Numerous species of the genus Staphylococcus other than Staphylococcus aureus are important pathogens in human clinical practice and veterinary medicine. With improved methods of identification and more precise classification, we have speciated over 500 strains of staphylococci representing 17 species and subspecies of non-S. aureus Staphylococcus. We have examined these strains for possible virulence factors which may play a role in their pathogenesis. Using transmission electron microscopy (TEM), we have demonstrated that small to large amounts of glycocalyx are found on staphylococcal cells. Animal models have shown that staphylococci cause abscess formation in the presence or absence of a foreign body implant. Molecular characterization of cell extracts of Staphylococcus intermedius show that this species elaborates a protein which is serologically similar to the enterotoxins of Staphylococcus aureus in ELISA tests, but differs markedly in other characteristics.

No / Streptococcus agalactiae is a significant pathogen causing invasive disease in neonates and thus an understanding of the molecular basis of the pathogenicity of this organism is of importance. N-terminal lipidation is a major mechanism by which bacteria can tether proteins to membranes. Lipidation is directed by the presence of a cysteine-containing 'lipobox' within specific signal peptides and this feature has greatly facilitated the bioinformatic identification of putative lipoproteins. We have designed previously a taxon-specific pattern (G+LPP) for the identification of Gram-positive bacterial lipoproteins, based on the signal peptides of experimentally verified lipoproteins (Sutcliffe I.C. and Harrington D.J. Microbiology 148: 2065-2077). Patterns searches with this pattern and other bioinformatic methods have been used to identify putative lipoproteins in the recently published genomes of S. agalactiae strains 2603/V and NEM316. A core of 39 common putative lipoproteins was identified, along with 5 putative lipoproteins unique to strain 2603/V and 2 putative lipoproteins unique to strain NEM316. Thus putative lipoproteins represent ca. 2% of the S. agalactiae proteome. As in other Gram-positive bacteria, the largest functional category of S. agalactiae lipoproteins is that predicted to comprise of substrate binding proteins of ABC transport systems. Other roles include lipoproteins that appear to participate in adhesion (including the previously characterised Lmb protein), protein export and folding, enzymes and several species-specific proteins of unknown function. These data suggest lipoproteins may have significant roles that influence the virulence of this important pathogen.

No / Streptococcus equi is the aetiological agent of strangles, one of the most prevalent diseases of the horse. The animal suffering and economic burden associated with this disease necessitate effective treatment. Current antibiotic therapy is often ineffective and thus recent attention has focused on vaccine development. A systematic understanding of S. equi virulence, leading to the identification of targets to which protective immunity can be directed, is a prerequisite of the development of such a vaccine. Here, the virulence factors of S. equi are reviewed.

Histophilus somni virulence factors include expression and antigenic variation of lipooligosaccharide (LOS). Phosphorylcholine (ChoP) is often expressed on H. somni LOS and also undergoes antigenic variation. In this study, five genes that play a role in expression and antigenic variation of ChoP, lic1ABCD and glpQ, were identified in the genome sequence of H. somni through sequence homology with Haemophilus influenzae genes. The open reading frame (ORF) of lic1A contained a variable number of tandem repeats of the tetranucleotide unit 5'-AACC-3'. Slipped strand mispairing in the repeat region during replication leads to shifting the downstream reading frame in and out of frame with the start codon, thus controlling phase variation of lic1A expression. Removal of the repeats from lic1A, cloning the gene in E. coli, and performing a functional assay on the product indicated that lic1A encodes a choline kinase and that the repeats were not required for expression of a functional gene product. Variation in the number of repeats in lic1A correlated with the antigenic variation of ChoP expression in strain 124P, but not in strain 738. This result supported previous findings that antigenic variation of ChoP expression in strain 738 is controlled through extension/truncation of the LOS outer core. Therefore, these results indicated that the lic1ABCD and glpQ genes control expression and antigenic variation of ChoP on the LOS of H. somni and that there are two possible mechanisms for ChoP antigenic variation. The role of H. somni expression of ChoP in colonization of the host respiratory tract was also examined. Experimental infection in the natural host showed that the population of H. somni that expresses ChoP was enriched in the bacteria that colonized the respiratory tract. In addition, bacteria expressing ChoP were able to aggregate bovine platelets through binding to the platelet activating factor receptor (PAF-R), which is also present on epithelial and endothelial cells. These results indicated that ChoP may play a role in the process of colonization and subsequent systemic invasion of host tissues, which may occur through binding of ChoP to PAF-R. Bacteria that did not express ChoP were more prevalent in systemic sites, indicating that ChoP expression may be disadvantageous for the organism during systemic dissemination. / Ph. D.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Vibrio vulnificus are marine bacteria causing fatal septicemia through wound infections or consumption of contaminated seafood. V. vulnificus is an excellent model for the study of surface polysaccharides, as it is capable of synthesizing capsular polysaccharide (CPS), lipopolysaccharide (LPS) and exopolysaccharide (EPS). V. vulnificus strains exhibit a multitude of carbotypes that evolve through unknown mechanisms. CPS is a confirmed virulence factor, but the genetics of its biosynthesis are unknown. The main objective of these experiments was to gain insight into the biosynthesis, regulation and evolution of ATCC 27562 outer surface polysaccharides. A miniTn10 transposon (Tn) system was used for mutagenesis and single insertions were confirmed through Southern analysis. A novel 25 kb CPS biosynthesis locus was identified through sequencing of regions surrounding Tn insertions; a region encoding putative LPS core biosynthetic functions was identified adjacent to the CPS cluster. The CPS locus contained features of O-antigen biosynthetic loci and was unusual in carrying characteristics of both group I and IV capsular biosynthetic loci. Mutations in this region resulted in elimination of CPS and LPS, and both were shown to be dependent on the activity of the polymerase Wzy. Evidence is presented here supporting horizontal transfer (HT) as a contributor to V. vulnificus CPS evolution. CPS regions of V. vulnificus 27562, YJ016 and CMCP6 contain strain specific genes surrounded by conserved regions, suggestive of HT. Moreover, a CPS locus virtually identical to that of 27562 was discovered in Shewanella putrefaciens strain 200. 27562 CPS is distinctive as it contains N-acetylmuramic acid. Genes encoding murA and murB activities were identified within the cluster and shown to be functionally redundant, supporting HT acquisition of this region. A screen of V. vulnificus gDNA library using CPS biosynthesis and transport mutants identified a cyclic diguanylate cyclase, dcpA. dcpA-mediated increase in cyclic diguanylate lead to EPS production, rugosity phenotypes and enhanced biofilm formation. Interestingly, virulence and motility were not affected suggesting complexity of cyclic diguanylate regulation in V. vulnificus, supported by the large number of cyclic diguanylate related proteins in Vulnificus strains.

Blastocystis spp. est un Straménopile parasite anaérobie fréquemment rencontré dans le tractus gastro-intestinal de l’homme et de divers animaux. Ce parasite est associé à des troubles gastro‐intestinaux aspécifiques, et semble impliqué dans des désordres fonctionnels tels que le syndrome de l’intestin irritable (IBS). Ce travail de thèse s’appuie sur le séquençage du génome de Blastocystis sp. ST7 réalisé en collaboration avec le Génoscope d’Evry, l’Université Nationale de Singapour, l’Institut Pasteur de Lille et l’Université de Provence. Ce génome est constitué d’un génome nucléaire de 18,8 Mpb pour 6020 gènes, et d’un génome mitochondrial de 29 kpb localisé dans des organites apparentés aux mitochondries. L’analyse de ce génome apporte des informations au niveau de l’évolution de ce microorganisme, de son adaptation à l’environnement intestinal et de ces facteurs de virulence potentiels. En effet, les analyses in silico de ce génome ont montré que Blastocystis sp. ST7 possède plusieurs gènes codant des protéines pouvant agir à l’interface entre l’hôte et le parasite et connues chez d’autres protozoaires pour être impliquées dans des phénomènes de pathogénie. Ce sont en particulier des PKS, des NRPS, et des hydrolases dont des protéases. D’autre part, des activités protéolytiques ont été mises en évidence expérimentalement dans les surnageants de culture du parasite. Deux protéases à cystéines (une cathepsine B et une légumaïne) pouvant être impliquées dans la physiopathologie du parasite, ont été identifiées et caractérisées dans les surnageants, confirmant ainsi nos analyses in silico. Ce travail ouvre de nombreuses pistes intéressantes à explorer pour évaluer l’impact de ce parasite en santé humaine. / Blastocystis spp. is a highly prevalent anaerobic Stramenopile parasite found in the intestinal tract of humans and various animals. This parasite is associated with non specific intestinal disorders, and could be involved in functional disorders such as the irritable bowel syndrome (IBS). In this work, the Blastocystis sp. ST7 genome sequencing project was carried out in collaboration with the Génoscope of Evry, the National University of Singapore, the Pasteur Institute of Lille and the University of Provence. This genome consists in a nuclear genome of 18,8 Mpb encoding 6020 genes, and a mitochondria‐like genome of 29 kpb localised in the mitochondrion‐like organelles. The analysis of this genome brings information about the evolution of this micro‐organism, its adaptation to the intestinal environment and its potential virulence factors. Blastocystis sp. ST7 was predicted to harbor several genes coding proteins that could act at the parasite‐host interface, and that are known to be involved in the pathogeny of many protozoa. They are PKS, NRPS, and hydrolases among them proteases. In addition, proteolytic activities were highlighted in the parasite culture supernatants. Two cysteine proteases (a cathepsin B and a legumain) were identified and characterized from the supernatants and could play a role in the physiopathology of the parasite, that confirm our in silico analyses. This work opens new ways to evaluate the impact of this parasite in human health.

Les champignons sont les principaux agents pathogènes des plantes. Leur étude est donc essentielle pour contrôler les maladies et maintenir un bon rendement de production agricole. La nutrition de ces pathogènes est basée sur l'absorption de nutriments, préalablement dégradés par un arsenal d'enzymes lytiques secrétées. La sécrétion des protéines est assurée par le trafic intracellulaire mettant en jeu de nombreuses vésicules. Chez les champignons filamenteux, ces vésicules ont été visualisées en microscopie électronique mais le processus mis en jeu pour leur biogénèse n'est toujours pas élucidé. L'identification de ce mécanisme est un donc un prérequis pour comprendre la sécrétion de facteurs de virulence. Dans ce but, un mutant non pathogène altéré au niveau de l'expression du gène codant la chaine lourde de la clathrine a été sélectionné parmi une banque de mutants générés chez le champignon nécrotrophe Botrytis cinerea. Le gène codant pour la chaine lourde de la clathrine est essentiel chez de nombreux organismes, ainsi un mutant dominant négatif de la chaine lourde de la clathrine a été généré et confirme la perte de pathogénicité. La caractérisation du mutant par une approche de protéomique a mis en évidence un défaut de sécrétion de 82 protéines incluant des facteurs de virulence connus. Un défaut de production de vésicules intracellulaires a également été constaté. Par ailleurs, le marquage de la clathrine à la GFP a permis de préciser sa localisation dans les cellules fongiques. Enfin, de façon surprenante, aucun défaut d'endocytose n'a été constaté au sein des mutants déficients en clathrine. Cette étude met en évidence pour la première fois le rôle essentiel de la clathrine dans le processus infectieux d'un champignon pathogène ainsi que son rôle dans a sécrétion de facteurs de virulence / Fungi are the most important plant pathogens on agricultural and horticultural crops. Study of fungal pathogens remains essential to understand pathogenic process and control plant diseases. These organisms secrete high amount of degrading enzymes involved in plant decomposition and they feed by absorption of degraded nutriments. Secretory proteins were described to be transported form Endoplasmic Reticulum and Golgi apparatus to extracellular space through intracellular vesicles. In filamentous fungi, intracellular vesicles were observed using electron microscopy but their biogenesis process is still unknown. Therefore, elucidation of the process and the identification of proteins involved in secretory vesicles biogenesis remains a challenge to understand virulence factors delivery. A nonpathogenic mutant altered in the expression of the gene coding for clathrin heavy chain was selected in a random mutant library generated in the necrotrophic pathogen Botrytis cinerea,. This gene is essential in many organisms, thus a clathrin dominant negative mutant was generated and confirming the nonpathogenic phenotype observed on several host plant. In eukaryotic cells, clathrin heavy chain is mainly described to be involved in endocytosis, but it is also essential for high density secretory vesicles formation in yeast. Characterization of the mutants using a proteomic approach revealed a secretion defect of 82 proteins including known virulence factors, as Plant Cell Wall Degrading Enzymes and elicitors. Furthermore, the clathrin mutant revealed a strong reduction of intracellular vesicles production. Clathrin was also localized in living cells using fluorescent GFP-tag protein. Endocytosis was also studied and surprisingly, any observable defect was observed for clathrin mutants. This study demonstrated for the first time the essential role of clathrin in the infectious process of a fungal pathogen and its role in virulence factors secretion

Um total de 123 isolados de Pasteurella multocida provenientes de suínos foi avaliado através da PCR para pesquisa de genes codificadores de capsula, toxina dermonecrótica e outros fatores de virulência. As amostras foram caracterizadas como tipo capsular A (78,8 %) e tipo capsular D (21%). Nenhum dos isolados foi positivo para presença de toxina dermonecrótica. Os genes codificadores dos fatores de virulência pesquisados foram observados nas freqüências de 93,5 % para nanB, 92,7% para psl, 91,9% para oma87 e nanH, 87,8% para sodA,87% para hghA,83,7% para ompH, 82,9% para sodC, 79,7% para ptfA e exbBD tonB,73,2% para hgbB, 14,6% para pfhA e 4,9% para tbpA. Estes achados são compatíveis com o descrito na literatura internacional. / A total of 123 Pasteurella multocida strains from swine was evaluated through PCR to detect capsular, dermonecrotic toxin codifying genes and others virulence factors. The strains were identified as capsular type A (78.8 %) and capsular type D (21%). None of isolates were positive to dermonecrotic toxin gene. The virulence factors genes were detected in the following frequency: 93.5 % to nanB, 92.7% to psl, 91.9% to oma87 and nanH, 87.8% to sodA, 87% to hghA, 83.7% to ompH, 82.9% to sodC, 79.7% to ptfA and exbBD tonB, 73.2% to hgbB, 14.6% to pfhA and 4.9% to tbpA. These findings were in accordance with international literature.

O presente trabalho teve como objetivo isolar e identificar os microrganismos aeróbicos e a frequência de isolamento de Escherichia coli patogênica ao homem em fezes de cães sem sintomas de colibacilose e assim averiguar a participação do cão como fonte de infeção de colibacilose humana. No período de setembro de 2002 a outubro de 2004, foram coletadas 220 amostras de fezes de animais capturados pelos CCZ de Guarulhos, Cotia e Barueri, cidades do Estado de São Paulo. O método de coleta foi através de swabs estéreis profundamente ao intestino dos animais anestesiados, mantidos sob refrigeração e em caldo BHI. As bactérias foram isoladas por semeadura em Mac Conkey, ágar sangue e Sabouraud. Houve crescimento de microrganismos em 100% delas. Foram isolados os seguintes microrganismos: 120 (54,54%) estirpes de E. coli em cultura pura e 76 (34,54%) estirpes em associação com outros agentes, Proteus mirabilis (2,27%), Klebsiella pneumoniae (2,27%), dentre outros microrganismos. Deve-se ressaltar ainda o isolamento de leveduras (Candida albicans) em associação com outros agentes a partir de 05 (2,27%) amostras de swabs retais. Uma vez isoladas, seus genes de virulência foram detectados por PCR. De um total de 196 estirpes de E. coli isoladas, 123 (62,75%) apresentaram um ou mais dos fatores de virulência estudados. Dessas 196 estirpes, 16 (8,16%) foram positivas para afa, 54 (27,55%) foram positivas para sfa, 38 (19,38%) foram positivas para pap, 66 (33,67%) foram positivas para aer, 31 (15,81%) foram positivas para cnf, 13 (6,63%) foram positivas para hly, 01(0,51%) foi positiva para VT2 e nenhuma das linhagens foi positiva para LT, STa e STb. Os cães aparentemente sadios, sem sintomas de colibacilose, poderiam estar participando da cadeia epidemiológica como reservatórios de E. coli uropatogênica ao homem, pois os genes encontrados com maior número foram aer, sfa e pap presentes em infecções extraintestinais, mais especificamente infecções urinárias. Os cães podem participar como reservatório de estirpes de E. coli resistentes a antimicrobianos. Das amostras de E. coli testadas, 85,64% apresentaram resistência à Cefalotina e 0% de resistência à Norfloxacina. Há a necessidade de mais pesquisas sobre o modo de transmissão das E. coli patogênicas entre o cão e o homem e dos fatores de virulência uropatogênicos encontrados com maior frequência tais como aer, sfa e pap. / The objective of the present study was to isolate and identify aerobic microorganisms and the isolation frequency of E. coli pathogenic to man in faeces of dogs without colibacillosis symptoms and thus to verify the participation of dog as a source of human colibacillosis infection. In the period between September 2002 to October 2004, 220 samples of faeces were collected from animals captured by the CCZ of Guarulhos, Cotia and Barueri, cities in the São Paulo state. The collection method used was barren swabs deeply to the intestine of anesthetized animals, which were kept under refrigeration and in a BHI broth. The bacteria were isolated through sowing in Mac Conkey, agar blood and Sabouraud. In 100% of them there was microorganism growth. The following microorganisms were isolated: 120 (54.54%) E. coli lineages in pure culture and 76 (34.54% lineages in association with other agents, Proteus mirabilis (2,27%), Klebsiella pneumoniae (2.27%,) amongst other microorganisms. The isolation of yeasts (Candida Albicans) in association with other agents from 05 (2.27%) rectal samples swabs must be emphasized. Once isolated virulence genes were detected by PCR. Of a total of 196 isolated lineages of E.coli, 123 (62.75%) presented one or more of the studied virulence factors. Of these 196 lineages, 16 (8.16%) were positive for afa, 54 (27.55%) positive for sfa, 38 (19.38%) positive for pap, 66 (33.67%) positive for aer, 31 (15.81%) positive for caf, 13 (6.63%) positive for hly, 01 (0.51%) positive for VT2 and none of the lineages were positive for LT, Sta and STb. Apparently healthy dogs, without colibacillosis symptoms could be participating in the epidemiological chain as an E. coli reservoir uropathogenic to man, as the genes found with high frequency were aer, sfa and pap present in extraintestinal infections, more specifically urinary infections. The dogs can participate as a reservior of E. coli lineages resistant to antimicrobials. Of the E. coli samples tested, 85,64% presented resistance to Cefalotina and 0% resistance to Norfloxacina. More research is needed on how the transmission of pathogenic E. coli between the dog and man happens and also on the uropathogenic virulence factors found more frequently, such as aer, sfa and pap.