Molecular Genetic and Structural Modeling Studies of Staphylococcus aureus RNA Polymerase and the Fitness of Rifampin Resistance Genotypes in Relation to Clinical Prevalence

Abstract

The adaptive and further evolutionary responses of Staphylococcus aureus to selection pressure with the antibiotic rifampin have not been explored in detail. We now present a detailed analysis of these systems. The use of rifampin for the chemotherapy of infections caused by S. aureus has resulted in the selection of mutants with alterations within the ␤ subunit of the target enzyme, RNA polymerase. Using a new collection of strains, we have identified numerous novel mutations in the ␤ subunits of both clinical and in vitro-derived resistant strains and established that additional, undefined mechanisms contribute to expression of rifampin resistance in clinical isolates of S. aureus. The fitness costs associated with rifampin resistance genotypes were found to have a significant influence on their clinical prevalence, with the most common clinical genotype (H 481 N, S 529 L) exhibiting no fitness cost in vitro. Intragenic mutations which compensate for the fitness costs associated with rifampin resistance in clinical strains of S. aureus were identified for the first time. Structural explanations for rifampin resistance and the loss of fitness were obtained by molecular modeling of mutated RNA polymerase enzymes.Resistance to antibiotics arising from point mutations in bacterial genes that encode drug targets is a well-recognized phenomenon (38), and expression of these mechanisms often confers a fitness cost that results from the decreased physiological activity of the altered target (1, 37). Nevertheless, increasing evidence obtained both from laboratory and from epidemiological studies indicates that intragenic compensatory mutations often act to maintain the long-term persistence of resistant bacteria by eliminating or reducing the fitness costs associated with the development of target-based resistance (27). Consequently, bacterial products that are the targets of antibiotic action present interesting systems for the study of structure-function relationships from the perspectives of resistance, fitness, and compensatory evolution (15,20). Furthermore, fitness costs and compensatory evolution are factors that can influence the prevalence of specific antibiotic resistance genotypes in the clinical setting (5,19,20,30,43).We have recently explored the genetic and structural basis of mupirocin resistance and fitness in Staphylococcus aureus and related this to the incidence of mupirocin resistant isoleucyltRNA synthetase genotypes arising in the clinic (19,20). A similar opportunity to examine these paradigms in relation to rifampin resistance now arises.Mutations that confer resistance to rifampin (Fig. 1) arise in the ␤ subunit (encoded by rpoB) of the target enzyme RNA polymerase (RNAP) and have been mapped to this location in all bacteria examined so far, including Escherichia coli (21), Mycobacterium tuberculosis (5, 36), and S. aureus (32,43). The fitness burdens and compensatory evolution associated with mutations in rpoB that confer resistance to rifampin have been studied in E. coli (37). However, the ...

Stepwise Decrease in Daptomycin Susceptibility in Clinical Staphylococcus aureus Isolates Associated with an Initial Mutation in rpoB and a Compensatory Inactivation of the clpX Gene

“…The RpoB A 477 D variation was previously reported to confer a severe fitness cost to S. aureus (38). As the deletion in clpX did not alter susceptibility to daptomycin, we speculated that the inactivation of clpX was selected because it compensates for a fitness cost conferred by the RpoB A 477 D variation.…”

“…rpoB mutations are common in DAP-NS strains, but to our knowledge, the A 477 D variation has not previously been selected after daptomycin exposure. However, the same rpoB mutation was selected following rifampin treatment in vitro, and the resulting strains exhibited decreased susceptibility to both rifampin and vancomycin (38,44). However, as only the rpoB gene was sequenced, the possibility that additional unidentified mutations contributed to the reported phenotypes in these two studies could not be ruled out (38,44).…”

“…Here, we demonstrate that the introduction of the single rpoB SNP in SADR-2 is enough to greatly increase the rifampin MIC and to double the vancomycin MIC (Table 2). As the A 477 D substitution is localized in the rifampin binding site of the RNAP ␤ subunit (38), this mutation presumably confers rifampin resistance by a direct mechanism. On the other hand, the decreased susceptibility to daptomycin, vancomycin, and ␤-lactams must be caused indirectly and likely involves the alterations observed in the cell wall.…”

“…However, the same rpoB mutation was selected following rifampin treatment in vitro, and the resulting strains exhibited decreased susceptibility to both rifampin and vancomycin (38,44). However, as only the rpoB gene was sequenced, the possibility that additional unidentified mutations contributed to the reported phenotypes in these two studies could not be ruled out (38,44). Here, we demonstrate that the introduction of the single rpoB SNP in SADR-2 is enough to greatly increase the rifampin MIC and to double the vancomycin MIC (Table 2).…”

Impact of rpoB Mutations on Reduced Vancomycin Susceptibility in Staphylococcus aureus

“…All of the mutations, except for one in M524rifR-10, were located within the rifampin resistance determining region (RRDR) that spans amino acid residues 463 to 550 (Table 2). Mutations in RRDR were reported to decrease the binding affinity of rifampin to RNA polymerase holoenzyme by lowering the hydrophobic interaction between RpoB and rifampin (21). It is interesting in this regard that a rifampin-selected mutant strain, M524rifR-10, possessed two rpoB mutations: one, R484C, within the RRDR, and the other, L887F, located outside the RRDR.…”