High Extracellular Levels of Mycobacterium tuberculosis Glutamine Synthetase and Superoxide Dismutase in Actively Growing Cultures Are Due to High Expression and Extracellular Stability Rather than to a Protein-Specific Export Mechanism

Abstract

Glutamine synthetase (GS) and superoxide dismutase (SOD), large multimeric enzymes that are thought to play important roles in the pathogenicity of Mycobacterium tuberculosis, are among the bacterium's major culture filtrate proteins in actively growing cultures. Although these proteins lack a leader peptide, their presence in the extracellular medium during early stages of growth suggested that they might be actively secreted. To understand their mechanism of export, we cloned the homologous genes (glnA1 and sodA) from the rapid-growing, nonpathogenic Mycobacterium smegmatis, generated glnA1 and sodA mutants of M. smegmatis by allelic exchange, and quantitated expression and export of both mycobacterial and nonmycobacterial GSs and SODs in these mutants. We also quantitated expression and export of homologous and heterologous SODs from M. tuberculosis. When each of the genes was expressed from a multicopy plasmid, M. smegmatis exported comparable proportions of both the M. tuberculosis and M. smegmatis GSs (in the glnA1 strain) or SODs (in the sodA strain), in contrast to previous observations in wild-type strains. Surprisingly, recombinant M. smegmatis and M. tuberculosis strains even exported nonmycobacterial SODs. To determine the extent to which export of these large, leaderless proteins is expression dependent, we constructed a recombinant M. tuberculosis strain expressing green fluorescent protein (GFP) at high levels and a recombinant M. smegmatis strain coexpressing the M. smegmatis GS, M. smegmatis SOD, and M. tuberculosis BfrB (bacterioferritin) at high levels. The recombinant M. tuberculosis strain exported GFP even in early stages of growth and at proportions very similar to those of the endogenous M. tuberculosis GS and SOD. Similarly, the recombinant M. smegmatis strain exported bacterioferritin, a large (ϳ500-kDa), leaderless, multimeric protein, in proportions comparable to GS and SOD. In contrast, high-level expression of the large, leaderless, multimeric protein malate dehydrogenase did not lead to extracellular accumulation because the protein was highly unstable extracellularly. These findings indicate that, contrary to expectations, export of M. tuberculosis GS and SOD in actively growing cultures is not due to a protein-specific export mechanism, but rather to bacterial leakage or autolysis, and that the extracellular abundance of these enzymes is simply due to their high level of expression and extracellular stability. The same determinants likely explain the presence of other leaderless proteins in the extracellular medium of actively growing M. tuberculosis cultures.Mycobacterium tuberculosis, the primary etiologic agent of tuberculosis, is one of the world's leading causes of death, killing 2 million persons annually worldwide (23). New modalities to combat M. tuberculosis and a greater understanding of the biology and immunology of this pathogen are high priorities of tuberculosis research.The extracellular proteins of M. tuberculosis have been the focus of many studies investigating ...

Regulation of nitrogen metabolism in Mycobacterium tuberculosis: A comparison with mechanisms in Corynebacterium glutamicum and Streptomyces coelicolor

“…It is possible that the high level of GS1 produced is required for efficient ammonium assimilation to support rapid growth under the restrictive conditions found in vivo (10). It has also been suggested that extracellular GS may manipulate phagosome pH modulation and consequently prevent phagosome-lysosome fusion (27,26). Furthermore, extracellular GS1 has been implicated in the synthesis of poly-L-glutamine-glutamate, a cell wall constituent of pathogenic mycobacteria (27)(28)(29), which may play a role in cell wall integrity.…”

“…The activity of glutamine synthetase in both C. glutamiucm, S. coelicolor, and M. tuberculosis is also regulated at the level of glnA1 transcription (27,30,31). In C. glutamicum, an increase in glnA1 transciption levels of between 1.2-and 3-fold under conditions of nitrogen limitation was detected through RNA hybridizations and real-time PCR analyses (19).…”

“…It has also been suggested that extracellular GS may manipulate phagosome pH modulation and consequently prevent phagosome-lysosome fusion (27,26). Furthermore, extracellular GS1 has been implicated in the synthesis of poly-L-glutamine-glutamate, a cell wall constituent of pathogenic mycobacteria (27)(28)(29), which may play a role in cell wall integrity. M tuberculosis GS1 lacks a leader peptide sequence and is considered to be an intracellular protein in most other bacteria, thus its abundance in the extracellular environment of M. tuberculosis and other pathogenic mycobacteria (26) has become a focus of study.…”

Mutually dependent secretion of proteins required for mycobacterial virulence

“…Therefore, we analyzed the culture filtrates of wild-type H37Rv and H37Rv⌬RD1 with the goal of identifying proteins present in wild-type but absent in mutant culture filtrates. To minimize the number of cytoplasmic proteins contaminating the culture filtrates because of background autolysis (20), very-short-term culture supernatants were collected (Ͻ24 h of culture). The proteins in these culture filtrates were identified by analyzing HPLC-resolved tryptic fragments by LCQ-MS͞MS.…”

The Role of Superoxide Dismutase in the Survival of <i>Mycobacterium tuberculosis</i> in Macrophages

“…It is well known that as a dependable vaccine, BCG belongs to an avirulent strain; compared with the virulent strain H37Rv, it loses the RD1 domain (29), but the loss of the virulent domain does not influence its invasion, survival, and long-term stimulation of the host immune system (30). Both the virulent strain H37Rv and the nonpathogenic mycobacterium secrete large amounts of SOD proteins (6). Related research also found that treatment with diethyldithiocarbamate, a potent inhibitor of SOD, increased Mycobacterium lepraemurium survival in murine splenic macrophages (31), notably suggesting that SOD proteins probably contribute to the long-term survival of mycobacterium in vivo.…”