Impact of staphylococcal protease expression on the outcome of infectious arthritis.

The exoproteases of Staphylococcus aureus have been proposed as virulence factors during S. aureus infections. To investigate this, we used the wild-type S. aureus strain 8325-4 and its mutants devoid of aureolysin, serine protease, and cysteine protease, respectively, in a well-established model of septic arthritis in mice. The inactivation of the exoprotease genes did not affect the frequency or the severity of joint disease. We conclude that in the model of haematogenously spread staphylococcal arthritis, the bacterial proteases studied do not act as virulence factors.

SarA is a repressor of hla (alpha-hemolysin) transcription in Staphylococcus aureus: its apparent role as an activator of hla in the prototype strain NCTC 8325 depends on reduced expression of sarS.

In most Staphylococcus aureus strains, inactivation of sarA increases hla transcription, indicating that sarA is a repressor. However, in S. aureus NCTC 8325 and its derivatives, used for most studies of hla regulation, inactivation of sarA resulted in decreased hla transcription. The disparate phenotype of strain NCTC 8325 seems to be associated with its rsbU mutation, which leads to sigma(B) deficiency. This has now been verified by the demonstration that sarA repressed hla transcription in an rsbU+ derivative of strain 8325-4 (SH1000). That sarA could act as a repressor of hla in an 8325-4 background was confirmed by the observation that inactivation of sarA in an agr sarS rot triple mutant dramatically increased hla transcription to wild-type levels. However, the apparent role of sarA as an activator of hla in 8325-4 was not a result of the rsbU mutation alone, as inactivation of sarA in another rsbU mutant, strain V8, led to increased hla transcription. Northern blot analysis revealed much higher levels of sarS mRNA in strain V8 than in 8325-4, which was likely due to the mutation in the sarS activator, tcaR, in 8325-4, which was not found in strain V8. On the other hand, the relative increase in sarS transcription upon the inactivation of sarA was 15-fold higher in 8325-4 than in strain V8. Because of this, inactivation of sarA in 8325-4 means a net increase in repressor activity, whereas in strain V8, inactivation of sarA means a net decrease in repressor activity and, therefore, enhanced hla transcription.

Reducing the fitness cost of antibiotic resistance by amplification of initiator tRNA genes.

Deformylase inhibitors belong to a novel antibiotic class that targets peptide deformylase, a bacterial enzyme that removes the formyl group from N-terminal methionine in nascent polypeptides. Using the bacterium Salmonella enterica, we isolated mutants with resistance toward the peptide deformylase inhibitor actinonin. Resistance mutations were identified in two genes that are required for the formylation of methionyl (Met) initiator tRNA (tRNAi)(fMet): the fmt gene encoding the enzyme methionyl-tRNA formyltransferase and the folD gene encoding the bifunctional enzyme methylenetetrahydrofolate-dehydrogenase and -cyclohydrolase. In the absence of antibiotic, these resistance mutations conferred a fitness cost that was manifested as a reduced growth rate in laboratory medium and in mice. By serially passaging the low-fitness mutants in growth medium without antibiotic, the fitness costs could be partly ameliorated either by intragenic mutations in the fmt/folD genes or by extragenic compensatory mutations. Of the extragenically compensated fmt mutants, approximately one-third carried amplifications of the identical, tandemly repeated metZ and metW genes, encoding tRNAi. The increase in metZW gene copy number varied from 5- to 40-fold and was accompanied by a similar increase in tRNAi levels. The rise in tRNAi level compensated for the lack of methionyl-tRNA formyltransferase activity and allowed translation initiation to proceed with nonformylated methionyl tRNAi. Amplified units varied in size from 1.9 to 94 kbp. Suppression of deleterious mutations by gene amplification may be involved in the evolution of new gene functions.

Natural human isolates of Staphylococcus aureus selected for high production of proteases and alpha-hemolysin are sigmaB deficient.

It has been reported that high production of proteases and alpha-hemolysin in the prototype Staphylococcus aureus strain 8325-4 was associated with its sigmaB deficiency. Here we analyzed one fresh clinical isolate (KS26) and two ancient human isolates (Wood46 and V8) selected for high production of proteases and alpha-hemolysin. All three strains lacked yellow pigment and showed a low level of expression of sigB-dependent promoters, indicating sigmaB deficiency. Nucleotide sequencing of the sigB operon revealed that KS26 and Wood46 had stop codons in rsbU and sigB, respectively, while V8 had an insertion of an IS element in rsbU. Complementation experiments with sigB on a plasmid reduced expression of proteases and alpha-hemolysin dramatically, indicating that the high production of these exoproteins was associated with sigmaB deficiency. Although sigmaB-deficient strains show attenuated virulence in some animal models, our results indicate that such strains can cause infection in humans.