Cloning and characterization of secretory tyrosine phosphatases of Mycobacterium tuberculosis

Two genes with sequence homology to those encoding protein tyrosine phospha tases were cloned from genomic DNA of Mycobacterium tuberculosis H(37)Rv. T he calculated molecular masses of these two putative tyrosine phosphatases, designated MPtpA and MPtpB, were 17.5 and 30 kDa, respectively. MPtpA and MPtpB were expressed as glutathione S-transferase fusion proteins in Escher ichia coli, The affinity-purified proteins dephosphorylated the phosphotyro sine residue of myelin basic protein (MBP), but they failed to dephosphoryl ate serine/threonine residues of MBP. The activity of these phosphatases wa s inhibited by sodium orthovanadate, a specific inhibitor of tyrosine phosp hatases, but not by okadaic acid, an inhibitor of serine/threonine phosphat ases, Mutations at the catalytic site motif, cysteine 11 of MPtpA and cyste ine 160 of MPtpB, abolished enzyme activity. Southern blot analysis reveale d that, while mptpA is present in slow-growing mycobacterial species as wel l as fast-growing saprophytes, mptpB was restricted to members of the M tub erculosis complex. These phosphatases were present in both whole-cell lysat es and culture filtrates of M tuberculosis, suggesting that these proteins are secreted into the extracellular medium. Since tyrosine phosphatases are essential for the virulence of several pathogenic bacteria, the restricted distribution of mptpB makes it a good candidate for a virulence gene of M. tuberculosis.