• mycobacterium
• tuberculosis
• esx
• attenuation
• macrophages

Mycobacterium tuberculosis (Mtb), the etiological agent of human tuberculosis, is the second leading infectious killer after Sars-CoV2 (above HIV). In 2020, the World Health Organization has estimated that M. tuberculosis has been responsible for about 9.9 million new tuberculosis cases and 1.5 million deaths worldwide. Nowadays, the main worries concerning tuberculosis are the emerging multidrug-resistant strains and the reactivation of pulmonary tuberculosis in immuno-compromised patients, especially HIV-co-infected or elderly population. Furthermore, the COVID-19 pandemic has forced a slowdown in TB treatment and an increase of misdiagnoses in settings where COVID-19 testing is unavailable.
The identification of improved antimycobacterial therapeutic regimens as well as the development of new vaccines are thus becoming increasingly urgent. The only currently licensed tuberculosis-vaccine, the Mycobacterium bovis Bacille de Calmette-Guerin (BCG), displays variable and partial protective efficacy. One of the main causes of BCG low immunogenicity is the loss of the functional type VII secretion system ESX-1 and its secreted key immunodominant antigens (ESAT-6 and CFP-10).
The main vaccination strategies involving attenuated microorganisms are based on recombinant BCG (e.g., VPM1002, a recombinant BCG strain expressing the listeriolysin O from Listeria monocytogenes) or attenuated strains of M. tuberculosis (e.g., MTBVAC, Mtb strain lacking two major virulence factors: PhoP and Fad26). However, neither of these strains owns a functional ESX-1 system. In the attempt to combine safety and optimal immune-system stimulation, new vaccination strategies are aimed at the development of alternative vaccine candidates, attenuated in virulence but still able to express functional ESX-1 and other type VII ESX secretion systems (ESX-3 and ESX-5), playing a key role in immunomodulation and host-pathogen interaction. In this scenario, genes associated to esx loci, encoding for proteins involved in mycobacterial virulence but not strictly related to the activity of the corresponding secretion system might represent interesting targets of deletion in Mtb, in order to obtain immunogenic and attenuated vaccine candidates.
This thesis was focused on the characterization of selected genes associated to ESX secretion systems. A panel of Mtb mutant strains deleted for selected esx genes has been characterized in terms of growth kinetics and virulence properties in different experimental models. Furthermore, the characterization of the expression profiles of selected cytokines (IL-1ß, INF-1, IL-6) in human THP1 cell line infected with mutant and wild-type Mtb strains has been performed.