Role of PknB kinase in antibiotic resistance and virulence in community-acquired methicillin-resistant Staphylococcus aureus strain USA300

Abstract

The regulation of cellular processes by eukaryote-like serine/threonine kinases is widespread in bacteria. In the last 2 years, several studies have examined the role of serine/threonine kinases in Staphylococcus aureus on cell wall metabolism, autolysis, and virulence, mostly in S. aureus laboratory isolates in the 8325-4 lineage. In this study, we showed that the pknB gene (also called stk1) of methicillin-resistant S. aureus (MRSA) strain COL and the community-acquired MRSA (CA-MRSA) strain USA300 is involved in cell wall metabolism, with the pknB mutant exhibiting enhanced sensitivity to beta-lactam antibiotics but not to other classes of antibiotics, including aminoglycosides, ciprofloxacin, bactrim, and other types of cell wall-active agents (e.g., vancomycin and bacitracin). Additionally, the pknB mutant of USA300 was found to be more resistant to Triton X-100-induced autolysis and also to lysis by lysostaphin. We also showed that pknB is a positive regulator of sigB activity, resulting in compromise in its response to heat and oxidative stresses. In association with reduced sigB activity, the expression levels of RNAII and RNAIII of agr and the downstream effector hla are upregulated while spa expression is downmodulated in the pknB mutant compared to the level in the parent. Consistent with an enhanced agr response in vitro, virulence studies of the pknB mutant of USA300 in a murine cutaneous model of infection showed that the mutant was more virulent than the parental strain. Collectively, our results have linked the pknB gene in CA-MRSA to antibiotic resistance, sigB activity, and virulence and have highlighted important differences in pknB phenotypes (virulence and sigB activity) between laboratory isolates and the prototypic CA-MRSA strain USA300.

FIG. 1.

Structural differences between the PknB kinase proteins of USA300 and COL. (A) Western blot analysis of PknB from USA300 (top panel) and COL (bottom panel). The pknB gene from each strain was cloned behind an N-terminal His tag. Expression of the fusion proteins after IPTG induction at 0, 2, and 4 h was monitored by Western blot analysis using a primary antibody directed toward the hexahistidine moiety. The schematic on the right shows the probable architecture of each PknB kinase protein on the basis of the estimated sizes of each fusion protein. Each of the PASTA domains is depicted as rectangles, while the kinase domain is represented by an oval. (B) Role of the PASTA domain in antibiotic resistance. COL and USA300 pknB mutants were complemented and cross-complemented with either their own or the heterologous kinase gene. The designations pknBu and pknBc correspond to the pkn genes of USA300 and COL, respectively. The antibiotic susceptibilities of the resultant strains were then compared between the parent and mutant strains by performing MIC assays using nafcillin and cloxacillin.

FIG. 2.

Cell wall phenotypes of the USA300 pknB mutant. Sensitivity of the pknB mutant to lysostaphin (A) and Triton X-100 (B). The pknB mutant was complemented with pEPSA5 containing pknB. The negative control for the mutant is the vector alone.

FIG. 3.

Decreased autolysin activity of the pknB mutant. (A) Bacteriolytic activities of secreted autolysins in the supernatants of stationary phase cultures, using heat-killed USA300 cells as the substrate. (B) Zymographic analysis of autolytic activity of the pknB mutant of USA300 using either M. lysodeikticus (top panel) or S. aureus 4220 (bottom panel) as the substrate. Note the decrease in the activity of the proenzyme for major autolysin Atl (130 kDa) and the processed autolysin (110 kDa; arrow) while the activities of the glucosaminidase (55 kDa) and amidase (63 kDa) cleaved from Atl are relatively unchanged. Numbers underneath the gel panels indicate the relative band intensities as determined by ImageJ.

FIG. 4.

Decreased atl transcription and increased protease activity in the pknB mutant contribute to its autolytic phenotype. (A) Northern blot analysis of stationary-phase cells showing the transcriptions of atl and spl (carrying a serine protease operon) in the pknB mutant in comparison with the levels in the parent and the complemented strain. (B) Protease activity in the supernatants of stationary-phase cells as assessed by an Enz-Chek Bodipy fluorescence kit. AU, arbitrary units. (C) Bacteriolytic activity of supernatants taken from stationary-phase cultures of different constructs grown in the presence of 1 mM PMSF using heat-killed USA300 as the substrate.

FIG. 5.

Decreased SigB activity in the pknB mutant. (A) Real-time PCR analysis of asp23 transcription in USA300, the pknB mutant, and the complemented mutant. (B) Growth of the pknB mutant at 42°C in comparison with that of the parent and complemented strains. (C) Growth of the pknB mutant complemented with the sigB gene in trans at 42°C in comparison with that of the parent and the mutant.

FIG. 6.

Expression of virulence determinants of the pknB mutant. (A) Northern blot analysis of sarA, RNAIII, RNAII, hla, and spa gene transcription in USA300, the pknB mutant, and the complemented mutant. (B) Relative hemolytic titers of the pknB mutant with sheep red blood cells in comparison to those of the parent and complemented strains.

FIG. 7.

Ability of the pknB mutant to cause murine skin infections. Six-week-old C57BL6 mice were injected with USA300, the pknB mutant, or the complemented strain. Weight changes during the course of the experiment were noted (A). After 3 days, the mice were sacrificed and their skin lesions were examined for size (B), bacterial burden (C), and histological changes in punch biopsy samples (D). In panels B and C, an asterisk represents P values of <0.05 as determined by the Kruskal-Wallace test. (D) Note the absence of the dermis and other subcellular structures (*) and the presence of bacteria (→) in the sample taken from the mouse infected with the pknB mutant.

FIG. 8.

Proposed model of gene regulation by pknB. In USA300, pknB acts as an activator of both atl and sigB. Its actions upon sigB are predicted to make the cell more resistant to harsh environmental conditions and less virulent based on the role of sigB in cell stress and its actions upon sarA and agr. In addition to reduced virulence, the decreased expression of the downstream effector splF in the pknB⁺ strain is predicted to reduce the degradation of the Atl proenzyme.