefotaxima mais expressiva nos isolados do esgoto hospitalar, com diferenças estatisticamente significante (p<0,05). O fenótipo multidroga resistente foi atribuído a 33,3%, nos isolados exclusivamente do esgoto hospitalar, com diferença estatisticamente significante (p = 0,0025) em relação aos isolados do esgoto da ETE. Genes de ?-lactamases foram encontrados em 35,6% das bactérias, sendo o blaKPC e blaTEM os de maiores ocorrências, ambos em 17,8% dos isolados, e os genes blaSHV e blaCTX-M em 13,3% e 8,9%. Somente em um isolado de Enterobacter cloacae no esgoto tratado da ETE foi identificado o gene blaSHV, os demais isolados portadores dos genes de ?-lactamases foram encontrados no esgoto hospitalar. Os dados obtidos neste estudo são importantes levando em consideração que no Brasil o esgoto hospitalar pode ser lançado in natura na rede coletora municipal, no entanto, acredita-se que tal permissão favorece a disseminação da multirresistência bacteriana, posto que, os resultados demonstram alta frequência de bactérias portadoras de genes de resistência a antibióticos no esgoto hospitalar estudado. Assim, a implementação do tratamento de efluentes hospitalares, especialmente os de hospitais terciários, e adicionalmente ao tratamento da ETE evitaria a propagação dessas bactérias no ambiente e de impactar negativamente os recursos hídricos / Antibiotic multi-resistance observed in Gram-negative bacilli is a serious public health problem due to high morbidity and mortality, especially in health care institutions. As a consequence of the intense use of antibiotics, multi-resistance to these drugs is mainly mediated by hydrolyzing enzymes, in which ?-lactamases, the main ?-lactam resistance mechanism observed in Gramnegative bacilli, are prominent. Hospital sewage and wastewater treatment plants (WWTP) are considered reservoirs of multiresistant bacteria by the presence of antibiotics that select these bacteria and favor the transmission of resistance determinants. In this sense, the present study aimed to evaluate the antibiotics multi-resistance and the production of ?-lactamase enzymes in Gram-negative bacilli isolated from hospital effluent and the wastewater treatment plants in Ribeirão Preto city, SP. In the tertiary hospital, sewage samples from the outpatient clinics, rooms patients and the hospital sewage junction were collected. In the WWTP, raw and treated sewage were collected. Ten microliters were seeded on MacConkey, Salmonella-Shigella, Cetrimide and TCBS agar and the identification of Gram-negative bacilli was performed by the Bactray® kit. Antibiotic susceptibility test was performed by agar-diffusion method. Phenotypic detection of ESBL-producing bacilli was performed by double-disc and discsynergy tests combined with clavulanic acid, and for the detection of carbapenemase-producing isolates the combined disk tests with phenylboronic acid and EDTA and Blue Carba test were used. PCR amplification of ESBL and carbapenemases-encoding genes was used. In total, 45 Gram-negative bacilli were isolated, and Klebsiella pneumoniae and Pseudomonas aeruginosa being the most prevalent. Extensive resistance was verified to ?-lactam antibiotics and resistance to aztreonam, cefepime and cefotaxime was more pronounced in hospital sewage isolates, with statistically significant differences (p<0.05). Multidrug-resistant phenotype was attributed to 33.3% in isolates exclusively from hospital sewage, with a statistically significant difference (p = 0.0025) in relation to the sewage isolates from the WWTP. ?-lactamase genes were found in 35.6% of the bacteria, with blaKPC and blaTEM having the highest occurrences, both in 17.8% of the isolates, and the blaSHV and blaCTX-M genes in 13.3% and 8, 9%. Only in an isolate of Enterobacter cloacae in the treated sewage from WWTP was the blaSHV gene identified, the other isolates carrying the ?-lactamases genes were found in hospital sewage. The data obtained in this study are important considering that in Brazil the hospital sewage can be released in nature in municipal collection network, however, it is believed that such permission favors the dissemination of bacterial multi-resistance, since, the results show high frequency of bacteria carrying antibiotic resistance genes in the hospital sewer studied. Thus, the implementation of treatment of hospital effluents, especially those in tertiary hospitals, and in addition to the treatment of WWTP would prevent the spread of these bacteria in the environment and negatively impact water resources

Orientador: Marcos Rogério André / Banca: Ana Patrícia Yatsuda Natsui / Banca: Rosangela Zacarias Machado / Resumo: Embora espécies de Anaplasma spp. sejam agentes Rickettsiales altamente difundidos em ruminantes domésticos e selvagens, com ampla distribuição mundial, poucos estudos foram realizados até momento com o intuito de detectar e/ou investigar a diversidade de Anaplasma spp. circulantes em bovinos em Moçambique. No presente estudo, ensaios sorológicos e moleculares foram utilizados para investigar a ocorrência de Anaplasma spp. em 219 bovinos amostrados nos distritos de Boane, Magude, Matutuíne, Moamba e Namaacha em Maputo, Moçambique. No teste iELISA para detecção de anticorpos IgG anti- A. marginale, 86,3% (189/219) das amostras mostraram-se soropositivas. Em ensaios de qPCR para os genes msp1β de A. marginale e msp2 para A. phagocytophilum, 97,3% (213/219) e 2,74% (6/219) dos animais mostraram-se positivos, respectivamente. A combinação de dois protocolos diferentes de cPCR baseados no gene 16S rRNA evidenciou 100% de amostras positivas para Anaplasma spp. Sequências de 16S rRNA filogeneticamente relacionadas a A. platys, A phagocytophilum, 'Candidatus Anaplasma boleense', A. centrale, A. marginale e A. ovis foram detectadas neste estudo. Inferências filogenéticas baseadas nos genes msp4 e msp5 posicionaram as sequências obtidas no clado de A. marginale, com a evidência de circulação de 1 e 2 haplótipos diferentes para cada gene, respectivamente. Anaplasma sp. filogeneticamente associado a A. platys foi evidenciado nas análises filogenéticas baseadas nos genes 16S rRNA e groEL.... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Although species of Anaplasma spp. are widespread Rickettsiales agents in domestic and wild ruminants, showing a worldwide distribution, few studies have been conducted in order to detect and/or investigate the diversity of Anaplasma spp. circulating in cattle in Mozambique. In the present study, serological and molecular tests were used to investigate the occurrence of Anaplasma spp. in 219 bovine sampled in the districts of Boane, Magude, Matutuíne, Moamba and Namaacha in Maputo, Mozambique. In the iELISA test for detection of IgG antibodies to A. marginale, 86.3% (189/219) of the samples were positive. In quantitative Real-time PCR tests targeting the msp1β genes of A. marginale and msp2 for A. phagocytophilum, 97.3% (213/219) and 2.74% (6/219) of the animals were positive, respectively. The combination of two different protocols of conventional PCR targeting the 16S rRNA gene evidenced 100% of positive samples for Anaplasma spp. Sequences of 16S rRNA phylogenetically related to A. platys, A. phagocytophilum, 'Candidatus Anaplasma boleense', A. centrale, A. marginale and A. ovis were detected in this study. Phylogenetic analysis based on msp4 and msp5 genes positioned the obtained sequences in the clade of A. marginale, with the evidence of circulation of 1 and 2 different haplotypes for each gene, respectively. Anaplasma sp. phylogenetically related to A. platys was evidenced in the phylogenetic analyses based on the 16S rRNA and groEL genes. In conclusion,a high diversit... (Complete abstract click electronic access below) / Mestre