title: Vancomycin tolerance induced by erythromycin but not by loss of vncRS, vex3, or pep27 function in Streptococcus pneumoniae.
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 DeltavncS, Deltavex3, DeltavncR, and Deltapep27 mutants, but not a DeltalytA strain, to vancomycin were indistinguishable from that of the parent strain. DeltavncS 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 DeltavncS strain, in the parent strain R6, and in two other strains bearing erythromycin resistance markers, namely, a DeltavncR strain and an unrelated DeltacomD 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 beta-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.
authors.affiliation: Infectious Diseases Research Division, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, USA.
authors.forename: Gregory T
authors.initials: GT
authors.lastname: Robertson
authors.forename: Jingyong
authors.initials: J
authors.lastname: Zhao
authors.forename: Bhushan V
authors.initials: BV
authors.lastname: Desai
authors.forename: William H
authors.initials: WH
authors.lastname: Coleman
authors.forename: Thalia I
authors.initials: TI
authors.lastname: Nicas
authors.forename: Raymond
authors.initials: R
authors.lastname: Gilmour
authors.forename: Leo
authors.initials: L
authors.lastname: Grinius
authors.forename: Donald A
authors.initials: DA
authors.lastname: Morrison
authors.forename: Malcolm E
authors.initials: ME
authors.lastname: Winkler
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000502:physiology*; D000818:Animals; D001329:Autolysis; D001426:Bacterial Proteins; D001483:Base Sequence; D004917:Erythromycin / Q000494:pharmacology*; D005260:Female; D051379:Mice; D008813:Mice, Inbred ICR; D008969:Molecular Sequence Data; D009154:Mutation; D011494:Protein Kinases / Q000502:physiology*; D013296:Streptococcus pneumoniae / Q000187:drug effects* / Q000235:genetics; D014157:Transcription Factors / Q000502:physiology*; D020713:Vancomycin Resistance
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D014157:Transcription Factors; C119633:VncR protein, Streptococcus pneumoniae; D004917:Erythromycin; D011494:Protein Kinases; C119671:VncS histidine kinase