Caracterización de los mecanismos de captación de hierro de Pasteurella Multocida
In the present work, the Pasteurella multocida iron uptake mechanisms have been characterised. These results could be use to the obtention of one vaccine against this pathogen.
The P. multocida fur gene has been identified and it has been demonstrated that the porine OmpH is under the control of the Fur protein. On the other hand, a P. multocida Fur mutant was constructed and its LD50 was calculated demonstrating that a mutation in this gene has no effect in the virulence of the strain.
Moreover, the region which contains P. multocida exbB, exbD and tonB genes has been characterised. The results obtained demonstrate that these three genes do not constitute a single transcriptional unit, but all these genes possess its own promoter which is under the Fur-Fe(II) regulation. It has been obtained a ExbB, ExbD and TonB mutants and these mutants present a decrease in their virulence to increase their LD50 by more than three orders of magnitude when were inoculated via intraperitoneal. These data demonstrate that exbB, exbD and tonB genes are essential to P. multocida infectious process.
Furthermore, the P. multocida Pm70 genome was analysed and nine putative haemin and haemoglobin-binding proteins have been identified by similarity with the same molecules of other bacterial pathogens. Quantitative binding assays have demonstrated that PM0040, PM0236, PM0300, PM0741, PM1081 and PM1428 bind both haemin and haemoglobin, whereas PM0576 and PM1282 only bind either haemoglobin or haemin, respectively. PM1078, despite presents similarity with a Yersinia enterocolitica haemin receptor, does not recognize none of these iron sources.
PM0300 was named hgbA and it has been demonstrate that this gene and the PM0298 and PM0299 ORFs, which present similarity with hugX and hugZ of Plesiomonas shigelloides, respectively, constitute a single transcriptional unit. PM0298 and PM0299 are essential to the viability of P. multocida cells. The hgbA gene is expressed in vivo within the two first hours post-inoculation, and in vitro is repressed by iron. Moreover, a HgbA mutant binds haemoglobin to the same extent as the wild-type strain and, in agreement with this, virulence of the P. multocida hgbA cells was no affected. On the other hand, the hgbA gene is present in almost all the strains of P. multocida analysed, independently of their serotype or their animal source. This result propose hgbA as a putative candidate to develop a fast and specific method to identify this pathogen.
Finally, the immumogenicity and the ability to induce protection of all these haemin and haemoglobin receptors were analysed. The results obtained have demonstrated that, despite PM0236, PM0741 and HgbA are immunogens, inoculation of mice with any single one of these binding proteins alone is not protective against P. multocida infection.
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Advisor:Barbe García, Jordi; Llagostera Casas, Monterrat
School:Universitat Autónoma de Barcelona
Source Type:Master's Thesis
Keywords:409 departament de genetica i microbiologia
Date of Publication:06/06/2003