Vancomycin Tolerance Induced by Erythromycin but Not by Loss ofvncRS,vex3, orpep27Function inStreptococcus pneumoniae

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Abstract
Vancomycin-tolerant Streptococcus pneumoniae is a growing problem among drug-resistant human pathogens. Some vancomycin-tolerant pneumococci have been reported to carry mutations in loci encoding a two-component regulatory system designated VncRS or in a proximal ABC transporter, Vex. A model was advanced proposing that the tolerance phenotype resulted from the inability of a vncS mutant to respond to the Vex-transported Pep27 “death peptide” signal and dephosphorylate VncR, thereby preventing relief of repression of autolytic and other cell death functions in response to antibiotics. To explore this hypothesis, we constructed mutations in vncS, vncR, vex3, and pep27 in S. pneumoniae strain R6 and two additional genetic backgrounds. The lytic responses of the isogenic ΔvncS, Δvex3, ΔvncR, and Δpep27 mutants, but not a ΔlytA strain, to vancomycin were indistinguishable from that of the parent strain. ΔvncS strains also failed to exhibit tolerance to vancomycin at various doses in multiple media and showed wild-type sensitivity to other classes of autolysis-inducing antibiotics. In contrast, addition of subinhibitory levels of the antibiotic erythromycin led to tolerance to vancomycin during late, but not early, exponential-phase growth in a ΔvncS strain, in the parent strain R6, and in two other strains bearing erythromycin resistance markers, namely, a ΔvncR strain and an unrelated ΔcomD strain that is defective in competence-quorum sensing. Thus, this tolerance effect resulted from changes in cell growth or other erythromycin-dependent phenomena and not inactivation of vncS per se. Consistent with these results, and in contrast to a previous report, we found that a synthetic form of Pep27 did not elicit lytic or nonlytic killing of pneumococci. Finally, microarray transcriptional analysis and β-galactosidase reporter assays revealed VncS-dependent regulation of the vex123 gene cluster but did not support a role for VncRS in the regulation of autolytic or other putative cell death loci. Based on these findings, we propose that vancomycin tolerance in S. pneumoniae does not result from loss of vncS function alone.