Biochemical and immunological characterization of a cpn60.1 knockout mutant of Mycobacterium bovis BCG.

Pathogenic mycobacteria possess two homologous chaperones encoded by cpn60.1 and cpn60.2. Cpn60.2 is essential for survival, providing the basic chaperone function, while Cpn60.1 is not. In the present study, we show that inactivation of the Mycobacterium bovis BCG cpn60.1 (Mb3451c) gene does not significantly affect bacterial growth in 7H9 broth, but that this knockout mutant (Δcpn60.1) forms smaller colonies on solid 7H11 medium than the parental and complemented strains. When growing on Sauton medium, the Δcpn60.1 mutant exhibits a thinner surface pellicle and is associated with higher culture filtrate protein content and, coincidentally, with less protein in its outermost cell envelope in comparison with the parental and complemented strains. Interestingly, in this culture condition, the Δcpn60.1 mutant is devoid of phthiocerol dimycocerosates, and its mycolates are two carbon atoms longer than those of the wild-type, a phenotype that is fully reversed by complementation. In addition, Δcpn60.1 bacteria are more sensitive to stress induced by H(2)O(2) but not by SDS, high temperature or acidic pH. Taken together, these data indicate that the cell wall of the Δcpn60.1 mutant is impaired. Analysis by 2D gel electrophoresis and MS reveals the upregulation of a few proteins such as FadA2 and isocitrate lyase in the cell extract of the mutant, whereas more profound differences are found in the composition of the mycobacterial culture filtrate, e.g. the well-known Hsp65 chaperonin Cpn60.2 is particularly abundant and increases about 200-fold in the filtrate of the Δcpn60.1 mutant. In mice, the Δcpn60.1 mutant is less persistent in lungs and, to a lesser extent, in spleen, but it induces a comparable mycobacteria-specific gamma interferon production and protection against Mycobacterium tuberculosis H37Rv challenge as do the parental and complemented BCG strains. Thus, by inactivating the cpn60.1 gene in M. bovis BCG we show that Cpn60.1 is necessary for the integrity of the bacterial cell wall, is involved in resistance to H(2)O(2)-induced stress but is not essential for its vaccine potential.

Biochemical analysis of the NAD+-dependent malate dehydrogenase, a substrate of several serine/threonine protein kinases of Mycobacterium tuberculosis.

PknD is one of the eleven eukaryotic-like serine/threonine protein kinases (STPKs) of Mycobacterium tuberculosis (Mtb). In vitro phosphorylation assays with the active recombinant PknD showed that the intracellular protein NAD+-dependent malate dehydrogenase (MDH) is a substrate of this kinase. MDH, an energy-supplying enzyme, catalyzes the interconversion of malate and oxaloacetate and plays crucial roles in several metabolic pathways including the citric acid cycle. The phosphorylation site was identified on threonine residues and the phosphorylation inhibited the MDH activity. In vitro, the recombinant MDH could also be phosphorylated by at least five other STPKs, PknA, PknE, PknH, PknJ, and PknG. Immunoprecipitation analysis revealed that MDH was hyperphosphorylated in the bacteria at the beginning of the stationary and under oxygen-limited conditions by STPKs other than PknD. On the contrary, when PknD-deficient mutant mycobacteria were grown in a phosphate-depleted medium, MDH was not detectably phosphorylated. These results suggest that although the MDH is a substrate of several mycobacterial STPKs, the activity of these kinases can depend on the environment, as we identified PknD as a key element in the MDH phosphorylation assay under phosphate-poor conditions.

Effect of PstS sub-units or PknD deficiency on the survival of Mycobacterium tuberculosis.

The membrane-associated phosphate-specific transporter (Pst) complex is composed of four different proteins: PstS, PstC, PstA and PstB. The PstS component detects and binds Pi with high affinity; the PstA and PstC form transmembrane pores for Pi entry, while PstB provides energy through ATP hydrolysis. In the Mycobacterium tuberculosis genome, four different gene clusters encode three PstS, and two of each of the other sub-units. We used RT-PCR to show that these clusters represent at least three distinct operons. The pstS3-containing operon was the only one induced by lack of environmental Pi. To study the physiologic role of the different PstS sub-units and that of another potential Pi receptor, PknD, we constructed and complemented their knockout (KO) mutants. In Sauton medium, the PstS1-3 KO grew faster than the Wt or the PknD KO. Following 24 h of complete starvation, the PstS3 or PknD deficient strains died if exposed to Pi poor conditions while the PstS1 and PstS2 KO survived and still grew faster than the Wt strain. These results suggest that PstS1-3 may play a role in the regulation of M. tuberculosis growth or metabolism while PstS3 and PknD contribute to the survival of the bacteria in phosphate poor conditions.

Mycobacterium tuberculosis with disruption in genes encoding the phosphate binding proteins PstS1 and PstS2 is deficient in phosphate uptake and demonstrates reduced in vivo virulence.

By measuring phosphate uptake by Mycobacterium tuberculosis strains with the pstS1 and pstS2 genes genetically inactivated, we showed that these pstS genes encode high-affinity phosphate binding proteins. In a mouse infection model, both mutants were attenuated in virulence, suggesting that M. tuberculosis encounters limiting phosphate concentrations during its intracellular life span.