Export of Recombinant Mycobacterium tuberculosisSuperoxide Dismutase Is Dependent upon Both Information in the Protein and Mycobacterial Export Machinery

Abstract

We have investigated the expression and extracellular release of enzymatically active superoxide dismutase, one of the 10 major extracellular proteins of Mycobacterium tuberculosis, both in its native host and in the heterologous host Mycobacterium smegmatis. We found that the M. tuberculosis superoxide dismutase gene, encoding a leaderless polypeptide of M r ϳ23,000 representing one of the four identical subunits of the enzyme, is expressed constitutively under normal growth conditions and at a 5-fold increased level under conditions of hydrogen peroxide stress. The highly pathogenic mycobacterium M. tuberculosis expresses 93-fold more superoxide dismutase than the nonpathogenic mycobacterium M. smegmatis, and it exports a much higher proportion of expressed enzyme (76 versus 21%); taking both expression and export into consideration, M. tuberculosis exports ϳ350-fold more enzyme than M. smegmatis. In M. smegmatis, recombinant M. tuberculosis superoxide dismutase is expressed at 8.4 times the level of the endogenous enzyme and the proportion exported (66%) approaches that in the homologous host; hence M. smegmatis exports up to 26-fold more of the recombinant than endogenous enzyme. Interestingly, subunits of the M. tuberculosis and M. smegmatis enzymes readily and stoichiometrically exchange with each other, forming five different complexes of four subunits, both when the enzymes are expressed in the recombinant host and when the purified enzymes are incubated together; however, each subunit retains its characteristic metal ion, iron for M. tuberculosis and manganese for M. smegmatis. Compared with the cell-associated enzyme, the supernatant enzyme of recombinant M. smegmatis is enriched for M. tuberculosis enzyme subunits, consistent with preferential export of the M. tuberculosis enzyme. Recombinant M. tuberculosis superoxide dismutase transcomplements a superoxide dismutase-deficient Escherichia coli, resulting in a reduction of sensitivity of the strain to oxidative stress, but the enzyme is not exported from this nonmycobacterial host. Our findings indicate that the information for export of the M. tuberculosis superoxide dismutase is contained within the protein but that export additionally requires export machinery specific to mycobacteria.Mycobacterium tuberculosis secretes or otherwise releases a large number of proteins into its extracellular milieu. Approximately 11 such proteins are released into broth cultures in sufficient abundance so as to merit designation as major extracellular proteins. In all cases examined thus far, these major extracellular proteins are also released into the phagosome of M. tuberculosis in human mononuclear phagocytes. The functions of several of these proteins are known, including glutamine synthetase (subunit molecular mass 58 kDa), mycolyl transferase (30/32-kDa complex), and iron-specific superoxide dismutase (subunit molecular mass 23 kDa).Extracellular proteins of M. tuberculosis are of great interest because of the likelihood that these proteins play critical role...

Cloning and Characterization of Secretory Tyrosine Phosphatases of Mycobacterium tuberculosis

“…The mechanism employed by mycobacteria in exporting these proteins is presently unclear. Other mycobacterial proteins such as glutamine synthetase and superoxide dismutase are also secreted into the extracellular medium in the absence of signal peptides at the N terminus (17). Chaperone proteins may be involved in the secretion of mycobacterial tyrosine phosphatases, as has been found previously with Y. pseudotuberculosis for export of a PTP (YopH) into the host macrophages (34).…”

Lipoamide dehydrogenase mediates retention of coronin-1 on BCG vacuoles, leading to arrest in phagosome maturation

“…Other mycobacterial proteins such as glutamine synthetase (Harth and Horwitz, 1997), superoxide dismutase (Harth and Horwitz, 1999) and mycobacterial tyrosine phosphatase (Koul et al, 2000) are also secreted into the extracellular medium in the absence of signal peptides at the N terminus and the mechanism employed in exporting these proteins is presently unclear.…”

Extracellular Mycobacterial DNA-binding Protein 1 Participates in Mycobacterium-Lung Epithelial Cell Interaction through Hyaluronic Acid

“…1) imply that some machinery is present that transports MDP1 outside the cell membrane rather than retention on the cell surface due to cell lysis of bacteria. Certain proteins that lack signal sequence and a transmembrane domain, such as ESAT-6 (37), CFP-10 (38), HSP65 and superoxide dismutase (SOD) (39,40), and glutamine synthetase (39), are actively exported from mycobacteria and play significant roles in the pathogenesis. Further study is needed to clarify the issue of transportation of MDP1.…”