Structure and function relationship of staphylocoagulase

The bacterial protein staphylocoagulase binds stoichiometrically to human prothrombin, resulting in a coagulant complex, staphylothrombin. The enzymatic properties of staphylothrombin differ from those of -thrombin in their substrate specificities toward natural and synthetic substrates, in addition to their interaction with protease inhibitors. In order to obtain information about the region of staphylocoagulase that interacts with human prothrombin, staphylocoagulase was cleaved by -chymotrypsin. Limited -chymotryptic cleavage of staphylocoagulase yielded three large fragments, of 43, 30, and 20 kD. The 43-kD fragment exhibited a high affinity for human prothrombin (K_d=1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (K_d=0.46 nM). A complex of the 43-kD fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. The 30-kD fragment exhibited weaker affinity for prothrombin (K_d=120 nM.) While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. The 20-kD fragment was found only to bind to prothrombin. The NH₂-terminal sequence analyses of these fragments revealed that the 43-kD fragment constitutes the NH₂-terminal portion of staphylocoagulase, and contains the 30-kD and 20-kD fragments. It is therefore concluded that the functional region of staphylocoagulase for binding and activation of prothrombin is localized in the NH₂-terminal region of the intact protein. The 43-kD fragment contained 324 amino acids with a molecular weight of 38,098. The 43-kD fragment had an unusual amino acid composition based on a sequence in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounted for more than 45% of the total. A comparison of the amino acid sequence of the 43-kD fragment with that of streptokinase did not reveal any obvious sequence homology. There was also no sequence homology with that of trypsin, -chymotrypsin, and elastase.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Heterogeneity and phylogenetic relationships of community-associated methicillin-sensitive/resistant Staphylococcus aureus isolates in healthy dogs, cats and their owners

Aims: To investigate the distribution of staphylococcal enterotoxin genes (se) and the molecular features of community‐associated methicillin‐sensitive/resistant Staphylococcus aureus (CA‐MSSA/MRSA) isolates in the nostrils of healthy pets and their owners. Methods and Results: A total of 114 Staph. aureus isolates were identified from 1563 nasal swab samples, and CA‐MRSA accounted for 20·2% (n = 23) of the total identified isolates. CA‐MRSA isolates (91·3%, 21/23) harboured higher percentage of se than did CA‐MSSA isolates (58·2%, 53/91) (P < 0·01), and the two highest se profiles of CA‐MRSA were seb‐sek‐seq (42·9%, 9/21) and seb‐sek‐seq‐sep (28·6%, 6/21). Of the MSSAs, 42·8% (39/91) were resistant to at least one antimicrobial drug and 8·8% (8/91) were multidrug resistant (MDR). We identified nine staphylocoagulase (SC) types (I–VIII and X) and three multilocus sequence types (ST59‐MRSA‐IV/V, ST‐239‐MRSA‐V and ST241‐MRSA‐V). SC VII (23·4%, 22/94), a staphylococcal food poisoning isolate found mainly in Japan, and ST‐59‐MRSA‐IV/V (85%, 17/20), a widespread CA‐MRSA clone found mainly in Taiwan, both were the most predominant types. Phylogenetic analysis together with se and molecular characteristics obtained using pulsed‐field gel electrophoresis showed that high levels of antimicrobial resistance and the se‐carrying clone ST59‐MRSA‐IV/V‐SC VII were all clustered in genogroup 5. Conclusions: The CA‐MRSA clone of se‐carrying‐MDR‐ST‐59‐IV/V‐SC VII was identified predominantly in this study, and this clone might play a significant role in staphylococcal food poisoning in community settings. Significance and Impact of the Study: To our knowledge, this is the first study focussing on enterotoxin‐carrying CA‐MRSA/MSSA in pets and their owners, and the results support the future warnings in animal–human bond caused by CA‐staphylococci in the commonwealth and the need to take cautions worldwide.     

Diversity of staphylocoagulase and identification of novel variants of staphylocoagulase gene in Staphylococcus aureus

Staphylocoagulase (SC) is a major phenotypic determinant of Staphylococcus aureus. Serotype of SC (coagulase type) is used as an epidemiological marker and 10 types (I-X) have been discriminated so far. To clarify genetic diversity of SC within a single and among different serotype(s), we determined approximately 1500 bp-nucleotide sequences of SC gene encoding D1, D2, and central regions (N-terminal half and central regions of SC; SC(NC)) for a total of 33 S. aureus strains comprising two to three strains from individual coagulase types (I-VIII, X) and 10 strains which were not determined as previously known SC serotypes (ND-strains). Amino acid sequence identities of SC(NC) among strains with a single coagulase type of II, III, IV, V, VI and X were extremely high (more than 99%), whereas lower identity (56-87%) was observed among different types. In contrast, within a single coagulase type of I, VII, or VIII, sequence divergence was found (lowest identity; 82%). SC(NC) sequences from the ND-strains were discriminated into two genetic groups with an identity of 71% to each other (tentatively assigned to genotypes [XI] and [XII]), and exhibited less than 86% sequence identities to those of most known coagulase types. All the types [XI] and [XII] strains were methicillin susceptible and belonged to different sequence types from those of coagulase types I-X strains reported so far by multilocus sequence typing. These findings indicated genetic heterogeneity of SC in coagulase types I, VII, and VIII strains, and the presence of two novel SC genotypes related to antigenicity of SC serotypes.