Evolutionary history of tuberculosis shaped by conserved mutations in the PhoPR virulence regulator

Abstract

Although the bovine tuberculosis (TB) agent, Mycobacterium bovis, may infect humans and cause disease, long-term epidemiological data indicate that humans represent a spill-over host in which infection with M. bovis is not self-maintaining. Indeed, human-to-human transmission of M. bovis strains and other members of the animal lineage of the tubercle bacilli is very rare. Here, we report on three mutations affecting the two-component virulence regulation system PhoP/PhoR (PhoPR) in M. bovis and in the closely linked Mycobacterium africanum lineage 6 (L6) that likely account for this discrepancy. Genetic transfer of these mutations into the human TB agent, Mycobacterium tuberculosis, resulted in down-regulation of the PhoP regulon, with loss of biologically active lipids, reduced secretion of the 6-kDa early antigenic target (ESAT-6), and lower virulence. Remarkably, the deleterious effects of the phoPR mutations were partly compensated by a deletion, specific to the animal-adapted and M. africanum L6 lineages, that restores ESAT-6 secretion by a PhoPR-independent mechanism. Similarly, we also observed that insertion of an IS6110 element upstream of the phoPR locus may completely revert the phoPR-bovis-associated fitness loss, which is the case for an exceptional M. bovis human outbreak strain from Spain. Our findings ultimately explain the long-term epidemiological data, suggesting that M. bovis and related phoPR-mutated strains pose a lower risk for progression to overt human TB, with major impact on the evolutionary history of TB.

Keywords: adaptation; evolution; phylogeny; zoonosis.

Fig. 1.

The phoPR allele from animal-adapted and M. africanum L6 strains is deficient. (A) Schematic global phylogenetic tree of the MTBC (1, 2). The length of the branches does not correlate with phylogenetic distance. Names of the strains used in this study are indicated. (B) Genome-wide transcriptional profiles of WT M. tuberculosis H37Rv, ΔphoPR mutant, and phoPR-bovis– or phoPR-TB–complemented strains. Fold-change values from individual probes for each gene were averaged. Those genes showing a statistically significant average fold-change > 2 or < 0.5 in the WT or the phoPR-TB–complemented strains relative to the ΔphoPR mutant were selected as positively or negatively regulated by PhoP, respectively. (C) qRT-PCR analysis of expression of main reporter genes of the PhoP regulon. (D) TLC analysis of lipids extracted from [¹⁴C] propionic acid-labeled cultures. The position of the major SL (the tetra-acylated sulfoglycolipids, Ac₄SGL) is highlighted (arrow). (E) Immunoblot of secreted and whole-cell fractions probed with ESAT-6– or GroEL2- (used as a lysis control) specific antibodies.

Fig. 2.

The phoPR-bovis allele impacts the interaction of M. tuberculosis with cellular or animal hosts. (A) Quantification of intracellular bacteria at 24-, 72-, or 144-h postinfection. Human-monocyte-derived macrophages (hMDM) were infected (at a multiplicity of infection of 2) for 2 h with indicated strains. Data are mean ± SD of three independent experiments performed in duplicate. (B and C) Seven-week-old BALB/c mice were infected intranasally with ∼200 cfu of the indicated strains. At the indicated time points, lungs (B) or spleens (C) were homogenized and plated for colony-forming unit determination. Data are means ± SD of four mice at days 14 and 56 and eight mice at days 1 and 28, from two independent experiments. The difference between experimental groups was evaluated by the two-tailed Student t test: P values, *P < 0.05, **P < 0.01, ***P < 0.005.

Fig. 3.

The phoPR-bovis allele is deficient but ESAT-6 is secreted in animal-adapted strains. (A) qRT-PCR analysis of expression of main reporter genes of the PhoP regulon in WT and recombinant M. bovis AN5 grown liquid culture. (B) TLC analysis of lipids extracted from [¹⁴C] propionic acid-labeled cultures. (C) Immunoblot of secreted and whole-cell fractions from WT or recombinant M. bovis strains probed with ESAT-6– or GroEL2-specific antibodies.

Fig. 4.

RD8 deletion allows ESAT-6 secretion independently from PhoPR. (A) Schematic representation of the genetic structure at the espACD locus in the M. tuberculosis sensu-stricto and animal-adapted or M. africanum L6 strains. Red lines indicate individual SNPs identified between pairwise-compared genomes. Blue lines indicate the boundaries of the RD8 deletion. (B) qRT-PCR analysis of espACD genes expression in five strains of the MTBC harboring a ΔphoPR deletion. (C) Immunoblot of secreted ESAT-6 from H37Rv WT, ΔphoPR mutant, and ΔphoPR mutant carrying the espACD fragment from M. bovis (espACD-bovis) or the control similar fragment from H37Rv (espACD-TB). Several transformants were analyzed. The bacterial supernatants and cell pellets were probed for ESAT-6 or GroEL2. (D) Quantification of the ESAT-6 signals in secreted protein fractions from the indicated strains (relative to H37Rv).

Fig. 5.

Insertion of IS6110 upstream phoPR in M. bovis B strain suppresses the phoPR-bovis deficiency. (A) Schematic representation of the genetic structure at the phoPR locus in the M. bovis B strain relative to “classic” M. bovis and M. tuberculosis strains. The positions of SNPs specific to the animal-adapted and M. africanum L6 and of the IS6110 insertion site are indicated in brackets. Red lines indicate individual SNPs identified between pairwise-compared genomes. Blue lines indicate the boundaries of IS6110. (B) qRT-PCR analysis of phoPR expression in M. tuberculosis H37Rv strain, the ΔphoPR mutant, and the recombinant M. tuberculosis strain expressing the phoPR-B allele. (C) Western blot analysis of PhoP from WT and phoPR-B expressing strains. (D) qRT-PCR analysis of expression of PhoP regulon genes in the indicated strains. (E) TLC analysis of lipids extracted from [¹⁴C] propionic acid-labeled cultures. (F) Survival of SCID mice infected with ∼4 × 10³ cfu of the indicated strains (five mice per group). Curves are significantly different between the phoPR-bovis expressing strain and the phoPR-TB– and phoPR-B–expressing strains (P < 0.005, according to the log-rank (Mantel– Cox) test).