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 ...

The interaction of a synthetic peptide representing the C-terminal 27 amino acids of the A1 domain of Shiga toxin (residues 220-246) with acidic phospholipid model membranes was characterized by FTIR spectroscopy. This peptide resembles a signal sequence and may mediate the translocation of the catalytic A1 chain of Shiga toxin to the cytoplasm following its retrograde transport to the lumenal compartment of the endoplasmic reticulum (ER). At pH 7 and 5, the peptide underwent a conformational change from random coil to alpha-helix upon addition of negatively charged phospholipids. Examination of the amide II band in the spectrum of the complex at pH 7 and pH 5 showed that in both cases, the N-H groups in the peptide backbone are largely protected from H/D exchange. Using polarized attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) measurements, the orientation of the alpha-helical portion of the peptide was found to be almost perpendicular with respect to the membrane plane at pH 7. However, at pH 5.0-5.4, the alpha-helix axis was preferentially oriented parallel to the membrane plane. The results suggest that at the neutral pH of the ER lumen, the peptide may insert into the membrane, while at the lower pH levels present in earlier endocytic compartments, the peptide would be less likely to traverse the bilayer. In summary, this putative signal peptide may not be able to cause a significant translocation of the A1 domain of Shiga toxin to the cytosol until it reaches the neutral pH of the ER compartment.

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