A SITE-DIRECTED STAPHYLOCOCCUS-AUREUS HEMB MUTANT IS A SMALL-COLONY VARIANT WHICH PERSISTS INTRACELLULARLY

Although small-colony variants (SCVs) of Staphylococcus aureus have be en recognized for many Sears, this phenotype has only recently been re lated to persistent and recurrent infections, Clinical S. aureus SCVs are frequently auxotrophic for menadione or hemin, two compounds invol ved in the biosynthesis of the electron transport chain elements menaq uinone and cytochromes, respectively. While this observation as well a s other biochemical characteristics of SCVs suggests a link between el ectron-transport-defective strains and persistent infections, the stra ins examined thus far have been genetically undefined SCVs, Therefore, we generated a stable mutant in electron transport by interrupting on e of the hemin biosynthetic genes, hemB, in S. aureus by inserting an ermB cassette into hemB. We isolated a hemB mutant, due to homologous recombination, by growth at a nonpermissive temperature and selection for erythromycin resistance. This mutant showed typical characteristic s of clinical SCVs, such as slow growth, decreased pigment formation, low coagulase activity, reduced hemolytic activity, and resistance to aminoglycosides. Additionally, the mutant was able to persist within c ultured endothelial cells due to decreased alpha-toxin production, Nor thern and Western blot analyses showed that expression of alpha-toxin and that of protein A were markedly reduced, at both the mRNA and the protein level, The SCV phenotype of the hemB mutant was reversed by gr owth with hemin or by complementation with intact hemB. Hence, a defec t in the electron transport system allows S. aureus SCVs to resist ami noglycosides and persist intracellularly.