ETD

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Tesi etd-03242019-213145


Tipo di tesi
Tesi di laurea magistrale
Autore
BERTOCCI, DANIELE
URN
etd-03242019-213145
Titolo
Characterisation of the ESX-2 secretion system in Mycobacterium tuberculosis
Dipartimento
BIOLOGIA
Corso di studi
BIOTECNOLOGIE MOLECOLARI
Relatori
relatore Dott.ssa Bottai, Daria
Parole chiave
  • Mycobacterium tuberculosis
  • ESX-2
Data inizio appello
08/04/2019
Consultabilità
Non consultabile
Data di rilascio
08/04/2089
Riassunto
The genus Mycobacterium comprises Gram-positive, straight or slightly curved non-motile rods, aerobic and nonsporeforming bacteria, and is the only member of the family Mycobacteriaceae, within the order Actinomycetales. This genus includes environmental mycobacteria, mostly saprophytic, and pathogenic mycobacteria that cause disease in animals and humans. One of the most important human pathogen is Mycobacterium tuberculosis, the causative agent of tuberculosis. The characteristic human pulmonary lesion is the tuberculous, where M. tuberculosis can survive in a metabolically inactive state even for decades, and reactivate as consequent of natural or induced immunologic failure.
Mycobacteria possess peculiar secretion systems named ESX, encoded by five paralogous esx loci (esx-1 - esx-5). Each gene cluster contains: i) a pair of esx genes, encoding for small proteins which represent the main substrates exported by the corresponding secretion system; ii) a set of genes called esx conserved components (ecc) genes, which encode for structural components of the secretion apparatus (the membrane-bound EccB, EccC, EccD, and EccE proteins), and a cytoplasmic ATPase (EccA); iii) additional genes encoding for mycobacteria-specific membrane-bound proteases (MycP proteins) or additional genes encoding for esx secretion-associated proteins (Esp proteins). A functional role of specific ESX secretion systems in nutrient uptake (ESX-3) or in virulence and host-pathogen interactions (ESX-1, ESX-4, ESX-5) has been demonstrated. In contrast, the functional role of ESX-2 in mycobacterial physiology or in host-pathogen interactions is still unknown. Microarray expression studies demonstrated that esx-2 genes are regulated by WhiB5, a mycobacteria-specific transcription factor, which is required for M. tuberculosis growth in the murine model of tuberculosis reactivation. In in vitro models, WhiB5 is induced under nutrient depletion conditions (incubation in PBS- Phosphate-buffered saline). In order to investigate the impact of ESX-2 in M. tuberculosis survival/growth under nutrient depletion conditions, two different M. tuberculosis ESX-2 knock-out mutants were generated. The Mtb eccC2-KO mutant was inactivated for the eccC2 gene, predicted to encode for the membrane-bound ATPase EccC2 involved in the ATP-mediated translocation of ESX substrates. The MtbΔesxC-KO strain lacks the esxC gene, which encodes for a putative ESX-2-secreted effector protein EsxC. Both mutants were obtained by using a “Recombineering-based” strategy. According to this approach, a phage-encoded recombination protein (Che9c), expressed under control of an acetamidase-inducible promoter, enables homologous recombination using linear DNA substrates. The eccC2 gene inactivation and the esxC deletion in the corresponding mutant strains were verified by PCR analysis and by sequencing. The in vitro analysis of growth/survival properties of both ESX-2 mutants in comparison with the wild-type M. tuberculosis strain in nutrient rich standard media and in PBS, will provide information on the role of ESX-2 and its secreted substrates in mycobacterial survival during starvation a condition that mimics and nutrient depletion occurring in the tubercle.
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