Inactivation of traP has no effect on the agr quorum-sensing system or virulence of Staphylococcus aureus

Abstract

The success of Staphylococcus aureus as a pathogen can largely be attributed to the plethora of genetic regulators encoded within its genome that temporally regulate its arsenal of virulence determinants throughout its virulence lifestyle. Arguably the most important of these is the two-component, quorum-sensing system agr. Over the last decade, the controversial presence of a second quorum-sensing system (the TRAP system) has been proposed, and it has been mooted to function as the master regulator of virulence in S. aureus by modulating agr. Mutants defective in TRAP are reported to be devoid of agr expression, lacking in hemolytic activity, essentially deficient in the secretion of virulence determinants, and avirulent in infection models. A number of research groups have questioned the validity of the TRAP findings in recent years; however, a thorough and independent analysis of its role in S. aureus physiology and pathogenesis has not been forthcoming. Therefore, we have undertaken such an analysis of the TRAP locus of S. aureus. We found that a traP mutant was equally hemolytic as the wild-type strain. Furthermore, transcriptional profiling found no alterations in the traP mutant in expression levels of agr or in expression levels of multiple agr-regulated genes (hla, sspA, and spa). Analysis of secreted and surface proteins of the traP mutant revealed no deviation in comparison to the parent. Finally, analysis conducted using a murine model of S. aureus septic arthritis revealed that, in contrast to an agr mutant, the traP mutant was just as virulent as the wild-type strain.

FIG. 1.

Secreted hemolytic activity of the traP mutant. S. aureus strains were streaked onto TSA sheep blood agar (Remel) and incubated for either 24 h (8325-4 lineage strains) (A) or 48 h (Newman lineage strains) (B). Tight zones of clearing around the immediate periphery of strains indicate secreted α-hemolysin, while the more diffuse zones around the strains correspond to secreted β-hemolysin.

FIG. 2.

Transcription profiling analysis. The transcriptional activities of hld (RNAIII) (A), α-hemolysin (hla) (B), V8 serine protease (sspA) (C), and surface protein A (spa) (D) were measured throughout growth in the 8325-4 (▪), 8325-4 traP::kan (□), Newman (•), and Newman traP::kan (○) backgrounds. All strains were grown at 37°C with shaking, and samples taken at the times indicated were assayed for β-galactosidase activity. The results are representative of at least three separate experiments.

FIG. 3.

Secreted and surface-associated protein profiles of the traP mutant. (A) Secreted protein profiles of 8325-4 (lane 1), 8325-4 traP::kan (lane 2), and 8325-4 agr::tet (lane 3). Protein samples are equivalent to 1.0 OD₆₀₀ unit of original culture. (B) Secreted protein profiles of Newman (lane 1), Newman traP::kan (lane 2), and Newman agr::tet (lane 3). Protein samples are equivalent to 2.0 OD₆₀₀ units of original culture. (C) Surface-associated protein profiles of 8325-4 (lane 1), 8325-4 traP::kan (lane 2), and 8325-4 agr::tet (lane 3). Protein samples are equivalent to 150 OD₆₀₀ units of original culture. (D) Surface-associated protein profiles of Newman (lane 1), Newman traP::kan (lane 2), and Newman agr::tet (lane 3). Protein samples are equivalent to 75 OD₆₀₀ units of original culture. The arrow denotes the 55-kDa band corresponding to surface protein A, which was subjected to densitometric analysis.

FIG. 4.

The presence of a functional traP locus is not required for the virulence of S. aureus in a murine model of septic arthritis. Female NRMI mice were inoculated with either S. aureus Newman or Newman traP::kan. Animals were continuously monitored over the course of the 10-day infection for weight loss (A) and for overt signs of arthritis in a double-blind fashion using an established arthritic index (B). Error bars indicate standard deviations. No statistically significance difference could be discerned between the two infection groups.