Secretion of an Acid Phosphatase (SapM) by Mycobacterium tuberculosis That Is Similar to Eukaryotic Acid Phosphatases

Abstract

Mycobacterium tuberculosis secretes a large number of polypeptides with broad biological and immunological functions. We describe here the characterization of a 28-kDa acid phosphatase of M. tuberculosis (SapM) localized to the culture filtrate. The mature protein demonstrated biochemical characteristics similar to those of the bacterial nonspecific acid phosphatases. However, SapM yielded significant sequence homology to fungal acid phosphatases and not those of bacteria. Thus, SapM may represent a new class of bacterial nonspecific acid phosphatases.The complete Mycobacterium tuberculosis genome was recently sequenced and annotated (8). However, of the 3,924 putative gene products, only a minority are assigned experimentally established physiological functions. Thus, continued analyses of the M. tuberculosis proteome are required to fully elucidate the biology of this intracellular pathogen. The most thoroughly studied gene products of M. tuberculosis are the culture filtrate proteins (CFPs) (reviewed in reference 6). The CFPs are proposed to participate in intracellular survival (6) and are a primary target of the host's protective T-cell response (21). Several laboratories have added some definition to the myriad of biological and enzymatic activities associated with the CFPs (4, 7, 15, 23). In the most comprehensive of these studies, Raynaud et al. (23) described 22 enzymatic activities. However, the molecular identities and characteristics of the enzymes responsible for these activities were not elucidated. One of the activities identified was that of an acid phosphatase.One mechanism of intracellular survival employed by M. tuberculosis is the modulation of host cell activities, such as intracellular vesicle fusion, phagosome maturation, and phagosome acidification (reviewed in reference 27). Although the specific proteins of M. tuberculosis that modulate macrophage activity remain elusive, recent studies of other intracellular pathogens demonstrate that acid phosphatases are important to this aspect of microbial pathogenicity (2,24,25,28). Thus, we believe that such an activity may also facilitate host cell modulation by M. tuberculosis. As a first step in testing this hypothesis, the presence of acid phosphatase activity in the culture filtrate of M. tuberculosis was confirmed and a 28-kDa protein possessing this activity was purified and characterized.Purification and identification of the acid phosphatase. To identify and isolate a secreted acid phosphatase, a culture filtrate of M. tuberculosis cells grown to late log phase was harvested (9) and dialyzed against 0.1 M sodium acetate (pH 6.0). Initial evaluation of this culture filtrate for phosphatase activity, using p-nitrophenyl phosphate (pNPP) as the substrate (5), indicated the presence of weak activity (750 nmol of pnitrophenol [pNP]/min/mg of protein) at pH 6.0. The culture filtrate was fractionated by cation-exchange chromatography, and a single asymmetric peak of acid phosphatase activity was eluted with about 0.3 M NaCl. Moreover, the acid ...

Porins Are Required for Uptake of Phosphates by Mycobacterium smegmatis

“…Three lipoproteins with phosphatase activity and the secreted acid phosphatase SapM were identified in M. bovis BCG (3) and M. tuberculosis (35), respectively. Our observation that the cell envelope of M. bovis BCG establishes a permeability barrier for phosphates indicates that at least one phosphatase with significant activity is localized in the periplasm.…”

Bacterial Virulence Factors: Secreted for Survival

“…under acidic conditions [17]. Two years later, the acid phosphatase from the culture filtrate protein fraction responsible for the observed activity was identified as a 28-kDa protein [18]. N-terminal sequencing unveiled the presence of a typical prokaryotic signal sequence and hence it was proposed that the protein would be secreted through the sec-dependent mechanism.…”

“…The protein was thus named secreted acid phosphatase of Mtb, SapM. Sequence analysis of SapM however showed no homology with prokaryotic acid phosphatases, instead SapM was shown to have a high homology with several acid phosphatases of fungal origin [18].…”

LspA inactivation in Mycobacterium tuberculosis results in attenuation without affecting phagosome maturation arrest

“…Phagosome maturation arrest is in part dependent on lipid components of the mycobacterial cell wall, such as lipoarabinomannan, phosphatidylinositol mannosides (PIMs) and cord factor (Anes et al, 2003;Fratti et al, 2001Fratti et al, , 2003Indrigo et al, 2003;Vergne et al, 2003Vergne et al, , 2004. Additional factors involved are the acid phosphatase SapM (Saleh & Belisle, 2000;Vergne et al, 2005), the bacterial serine/threonine kinase PknG (Walburger et al, 2004), and the zinc-metalloprotease Zmp1 (Master et al, 2008).…”