Resistance Determinants for β-Lactam Antibiotics in Laboratory Mutants ofStreptococcus pneumoniaeThat Are Involved in Genetic Competence

Abstract

Laboratory mutants of Streptococcus pneumoniae resistant to either cefotaxime or piperacillin reveal defects in competence development independent of the selective beta-lactam. A resistance determinant ciaH encoding a putative histidine kinase of a two-component signal-transducing system that is also involved in competence regulation was recently identified in cefotaxime-resistant mutants. We show now that the CiaH protein can be phosphorylated by ATP in vitro, and that it also phosphorylates the cognate response regulator CiaR. The mutant C306 containing the CiaH mutation Thr-230-Pro is completely noncompetent. It does not release competence-inducing activity (competence factor) into the medium nor can such an activity be released from the cells. Competence in C306 cannot be induced upon addition of external competence factor, in contrast to the competence-defective piperacillin-resistant mutants P506 and P408. A novel resistance determinant cpoA specific for piperacillin was identified in piperacillin-resistant mutants. CpoA is responsible for the competence defect in P506 but not in P408. The results document a tight link between the action of beta-lactams and competence development in the pneumococcus and confirm that the two beta-lactams piperacillin and cefotaxime act via different primary targets.

TheStreptococcus pneumoniae ciaRegulon: CiaR Target Sites and Transcription Profile Analysis

“…We do not know yet whether the phenotype of ciaH mutants-increased beta-lactam resistance-is due to one particular gene product or is a reflection of an overall change in cell wall biochemistry mediated by the cia regulon. The twofold increase in the MIC of cefotaxime for ciaH mutants was observed in the wild-type background (from 0.02 to 0.04 g/ml) and in mutants where pbp2x mutations had already resulted in elevated MICs (from 0.2 to 0.4 g/ml), and these values are in the range conferred by single point mutations in penicillin binding protein genes (17,58). Homologues of the enzymes pyrimidine-nucleoside phosphorylase and deoxyribose phosphate aldolase are required for pyrimidine biosynthesis and feed into the precursor FIG.…”

“…Curiously, ciaH mutants were affected in the development of genetic competence as well (19,20), an effect which has since been confirmed in several recent publications (14,34,58). The comCDE system, which is responsible for the induction of genetic competence, is a complex regulatory network: the CSP peptide, the processed secreted product of comC, is recognized by the histidine protein kinase ComD, and the response regulator ComE is responsible for autoinduction of the comCDE operon and other early genes, among which are the transcriptional activators ComX1 and ComX2, which in turn induce the late competence genes (43).…”

“…R6 Cm r transformants were isolated, and a blue colony was used for the introduction of cia mutations. Insertion-duplication mutants in ciaR produced dark-blue colonies, whereas introduction of the ciaH C306 allele resulted in white colonies; no mixed populations were observed for the cia mutants as has been described for strains grown under competence-preventing conditions (58). Chromosomal fusion of the lacZЈ gene under the control of the lic and dlt promoter regions was performed using the lacZ reporter plasmid pEVP3 (11).…”

A novel resistance mechanism against beta-lactams in Streptococcus pneumoniae involves CpoA, a putative glycosyltransferase

“…The increase in resistance may appear only marginal; i.e., the MICs for the mutants increased only by a factor of two compared to the MIC for the parental strain. One should be reminded, however, that a single point mutation in PBP2x confers generally at most a fourfold increase in MIC for cefotaxime, and with PBP2b, the piperacillin MIC increases just twofold or even less (11,52). The high-level resistance phenotype of laboratory mutants or clinical isolates is due to a combination of mutations in several genes.…”

“…The competence deficiency appears to be due to distinct mechanisms: the ciaH C306 allele conferred complete loss of transformability, and the mutant did not produce CF. Neither the growth medium nor wash fractions of C306 contained detectable amounts of competence-inducing activity (12,52). In contrast, cpoA mutants have residual competence activity, and CF activity was detected in the growth medium of P506 containing the cpoA-P106 allele although very late during the growth cycle (not shown [10]), showing that the cpoA mutation allows for CSP synthesis but not necessarily at the normal time during the growth cycle.…”