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| Author(s): |
Ana Laura Boechat Borges
Total Authors: 1
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| Document type: | Master's Dissertation |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ) |
| Defense date: | 2008-10-15 |
| Examining board members: |
Regina Lúcia Baldini;
Frederico José Gueiros Filho;
Julio Cezar Franco de Oliveira
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| Advisor: | Regina Lúcia Baldini |
| Abstract | |
Pseudomonas aeruginosa is a ubiquitous gammaproteobacteria able to infect immunocompromised individuals and to cause nosocomial infections. Between two direct repeats in the PAPI-1 pathogenicity island present in strain PA14, there are two gene clusters transcribed in opposite directions. The first, composed of pvrS, pvrR, rcsC and rcsB, encodes two-component systems proteins and is implicated in virulence, whereas the second comprises five genes (cupD1-D5) encoding a chaperone-usher fimbria with high similarity to cupA, a gene cluster involved in biofilm formation in other strains of P. aeruginosa. Fimbriae belonging to the same family of Cup are related to pathogenicity of other bacteria. In order to study the relationship between these two clusters, the organization of the genes in operons was characterized using RT-PCR, which results lead to the conclusion that the twocomponent systems genes are arranged into two different operons (pvrSR and rcsCB) and that cupD1-D2 share the same promoter, with some evidences that the operons extends from cupD1 to cupD5. The transcription start sites and the promoter regions of each operon were characterized with RACE 5 and primer extension assays to look for sequences that could be relevant to the activation of expression of these genes. Quantitative RT-PCR assays were carried out to investigate whether the expression of CupD fimbriae is regulated by the twocomponent systems encoded by the adjacent genes and the results showed a lower expression of cupD in the rcsB mutant strain than in the wild-type. RcsB bears a DNA-binding domain and, although our assays of DNA-protein interactions have failed, data obtained by qRT-PCR 1 strongly indicate that this protein functions as a transcription activator of fimbrial genes. These findings were corroborated by the primer extension assay, in which the band corresponding to the transcriptional start of cupD1-D2 was visible only when the reaction was performed with the RNA extracted of P. aeruginosa overexpressing RcsB. Unlike the effect observed for RcsB in cupD1, cupD2 and cupD5 transcription, the histidine quinase RcsC acts negatively in the fimbrial genes expression, suggesting that it might function predominantly as a RcsB phosphatase. PvrS and PvrR seem to regulate cupD positively and indirectly. As a second aim of this work, virulence assays of P. aeruginosa in the model host Dictyostelium discoideum were optimized, and a technique for testing genes studied in the laboratory that could be important for Pseudomonas virulence was established. These assays confirmed the attenuation-in-virulence of strains mutant in a putative methyltransferase gene, as observed before in plant, mouse and drosophila models. The results obtained in this work may contribute to P. aeruginosa infection control research, since this bacterium depends on fimbriae to successfully colonize abiotic surfaces that act as a dissemination vehicle, and to allow the bacteria to persist into the host organism. (AU) | |
| FAPESP's process: | 06/02163-0 - Genes involved with Pseudomonas aeruginosa PA14 pathogenicity: characterization of promoter regions and virulence assays in Dictyostelium discoideum host model |
| Grantee: | Ana Laura Boechat Borges |
| Support Opportunities: | Scholarships in Brazil - Master |