Genetic and biochemical analysis of a class C non-specific acid phosphatase (NSAP) of Clostridium perfringens

Abstract

Clostridium perfringens, a Gram-positive anaerobe, is a human pathogen that causes gas gangrene in muscle tissues. Its ability to grow and survive in the host is believed to be due to the production of numerous enzymes that enable the organism to obtain essential nutrients from the host. In this study, CPE0201, a putative acid phosphatase gene deduced by genome analysis, was shown to encode a non-specific acid phosphatase inC. perfringens. Multiple alignments of the amino acid sequence showed that CPE0201 shares two signature motifs that belong to a class C acid phosphatase family. Expression of CPE0201 was shown to be positively regulated by the global VirR/VirS-VR-RNA regulatory cascade at the transcriptional level. To determine the acid phosphatase activity of the CPE0201-encoded protein, cloning, expression, purification and several biochemical characterizations were carried out. The optimum pH for activity of the CPE0201 enzyme was 4.8, and itsVmaxandKmwere 3.08 nmol ml−1min−1and 2.84 mM, respectively, withp-nitrophenyl phosphate (PNPP) as substrate. A CPE0201 mutant did not grow in a minimal medium containing PNPP, while it showed normal growth when Na2HPO4was added to the medium. The enzyme appears to be associated with the surface of the cell, where it may function to acquire inorganic phosphate from organic phosphomonoesters in acidic conditions, which could play an important role in the survival and growth ofC. perfringensin the host tissue.