Caratterizzazione dei fattori sigma micobatterici SigE e SigF Caratterizzazione del dominio PPE della proteina PPE17 di Mycobacterium tuberculosis

Abstract Mycobacterium tuberculosis (MTB) is the causative of tuberculosis, a disease, which causes 2 millions of death every year, with a dramatic incidence especially in developing countries. To find new drug and vaccine strategies against MTB, it is of fundamental importance to study the mechanisms, that allow its survival to environmental stresses, to which it is subjected during the period of infection and latency in the host macrophages. The fine transcriptional regulation of specific genes in response to stress conditions and the peculiar structure of its wall play a key role on this. In the first part of the PhD project two mycobacterial sigma factors, SigE and SigF, which regulate the transcription of specific genes in response to various environmental stresses such as surface stress, oxidative stress, alkaline pH and thermal shock, have been characterized. First the transcriptional regulation, translational and post-translational regulation of the extracytoplasmic function (ECF) transcription factor SigE were studied. Regarding the study of the transcriptional regulation, it was possible to confirm by 5'RACE PCR and RT-PCR experiments the presence of three promoters of sigE, and to determine the contribution of each promoter in the transcription of this gene, depending on the environmental conditions of bacterial growth. The fact, that the transcriptional start codon of one of these promoters is located 63 base pairs downstream of the start codon annotated in MTB genome opened the possibility of the existence of two isoforms of Sige. By translational fusions between specific sequences of sigE with lacZ, deprived of its own translational initiation codon, and subsequent site-specific mutagenesis, it was possible to confirm, based on further beta-galactosidase activity detection, the existence of two alternative start codons, an ATC and a TTG, coding for an isoform of respectively 218 and 215 of amino acids, in addition to the ATG already annotated in MTB genome, which encodes for an isoform of 257 amino acids. Finally, it was possible to confirm, that the gene downstream sigE encodes for the anti-sigma factor of SigE, called RseA, capable of binding both isoforms of SigE. In a second project also the role of the factor SigF M. smegmatis in the biosynthesis of carotenoid pigments, resistance to hydrogen peroxide and in the efficiency of bacterial transformation was studied. By RT-PCR it has been shown, that SigF controls the transcription of genes involved in the biosynthesis of carotenoid pigments, and, assuming that they serve as protection against free radicals, it was verified that the sigF mutant strain is actually more sensitive compared to the wild type strain to treatment with hydrogen peroxide. Finally, we also demonstrated, that the mutant strain has a higher transformation efficiency than the wild type strain, indicating that SigF regulates the transcription of genes possibly involved in the permeability of the cell wall. In the second part of the project, the localization of the protein on the surface PPE17 mycobacteria was characterized. Like other members of the PPE family, the PPE17 has a highly conserved N-terminal domain, which, based on different evidences in literature, is assumed to play an important role in their translocation to the mycobacterial surface. Moreover, it was investigated the possible influence of the presence of PE11 in the translocation process or in the stability of PPE17, as the PE11 coding sequence is in tandem and co-transcribed with that encoding the PPE17, and there is a specific interaction between these two proteins. The data obtained by proteinase K sensitivity assays performed on M. smegmatis strains, expressing the entire PPE17 or only its domain PPE (dPPE17) fused with the HA epitope, confirm, that the entire PPE17 is exposed on the surface, both in the presence and absence of PE11. According to data obtained, the possibility to translocate the MTB model antigen (Mpt64) on the surface of the vaccine strain M. bovis BCG, by fusing them with the dPPE17 was tested. Proteinase K and whole cell ELISA assays performed on cultures of M. bovis BCG expressing this chimeric protein indicate, that it is indeed localized at the mycobacterial surface. Similarly, another two fusions with dPPE17 were constructed to express on the mycobacterial surface the multimeric MTB antigen AG85-ESAT6 of MTB and the Csp C3 antigen of Plasmodium bergii. According to the proteinase K sensitivity assays carried out on strains of M. smegmatis expressing the two chimeric proteins indicate that also in this case both are localized at the surface. The strains of M. bovis BCG expressing these antigens on their surface will be tested in future in the mouse model to measure any increase in protection compared to the wild type strain.