Characterization of a unique class C acid phosphatase from Clostridium perfringens

Abstract

Clostridium perfringens is a gram-positive anaerobe and a pathogen of medical importance. The detection of acid phosphatase activity is a powerful diagnostic indicator of the presence of C. perfringens among anaerobic isolates; however, characterization of the enzyme has not previously been reported. Provided here are details of the characterization of a soluble recombinant form of this cell-associated enzyme. The denatured enzyme was approximately 31 kDa and a homodimer in solution. It catalyzed the hydrolysis of several substrates, including para-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 3' and 5' nucleoside monophosphates at pH 6. Calculated K(m)s ranged from 0.2 to 0.6 mM with maximum velocity ranging from 0.8 to 1.6 micromol of P(i)/s/mg. Activity was enhanced in the presence of some divalent cations but diminished in the presence of others. Wild-type enzyme was detected in all clinical C. perfringens isolates tested and found to be cell associated. The described enzyme belongs to nonspecific acid phosphatase class C but is devoid of lipid modification commonly attributed to this class.

FIG. 1.

Amino acid sequence alignment of bacterial NSAP class C proteins. The top amino acid sequence of the alignment is that of the H. influenzae class C acid phosphatase lipoprotein e (P4) designated Hi. Directly above the H. influenzae sequence are black numbers (from 1 to 270 in multiples of 10) designating the residue number of this prototype class C enzyme. Above the residue numbers are symbols designating stretches of α-helices and β-strands as determined by structural analysis (8). Other sequences, including that from C. perfringens CppA (Cp), are Pasteurella multocida (Pm), Helicobacter pylori (Hp), Bacillus anthracis (Ba), Staphylococcus aureus (Sa), Streptococcus equisimilis (Se), Elizabethkingia (Chryseobacter) meningosepticum (Em), Clostridium novyi (Cn), and Francisella tularensis (Ft). Residues identically conserved in all sequences are denoted by white letters on a red background. Other regions of high sequence conservation are indicated by boxed red letters on a white background. Black bars under the alignment indicate residues of the DDDD motif. This figure was created with ESPript (12). Gaps introduced to maximize alignment are indicated by periods.

FIG. 2.

SDS-PAGE separation and detection of rCppA and active site mutant purification samples. All sample lanes contain ∼25 μg protein. (A) Lane 1, Novagen Perfect Protein standards (150, 100, 75, 50, 35, 25, and 15 kDa); lane 2, E. coli BL21 AI transformed with pRCppA, after autoinduction; lane 3, preparation after treatment with a French press; lane 4, supernatant after ultracentrifugation; lane 5, pooled fractions after Ni-chelate chromatography; lane 6, pooled fractions after Q-Sepharose chromatography; lane 7, purified rCppA D100A. (B) Western blot detection of active recombinant rCppA (lane 6) and mutant rCppA D100A (lane 7) with anti-His tag (α-His tag) and detection of active rCppA (lane 6) and mutant rCppA D100A (lane 7) with anti-rCppA (α-rCppA) (1:25,000 dilution). (C) 4MUP-mediated zymogram detection of acid phosphatase activity of rCppA purification samples, following SDS-PAGE and renaturation. Lanes are identical to those in panel A.

FIG. 3.

Effect of pH on the phosphomonoesterase activity of rCppA. Effect of pH on the phosphomonoesterase activity of rCppA for pNPP (•) and 5′ UMP (□) both at a final concentration of 1 mM. Max, maximum. (Inset A) Effect of acetate concentration on rCppA activity for pNPP at pH 5.0 and 6.0. (Inset B) Effect of pH on the K_m of pNPP for rCppA at six pHs as assessed by standard kinetic assays as described in Materials and Methods.

FIG. 4.

Immunological detection and localization of C. perfringens CppA. (A) SDS-PAGE and Western blot detection (rabbit anti-rCppA [1:25,000]) of recombinant CppA (lane 1) and wild-type C. perfringens (lanes 2 to 9) in eight clinical isolates. (B) Western blot detection (rabbit anti-rCppA [α-rCppA] [1:25,000]) of CppA in the ultracentrifugation pellet (CP ultra pellet) (lane 1) and rCppA (lane 2) after SDS-PAGE separation.

FIG. 5.

Transmission electron micrographs depicting detection and localization of acid phosphatase activity of C. perfringens type strain ATCC 10543. Thin sections of gelatin-encased whole-cell suspensions treated with AMP as a substrate and then with lead nitrate as a phosphate capture agent prior to processing for TEM. The images are from a standard fixation and processing method with no additional staining after thin sectioning (inset, without lead) (A) and a microwave-assisted immunofixation protocol with staining after thin sectioning (inset, minus substrate) (B). Bars, 0.2 μm.