Mutations in ppe38 block PE_PGRS secretion and cause hypervirulence in Mycobacterium tuberculosis

Mycobacterium tuberculosis requires a large number of secreted and exported proteins for its virulence, immune modulation and nutrient uptake. Most of these proteins are transported via the different typeVII secretion (T7S) systems that are present in this pathogen. The most recently evolved T7S system, ESX-5, is responsible for the secretion of dozens of substrates belonging to the PE and PPE families, which are named for conserved proline and glutamic acid residues close to the N-terminus. However, the role of these proteins remains largely elusive. Here we show that mutations in ppe38 lead to a complete block in the secretion of the two largest subsets of ESX-5 substrates, i.e. PPE-MPTR and PE-PGRS. Importantly, hypervirulent clinical M. tuberculosis strains of the Beijing lineage have a similar mutation and a concomitant loss of secretion. Restoration of PPE38-dependent secretion reverted the hypervirulence phenotype of such a Beijing strain, whereas mutation of the ppe38-locus in a moderately virulent M. tuberculosis strain resulted in increased virulence. Phylogenetic analysis of revealed that this mutation occurred at the branching point of ‘modern’ Beijing sublineages and is present in all analysed outbreak strains. Our results demonstrate that loss of secretion of specific PE/PPE subsets and increased virulence of M. tuberculosis strains are both caused by mutations in the ppe38-locus. These results indicate that these ESX-5 substrates play an important role in virulence attenuation. These observations could contribute to the success of the ‘modern Beijing’ lineage strains that cause major tuberculosis outbreaks worldwide.