Periodic Reporting for period 1 - Slow growth (Deciphering the molecular mechanism(s) behind the evolution of Mycobacterium tuberculosis towards slow growth, and the impact on virulence and persistence)
Período documentado: 2021-06-01 hasta 2023-05-31
Tuberculosis (TB) remains a major public health problem, with more than 1.5 million deaths per annum. An estimated further 1.7 billion people are latently infected and at risk of disease later in life. Worryingly, multidrug-resistant Mycobacterium tuberculosis strains resistant to first-line anti-TB drugs are continually emerging. The treatment of TB is very long: 6 months combining 4 antibiotics, and up to 2 years for a multidrug-resistant TB. Despite extensive drug discovery projects lead by companies and large public- private partnerships, TB remains one of the top 10 causes of human death worldwide and new strategies/treatments are needed to fight this disease.
The remarkable pathogenic success of M. tuberculosis is still not fully uncovered yet but is so far mainly attributed to: (i) its immune evasion capacity, (ii) its ability to persist in the host and cause latent infection and (iii) its aerogenic transmission to other hosts from diseased individuals. Recently, it has been described that ancestral mycobacteria were first fast-growing mycobacteria, with a single major evolutionary separation into fast- and slow-growing mycobacteria. Importantly, all the major human mycobacterial pathogens belong to the slow-growing mycobacteria, compared to avirulent and environmental fast-growing mycobacteria.
In this context, my project aimed to identify new mechanisms on mycobacterial virulence and persistence that are linked to the slow growth rate of M. tuberculosis, gaining fundamental insights and perspectives into host-mycobacterial interaction. This project may lead to innovative approaches to treat TB. Mycobacterium canettii appeared to be a unique resource to elucidate this question, as it presents a faster growth rate, a reduced virulence/persistence, and is known to be the closest ancestor of M. tuberculosis.
To investigate how M. tuberculosis has evolved towards a slow growth lifestyle and if slow growth promotes virulence and persistence, this proposal was divided into 3 work packages:
• The design of high-resolution single cell tools required to investigate growth rate, virulence and persistence of M. canettii.
• A global approach to identify slow grower M. canettii mutants, before assessing their virulence and persistence.
• A focused approach where mycobacterial strains are genetically modified to modulate their growth rate, before assessing their virulence and persistence.
The remarkable pathogenic success of M. tuberculosis is still not fully uncovered yet but is so far mainly attributed to: (i) its immune evasion capacity, (ii) its ability to persist in the host and cause latent infection and (iii) its aerogenic transmission to other hosts from diseased individuals. Recently, it has been described that ancestral mycobacteria were first fast-growing mycobacteria, with a single major evolutionary separation into fast- and slow-growing mycobacteria. Importantly, all the major human mycobacterial pathogens belong to the slow-growing mycobacteria, compared to avirulent and environmental fast-growing mycobacteria.
In this context, my project aimed to identify new mechanisms on mycobacterial virulence and persistence that are linked to the slow growth rate of M. tuberculosis, gaining fundamental insights and perspectives into host-mycobacterial interaction. This project may lead to innovative approaches to treat TB. Mycobacterium canettii appeared to be a unique resource to elucidate this question, as it presents a faster growth rate, a reduced virulence/persistence, and is known to be the closest ancestor of M. tuberculosis.
To investigate how M. tuberculosis has evolved towards a slow growth lifestyle and if slow growth promotes virulence and persistence, this proposal was divided into 3 work packages:
• The design of high-resolution single cell tools required to investigate growth rate, virulence and persistence of M. canettii.
• A global approach to identify slow grower M. canettii mutants, before assessing their virulence and persistence.
• A focused approach where mycobacterial strains are genetically modified to modulate their growth rate, before assessing their virulence and persistence.
During this project, we quickly realized that using M. canettii would not be successful, especially as the transposon mutant library showed already size heterogeneity on solid medium. Moreover, one of the high-resolution single cell tool could not be cloned into M. canettii. Therefore, we decided to mainly focus on the last work package to investigate if mycobacterial growth rate was linked to persistence/virulence. We successful engineered mycobacterial strains expressing differently the ribosomal RNA operon (coding for major components of the ribosome, involved in protein synthesis, and known to be a limiting factor for growth rate). After a deeper investigation, we realized that the growth rate of the mutants was not affected, nor the virulence and persistence.
Interestingly, although the growth rate of the mutants was identical to the wild type's, mycobacterial cell size was affected in the mutants, with bacteria being smaller than the wild-type. Following a transcriptomics analysis, we showed that one third of the genes that are differentially expressed in the mutants, compared to the wild-type belongs to the cell wall biogenesis, cell division and elongation families. This project therefore unraveled an unexpected link between the ribosomal RNA operon and mycobacterial size regulation. Further investigations will be necessary to fully understand the impact of mycobacterial cell size modulation at a host-pathogen level is.