Natural mutations in the sensor kinase of the PhoPR two-component regulatory system modulate virulence of ancestor-like tuberculosis bacilli.

The molecular factors and genetic adaptations that contributed to the emergence of Mycobacterium tuberculosis (MTB) from an environmental Mycobacterium canettii-like ancestor, remain poorly investigated. In MTB, the PhoPR two-component regulatory system controls production and secretion of proteins and lipid virulence effectors. Here, we describe that several mutations, present in phoR of M. canettii relative to MTB, impact the expression of the PhoP regulon and the pathogenicity of the strains. First, we establish a molecular model of PhoR and show that some substitutions found in PhoR of M. canettii are likely to impact the structure and activity of this protein. Second, we show that STB-K, the most attenuated available M. canettii strain, displays lower expression of PhoP-induced genes than MTB. Third, we demonstrate that genetic swapping of the phoPR allele from STB-K with the ortholog from MTB H37Rv enhances expression of PhoP-controlled functions and the capacities of the recombinant strain to colonize human macrophages, the MTB target cells, as well as to cause disease in several mouse infection models. Fourth, we extended these observations to other M. canettii strains and confirm that PhoP-controlled functions are expressed at lower levels in most M. canettii strains than in M. tuberculosis. Our findings suggest that distinct PhoR variants have been selected during the evolution of tuberculosis bacilli, contributing to higher pathogenicity and persistence of MTB in the mammalian host.

Host phospholipid peroxidation fuels ExoU-dependent cell necrosis and supports Pseudomonas aeruginosa-driven pathology.

Regulated cell necrosis supports immune and anti-infectious strategies of the body; however, dysregulation of these processes drives pathological organ damage. Pseudomonas aeruginosa expresses a phospholipase, ExoU that triggers pathological host cell necrosis through a poorly characterized pathway. Here, we investigated the molecular and cellular mechanisms of ExoU-mediated necrosis. We show that cellular peroxidised phospholipids enhance ExoU phospholipase activity, which drives necrosis of immune and non-immune cells. Conversely, both the endogenous lipid peroxidation regulator GPX4 and the pharmacological inhibition of lipid peroxidation delay ExoU-dependent cell necrosis and improve bacterial elimination in vitro and in vivo. Our findings also pertain to the ExoU-related phospholipase from the bacterial pathogen Burkholderia thailandensis, suggesting that exploitation of peroxidised phospholipids might be a conserved virulence mechanism among various microbial phospholipases. Overall, our results identify an original lipid peroxidation-based virulence mechanism as a strong contributor of microbial phospholipase-driven pathology.

Parallel in vivo experimental evolution reveals that increased stress resistance was key for the emergence of persistent tuberculosis bacilli.

Pathogenomic evidence suggests that Mycobacterium tuberculosis (MTB) evolved from an environmental ancestor similar to Mycobacterium canettii, a rare human pathogen. Although the adaptations responsible for this transition are poorly characterized, the ability to persist in humans seems to be important. We set out to identify the adaptations contributing to the evolution of persistence in MTB. We performed an experimental evolution of eight M. canettii populations in mice; four populations were derived from the isolate STB-K (phylogenomically furthest from MTB) and four from STB-D (closest to MTB), which were monitored for 15 and 6 cycles, respectively. We selected M. canettii mutants with enhanced persistence in vivo compared with the parental strains, which were phenotypically closer to MTB. Genome sequencing of 140 mutants and complementation analysis revealed that mutations in two loci were responsible for enhanced persistence. Most of the tested mutants were more resistant than their parental strains to nitric oxide, an important effector of immunity. Modern MTB were similarly more resistant to nitric oxide than M. canettii. Our findings demonstrate phenotypic convergence during experimental evolution of M. canettii, which mirrors natural evolution of MTB. Furthermore, they indicate that the ability to withstand host-induced stresses was key for the emergence of persistent MTB.

Protein O-mannosylation deficiency increases LprG-associated lipoarabinomannan release by Mycobacterium tuberculosis and enhances the TLR2-associated inflammatory response.

Protein O-mannosylation is crucial for the biology of Mycobacterium tuberculosis but the key mannosylated protein(s) involved and its(their) underlying function(s) remain unknown. Here, we demonstrated that the M. tuberculosis mutant (Δpmt) deficient for protein O-mannosylation exhibits enhanced release of lipoarabinomannan (LAM) in a complex with LprG, a lipoprotein required for LAM translocation to the cell surface. We determined that LprG is O-mannosylated at a unique threonine position by mass spectrometry analyses of the purified protein. However, although replacement of this amino acid by an alanine residue completely abolished LprG O-mannosylation, the increased release of the LAM/LprG complex was preserved. We found that the increased secretion of this complex is due to enhanced LAM production in the Δpmt M. tuberculosis and M. smegmatis mutants relative to their wild-type counterparts. This abnormal release of LAM/LprG has functional consequences on the induction of inflammatory responses and provides a possible explanation for the reduced virulence of the M. tuberculosis Δpmt mutant.