Pseudomonas aeruginosa is an opportunistic bacterium that can infect immunocompromised people, and is especially prevalent in patients with cystic fibrosis. Treatment of P. aeruginosa is complicated due to resistance to many classes of antibiotics. This is partly due to biofilm formation, which is not simply a diffusion barrier, but also has a distinct mechanism for resisting the action of antibiotics. P. aeruginosa quinolone signal (PQS) has an important role in quorum sensing, which is involved in biofilm formation. PqsBC is a condensing enzyme in the biosynthesis of the PQS. The crystal structures of PqsBCC129A and PqsBCC129A-Fe3+ were collected to 2.04 Å and to 2.3 Å, respectively. The crystal structure showed that PqsB and PqsC have a pseudo 2-fold symmetry that mimics the FabH homodimer as well as the presence of a catalytic diad instead of the typical catalytic triad in PqsBC, seen in other FabH family enzymes. The PqsCC129A active site volume is twice the volume as those of FabH enzymes or PqsD, with a calculated volume of 761Å3, compared to 389Å3 for PqsD and 367Å3 for FabH.The PqsBCC129A-Fe3+ crystal structure shows that Fe3+ binds to nitrogen atoms from PqsB His282 and PqsC His2 along with oxygen atoms from PqsB Glu48 and Glu280. Therefore, there are four bonds involved in the interaction between Fe3+ and PqsBCC129A. These bond lengths are very similar to those observed in the structures of Azurin and FutA1 complexed with iron. These crystal structures of PqsBC provide a unique insight into substrate recognition and establish a scaffold for structure guided drug design of novel antimicrobial agents.

No description available.

This thesis details research in three areas, in attempts to produce more effective inhibitors of the staphylococcal agr quorum sensing system. The non-ribosomal peptide synthase biosynthetic cluster responsible for the production of the aureusimines represents roughly 0.5% of the S. aureus genome. However, their function is yet to be elucidated. Research detailed herein develops a new reliable method for synthesis for these natural products. Efforts to discover the biological target or function of these compounds using affinity chromatography is reported. Further biological investigations revealed that the aureusimines are mild antagonists of the agr system. Weak inhibition of CCL-2 mediated chemotaxis of monocytes and staphylococcal biofilm formation is also observed. However, the main biological function of these natural pyrazinones is yet to be discovered. The staphylococcal bioreporter assays were used to eludicdate the structure-activity relationship of a series of truncated AIP-based antagonists against the AgrC1 receptor. Promising inhibitors are then evaluated against AgrC2, AgrC3 and AgrC4. Several compounds were found to be potent low nanomolar inhibitors across all four agr groups. A bioreporter assay based on the mutant receptor A101T T104V AgrC1 in which (Ala5)AIP1 is an agonist was also used to evaluate the panel of compounds. This revealed that most of these truncated AIP-based compounds are agonists of the mutant receptor, similar to (Ala5)AIP1. However, (Ala2, Leu4, Tfh5)trAIP1 (3.23) effectively inhibited activation of this bioreporter by AIP1. Compound 3.23 was also a sub-nanomolar inhibitor of AgrC1 and a low nanomolar inhibitor in other agr groups. Compound 3.23 is the most potent AgrC1 inhibitor discovered to date and, furthermore, its effects are likely to be less susceptible to mutations within the AgrC receptor. The depsipeptide natural products, solonamides, were synthesised using two uniquely different strategies. Development of a new synthetic strategy produced analogues with a high yield and diastereomeric excess in contrast with previous low yielding or non-stereoselective strategies. Their previously reported inhibition of the agr system was confirmed and fully quantified. Solonamide A and B inhibit activity in all four agr groups. Expression of toxic shock syndrome toxin-1 (TSST-1) and α-hemolysin were reduced in Staphylococcus aureus KH1187A. Schild analysis of data from agr bioreporters revealed that the inhibition is not competitive (as previously reported), but the solonamides act as negative allosteric modulators of both the AgrC1 and AgrC2 receptors, interacting with a new putative conserved allosteric binding site. Weak agonism at high concentrations was also discovered, which has not been previously observed. A panel of analogues was produced to assess the SAR with AgrC1. Modifications of the solonamide scaffold achieved mild improvements of physical characteristics and potency.

Trifluralina (a, a, a,trifluoro-2,6-dinitro-N, N-dipropil-p-toluidina) (TFL) é um herbicida pré-emergente, incorporado ao solo que tem sido usado na agricultura desde a década de sessenta; ele é moderadamente persistente em vários tipos de solos do Brasil. O objetivo deste estudo foi isolar - de um solo agrícola contaminado por quatro décadas - e caracterizar bactérias resistentes a TFL, determinar suas habilidades em degradar a TFL, investigar a presença de gens degardadores que possam estar envolvidos na degradação da TFL e propor um método de ensino teórico prático sobre a biodegradação, para cursos de graduação. Oito bactérias foram isoladas, pela técnica de subculturas repetidas em meio contendo TFL como única fonte de carbono, e identificadas, pelo método bioquímico e seqüenciamento do rDNA 16S como Klebsiella oxytoca, Herbaspirillum seropedicae, 3 strains of Bacillus simplex, 2 de Pseudomonas montelli e uma outra Pseudomonas sp. Uma terceira bactéria (iaolado #9), não identificada, que crescia ao redor de cristais de TFL em meio sólido, foi isolada; esta é uma técnica nova que poderá ser útil no isolamento de bactérias que são resistentes a outros compostos pouco solúveis em água. Todas as bactérias isoladas foram submetidas ao teste de biodegradação, em um meio contendo sais minerais, 0,1% succinato, 0,1 % de extrato de leveduras e 50 mg. L-1 de TFL Cinco bactérias reduziram a concentração de TFL no meio, após trinta dias de incubação: Klebsiella oxytoca (24,6 %), Herbaspirillum seropedicae (16,4 %), Bacillus simplex 2 (25.0 %), Bacillus simplex 3 (16.0 %) e isolado 9 (21.0 %). Uma bactéria conhecida como degradadora da TFL, Brevundimonas diminuta (NCIMB 10329) degradou a TFL, neste meio, de maneira semelhantes ao das bactérias isoladas. Os DNAs extraídos das quatro bactérias identificadas degradadoras da TFL, foram sondados para os gens catbólicos ndoB, todC, xylX, catA e xylE, os quais codificam as enzimas naftaleno 1,2-dioxigenase, toluene dioxigenase, toluate 1,2-dioxigenase, catecol 1,2-dioxigenase e catecol 2,3-dioxigenase, respectivamente. Técnicas de PCR e hibridização demonstraram que os DNAs de todas estas quatro bactérias foram fortemente hibridizadas para o gen ndoB, contudo, usando a técnica de ¨zonas claras¨, observou-se que nenhuma delas degradou naftaleno. Estes resultados indicam a presença de gens dioxigenases, nestas bactérias degradadoras da TFL, que poderiam estar transformando a TFL como substrato principal, ou como cometabolismo. O conhecimento sobre processos de biodegradação é necessário para os graduados dos cursos de agronomia, química, biologia, tecnologia de alimentos, etc. Neste trabalho, também, propomos o estudo teórico e prático da compostagem o qual estimula o interesse dos estudantes em aprender sobre o metabolismo envolvido neste, e em outros, processos biotecnológicos.

A canela-preta, causada por Pectobacterium carotovorum subsp. brasiliensis (Pcbr), está entre as principais doenças bacterianas da batata. Estudos epidemiológicos desta doença dependem de métodos eficientes de detecção. Como a principal característica das pectobactérias é a produção de enzimas pectolíticas em grande quantidade, os genes pnl e rdg, relacionados à pectina liase foram selecionados para a projeção de oligonucleotídeos iniciadores (primers) específicos para Pcbr. Os alinhamentos das seqüências de nucleotídeos dos genes pnl e rdg mostraram a existência de seqüências conservadas entre as estirpes de Pcbr, a partir das quais foram projetados os primers PcbrPnlF/ PcbrPnlR e PcbrRdgF/PcbrRdgR, que geraram produtos de 130 e 180 pb, respectivamente. Para a avaliação dos primers e métodos de extração de DNA Total, 10 hastes de plantas de batata com sintomas de canela-preta, foram coletadas de cada uma de quatro lavouras (Ágata: 3, Asterix: 1) no município de São Francisco de Paula, RS. Pcbr foi detectada através de PCR com PcbrRdgF/PcbrRdgR e DNA extraído por fervura e FTACard em 25 e 55% das amostras, e com PcbrPnlF/ PcbrPnlR em 12 e 45%, respectivamente. Estes resultados indicaram que os primers PcbrRdgF/PcbrRdgR foram mais eficientes, tanto na extração com FTACard (22%) quanto por fervura (108%). A detecção por PCR com ambos pares de primers foi maior quando o DNA foi extraído pelo método FTACard. / Blackleg, caused by Pectobacterium carotovorum subsp. brasiliensis (Pcbr), is among the major bacterial diseases of potato. Epidemiological studies of this disease depend on efficient methods of detection. As the main trait of pectobacteria is the production of large amounts of pectolytic enzymes, genes pnl and rdg, related to pectin liase, were chosen to project primers specific to Pcbr. The nucleotide sequence alignments of the genes pnl and rdg showed the existence of conserved sequences among Pcbr strains. Primers PcbrPnlF/PcbrPnlR (130 bp product) and PcbrRdgF/PcbrRdgR (180 bp product) were designed based on them. To evaluate them and the total DNA extraction methods, 10 stems of potato plants, with blackleg, were harvested from each of four fields (Ágata: 3; Asterix: 1) in the São Francisco de Paula county, RS. Pcbr was detected through PCR with PcbrPnlF/ PcbrPnlR and PcbrRdgF/PcbrRdgR primers and DNA extracted by boiling and FTACard in 25 and 55%, and in 12 and 45% of stems, respectively. These results indicated the PcbrRdgF/PcbrRdgR primers were more efficient with DNA extracted by both boiling (108%) and FTACard (22%). Pcbr detection using both primer sets was higher with DNA extracted by FTACard method.

O gênero Paenibacillus, anteriormente pertencente ao gênero Bacillus, tem como representantes bastonetes produtores de esporos que se diferenciam destes pelos padrões encontrados através da amplificação e seqüenciamento de um fragmento da subunidade 16S rDNA. Esse gênero é extremamente diverso sendo encontrado tanto em ambientes aquáticos como em ambientes terrestres, e seus representantes podendo ser desde patógenos de animais a fixadores de nitrogênio (associados ou não a plantas). Dos oitenta bastonetes Gram positivos esporulados isolados dos balneários de Ipanema, Belém Novo e Lami (Porto Alegre/RS), trinta e cinco foram identificados através de PCR como sendo do gênero Paenibacillus e dos sessenta isolados de solo provenientes de Belém Novo (Porto Alegre/RS) e Osório (RS), vinte e nove são pertencentes a este gênero. Nos isolados provenientes da água foram encontrados representantes de P. validus (8), P. azotofixans (6), P. illinoisensis (3), P. chibensis (3), P. glucanolyticus (3), P. borealis (2), P. koreensís (2), P. brasiliensis (2), P. odorífer (1), P. apiarius (1), P. graminís (1), P. macerans (1), P. pabuli (1) e P. thiaminolyticus (1). Entre os indivíduos provenientes de amostras de solo foram identificados P. alginolyticus (7), P. pabuli (5), P. azotofixans (3), P. validus (4), P. thiaminolyticus (2), P. chibensis (2), P. apiarius (2), P. glucanolyticus (2), P. macquariensis (1) e P. peoriae (1). A caracterização genética através de BOX-PCR produziu fragmentos de 4365 a 445bp e revelou vinte e oito grupamentos em um nível de similaridade de 73% nos isolados provenientes da água, e dezoito grupamentos a 70% nos isolados do solo. Pelas análises foi possível identificar um elevado grau de polimorfismo, com diferenças inter e intraespecíficas entre os isolados de Paenibacillus, isso sendo evidenciado pelo alto número de grupamentos encontrados. Os isolados de Paenibacillus provenientes da água e do solo puderam ser separados em grupamentos distintos através do BOX-PCR, sugerindo que populações distintas estejam estabelecendo-se nestes ambientes. / Paenibacillus genus, formerly Bacillus genus, are spore producer rod-shape that differ from Bacillus for the patterns found through 16S rDNA subunit amplification and sequencing. This genus is extremely diverse, being found both on aquatic and land environment, and it can be from animal pathogens to nitrogen fixer (associated or not to piants). From the eighty sporulated Gram positive rod-shape isolated from Ipanema, Belém Novo and Lami (Porto Alegre/RS), tirthy-five were identified through PCR as being Paenibacillus genus and, from the sixty isolated soul samples collected in Belém Novo (Porto Alegre/RS) and Osório (RS), twenty-nine belonged to this genus. Isolates of P. validus (8), P. azotofixans (6), P. iffinoisensis (3), P. chibensis (3), P. glucanolyticus (3), P. borealis (2), P. koreensis (2), P. brasiliensis (2), P. odorifer (1), P. apiarius (1), P. graminis (1), P. macerans (1), P. pabuli (1) and P. thiaminolyticus (1) were found on the isolated coming from water. Among the isolates coming from soil samples, it was identified P. alginolyticus (7), P. pabuli (5), P. azotofixans (3), P. validus (4), P. thiaminolyticus (2), P. chibensis (2), P. apiarius (2), P. glucanolyticus (2), P. macquariensis (1) and P. peoriae (1). The genetic characterization through BOX-PCR produced fragments from 4365 to 445bp and revealed twenty-eight groups with 73% similarity levei on water isolates, and eighteen groups with 70% levei on soil isolates. Through the ran out analysis a high degree of polymorphism were identified, with inter and intraspecific differences between the Paenibacillus isolates, what was observed with the high amount of groups found. The Paenibacillus isolates from water and soil could be separated in distinct groups through BOX-PCR, what suggests that different populations are being established in this environments.

A incidência de infecções microbianas relacionadas ao uso de cateteres vem aumentando. Uma característica importante dos microrganismos envolvidos é a capacidade de aderência à superfícies com conseguinte formação de colônia, produção de substâncias extracelulares, e subsequente formação de uma matriz compacta, amorfa, de múltiplas camadas, o biofilme. A atividade de bacteriocinas produzidas por Staphylococcus aureus e Staphylococcus epidermidis sobre microrganismos isolados de dispositivos intravasculares foi avaliada. As bacteriocinas Pep5 e Epidermina inibiram um maior número de isolados clínicos quando avaliadas in vitro por antagonismo direto, apresentando halos de inibição de 20 a 36 mm. A adição de Pep5 e Epidermina durante o crescimento de S. epidermidis e Corynebacterium sp resultou na redução do número de células viáveis. O efeito das bacteriocinas sobre a adesão bacteriana à cateteres de silicone foi avaliado através de três protocolos onde variou-se o momento de adição da bacteriocina: (A) simultaneamente, (B) previamente, e (C) posteriormente ao inóculo microbiano. As bacteriocinas Pep5 e Epidermina reduziram significativamente o número de células de S. epidermidis e Corynebacterium sp. aderidas aos cateteres após 6 h e 12 h de incubação. Não foram observadas diferenças importantes entre os três protocolos A, B, ou C. O efeito de Pep5 sobre Corynebacterium sp. foi verificado por microscopia eletrônica de varredura, podendo ser observadas áreas com resíduos celulares na superfície do cateter. / The in cidence of catheter-related microbial infections has been increasing. One important characteristic of related microorganisms is the adhesion to surfaces with colony formation, production of extracellular substances, and subsequent development of a compact, amorphous, multiple layer matrix, the biofilm. The activity of bacteriocins produced by Staphylococcus aureus and Staphylococcus epidermidis against microorganisms isolated from intravascular catheters was studied. The bacteriocins Pep5 and Epidermin inhibited a higher number of clinical isolates when evaluated by direct antagonism, showing inhibition zones of 20 to 36 mm. The addition of Pep5 and Epidermin during growth of S. epidermidis and Corynebacterium sp. resulted in a decrease of viable cell counts. The effect of bacteriocins on bacterial adhesion to silicon catheter was evaluated through three protocols differing in the time of bacteriocin addition: (A) simultaneously, (B) before, and (C) after the microbial inoculum. The bacteriocins Pep5 and Epidermin caused a significant reduction of the number of adhered cells of S. epidermidis and Corynebacterium sp. after 6 h and 12 h. No important differences among the protocols A, B or C were observed. The effe tions ct of Pep5 on Corynebacterium sp. was studied by scanning electron microscopy. Sec with cellular residues were observed on the catheter surface.

Rhodococcus equi is a soil-dwelling actinomycete with the ability to cause pyogranulomatous infections in different animal species and people. Young foals are particularly susceptible and develop a severe pulmonary illness known as rhodococcal pneumonia. The infection is endemic in many horse-breeding farms worldwide and poses a major challenge to the equine industry, as there is no commercial vaccine available. R. equi is a facultative intracellular pathogen. Intracellular survival in macrophages and hence virulence depends on the presence of large plasmids that carry a set of genes encoding virulence-associated proteins (Vaps) of largely unknown functions. Virulence plasmids are of different types and appear to determine host-specific infectivity for horses, pigs and cattle. In this thesis, I explored bacterial chromosomal factors that contribute to the virulence of R. equi. Previous microarray transcription profiling work from the laboratory showed that housekeeping metabolic genes from the R. equi chromosome were co-opted to serve a virulence function via co-regulation with plasmid virulence genes. Here, I identified a further virulence plasmid-co-expressed metabolic chromosomal locus with a key role in R. equi pathogenesis. The identified locus, gltAB1, encodes an NADPH-dependent glutamate synthase required for ammonia assimilation under low nitrogen conditions. Reverse-transcription quantitative rea-ltime PCR confirmed that gltAB1 was co-expressed with the vap genes from the plasmid whereas a homologous chromosomal locus encoding a second NADPH-dependent glutamate synthase, gltAB2, was not. In-frame deletion mutants were constructed and their virulence analysed. gltAB1 but not gltAB2, was found to be involved in virulence and required for intracellular proliferation in J774A.1 macrophages. The ΔgltAB1 mutant showed significant attenuation in vivo in a mouse infection model, in contrast to the ΔgltAB2, which behaved like the wild type. The ability of the ΔgltAB1 mutant strain to act as a live attenuated vaccine was tested in experiments in BALB/c mice. The mutant conferred protection against subsequent challenge of the animals with wild-type virulent bacteria, thus identifying a novel candidate vaccine for the control of R. equi pneumonia in foals. Furthermore, this thesis describes studies of the bovine-type plasmid, previously sequenced in our laboratory. The purpose of this work was to determine if VapN, the bovine-type allelic variant of the VapA protein encoded in the equine-type plasmid, was also essential for R. equi virulence. A plasmid-less derivative strain and a deletion mutant in the vapN gene were examined for their ability to proliferate in two different cell lines and to persist in BALB/c mice. These strains showed the same strong virulence attenuation observed with plasmid-less and ΔvapA strains derived from the equine isolate R.equi 103S, demonstrating that the bovine-type VapN protein also plays a central role in R. equi virulence. Additionally, the thesis includes preliminary work on approaches to explore the role and mechanism of Vap proteins in R. equi virulence. It also describes the construction of GFP-tagged R. equi strains for use in cell biological experiments and live imaging of infected cells.

Burkholderia pseudomallei (Bp) is the causative agent of melioidosis, a disease with a mortality rate of over 40%, and is a major public health concern in the endemic regions of Thailand and northern Australia. Bp is a resilient pathogen capable of surviving in diverse environments including soil, fresh and seawater, and plant and animal tissues for extended lengths of time. Bp is intrinsically resistant to antibiotics, which contributes to persistence and relapse in over 25% of melioidosis patients, and there is currently no vaccine. Our lab has previously shown that immunization with Bp outer membrane vesicles (OMVs) derived from Bp grown in Luria Burtani broth provides significant protection against melioidosis in mice. However, this protection was limited to the acute phase of infection and animals immunized with OMVs were unable to clear the bacteria. In this work, we show that by manipulating the growth media to simulate various bacterial niches, including the natural hypertonic soil environment (NaCl-supplemented), the limited-nutrient host macrophage intracellular environment (M9CG minimal media), and quorum sensing conditions (QS-molecule supplemented), OMV protein content can be modified to include those proteins potentially important for Bp survival and may contribute to protection against chronic or persistent infection. Here, we characterize the composition of selectively enriched Bp OMVs and demonstrate that enriched OMVs are non-toxic and well-tolerated both in vitro and in vivo. Immunization of BALB/c mice with QS OMVs elicits significantly greater OMV-, CPS-, and LPS- serum IgG along with cell-mediated immune responses compared to mice immunized with LB OMVs. LB, M9CG, and QS OMV immunization provided equal protection against aerosolized Bp through the acute phase of infection, and M9CG OMV-immunized mice demonstrated fewer signs of morbidity and less weight-loss over the course of infection, indicating potential control of the bacteria. These results suggest that immunizing with OMVs selectively enriched with intracellular proteins may elicit the necessary immune responses to protect against persistent melioidosis. / Nicole L Kikendall

No description available.