Non-spa-typeable clinical Staphylococcus aureus strains are naturally occurring protein A mutants

Abstract

Staphylococcus aureus is a major human pathogen responsible for increasing the prevalence of community- and hospital-acquired infections. Protein A (SpA) is a key virulence factor of S. aureus and is highly conserved. Sequencing of the variable-number tandem-repeat region of SpA (spa typing) provides a rapid and reliable method for epidemiological studies. Rarely, non-spa-typeable S. aureus strains are encountered. The reason for this is not known. In this study, we characterized eight non-spa-typeable bacteremia isolates. Sequencing of the entire spa locus was successful for five strains and revealed various mutations of spa, all of which included a deletion of immunoglobulin G binding domain C, in which the upper primer for spa typing is located, while two strains were truly spa negative. This is the first report demonstrating that nontypeability of S. aureus by spa sequencing is due either to mutation or to a true deficiency of spa.

FIG. 1.

(A) Amplification of the entire spa locus. By use of primers flanking the entire spa locus, spa could be amplified in five of eight non-spa-typeable strains: NT935, NT937, NT938, NT939, and NT941. (B) Results of sequencing of the spa locus for five non-spa-typeable S. aureus strains: NT935, NT937, NT938, NT939, and NT941. The deletions are indicated by gray rectangles. Strain NT935 revealed a deletion from the middle part of IgG-binding domain C up to the beginning of the repeat region X_R, resulting in a frameshift mutation, presumably leading to a premature stop codon. In strain NT937, a deletion of the three IgG-binding domains A, B, and C occurred; in strains NT938 and NT939, a deletion of IgG-binding domain C was observed; and in strain NT941, a deletion of the four IgG-binding domains D, A, B, and C was demonstrated.

FIG. 2.

(A) Southern blot hybridization analysis. SmaI-digested chromosomal DNAs of non-spa-typeable strains were probed with a 252-bp fragment of spa, which detects the S and E regions. As positive controls, DNAs of the S. aureus strains Cowan I and 8325-4 were used. (B and C) Western blot analyses. Cell wall extracts (B) and secreted proteins (C) of the non-spa-typeable strains were incubated with affinity-purified immunoglobulins from rabbit serum as primary antibodies and with alkaline phosphatase-conjugated goat anti-rabbit immunoglobulins as secondary antibodies. As a marker, the PageRuler Plus prestained protein ladder from Fermentas GmbH was used. Protein A P3838 (Sigma-Aldrich Chemie GmbH) was used as a positive control.

FIG. 3.

Phylogenetic tree of the non-spa-typeable S. aureus strains. (A) PFGE results. The molecular weight marker used consists of concatemers of lambda phage DNA. (B) Deduced phylogenetic tree after SmaI digestion of chromosomal DNA from the non-spa-typeable strains.