tcaA inactivation increases glycopeptide resistance in Staphylococcus aureus

Abstract

The experimental deletion of the tcaRAB region has been shown to increase teicoplanin resistance in Staphylococcus aureus. By sequential genetic complementation of a tcaRAB mutant, we identified tcaA as the key gene within tcaRAB that is responsible for changes in glycopeptide resistance levels. Northern blot analysis of the tcaRAB region showed that the tcaA gene is expressed only weakly over the growth cycle and is strongly inducible by teicoplanin. Among some clinical isolates tested, glycopeptide-intermediate-resistant (GISA) strains Michigan and SA137/93G were found to have truncated tcaA genes. While the former carries a nucleotide insertion that creates a premature stop codon, the latter was found to harbor an IS256 insertion. Complementation of these two GISA strains with a functional tcaA allele reduced their levels of teicoplanin and vancomycin resistance five- to eightfold and twofold, respectively. The data presented here indicate that inactivation of tcaA contributes to and plays a relevant role in glycopeptide resistance in S. aureus clinical isolates.

FIG. 1.

Complementation of tcaRAB mutant BB1372. The nucleotide positions shown correspond to the sequence registered in GenBank and EMBL (accession number AY008833). Bold lines, fragments inserted into vector pAW17; dotted lines, deleted ClaI-ClaI fragment; asterisk, the BglII site, which was used to remove part of tcaA from pAW17-tcaAB to create pAW17-tcaB. The MICs of teicoplanin and vancomycin on BHI agar for strain BB1372 complemented with the indicated plasmids are shown on the right. The positions of the probes used for Northern blot analysis are indicated as bars above the open reading frames.

FIG. 2.

Northern blot analysis of tcaRAB transcription. (A) Transcripts hybridizing to the probes specific for tcaR, tcaA, and tcaB; the positions of the probes within the corresponding open reading frames are shown in Fig. 1. Total RNA was harvested from COL at growth stages corresponding to the OD₆₀₀ shown. (B) Total RNA extracted from COL, uninduced (−) and induced (+) with teicoplanin, probed with tcaA. Each lane contains 10 μg of RNA. The positions of 16S rRNA (1.5 kb) and 23S rRNA (2.9 kb) and the approximate sizes (in nucleotides) of potential transcripts are shown, followed by the putative compositions of the transcripts in parentheses. As an indication of RNA loading, the 16S rRNA band from the ethidium bromide-stained gel is shown.

FIG. 3.

Population analysis profiles. Resistance profiles represent the number of CFU from an overnight culture plated on increasing concentrations of vancomycin after 48 h of incubation at 35°C.

FIG. 4.

tcaA inactivation in GISA strains. The nucleotide sequence resulting from a nucleotide insertion in strain MI is shown in the box. The insertion of an additional adenine nucleotide, highlighted in bold, generated a stop codon (asterisk). The position of the IS256 insertion in strain SA137/93G is shown. This was accompanied by an 8-bp duplication at both ends of the element. The arrow indicates the direction of tnp within IS256. Numbers indicate the nucleotide positions from the 5′ terminus of tcaA.