The Streptococcus pneumoniae pezAT Toxin-Antitoxin System Reduces β-Lactam Resistance and Genetic Competence

Abstract

Chromosomally encoded Type II Toxin-Antitoxin operons are ubiquitous in bacteria and archaea. Antitoxins neutralize the toxic effect of cognate Toxins by protein-protein interactions and sequestering the active residues of the Toxin. Toxins target essential bacterial processes, mostly translation and replication. However, one class apart is constituted by the PezAT pair because the PezT toxin target cell wall biosynthesis. Here, we have examined the role of the pezAT toxin-antitoxin genes in its natural host, the pathogenic bacterium Streptococcus pneumoniae. The pezAT operon on Pneumococcal Pathogenicity Island 1 was deleted from strain R6 and its phenotypic traits were compared with those of the wild type. The mutant cells formed shorter chains during exponential phase, leading to increased colony-forming units. At stationary phase, the mutant was more resilient to lysis. Importantly, the mutant exhibited higher resistance to antibiotics targeting cell walls (β-lactams), but not to antibiotics acting at other levels. In addition, the mutants also showed enhanced genetic competence. We suggest that PezAT participates in a subtle equilibrium between loss of functions (resistance to β-lactams and genetic competence) and gain of other traits (virulence).

Keywords: Streptococcus pneumoniae; antibiotic resistance; genetic competence; genetic transformation; pezAT; toxin–antitoxin.

FIGURE 1

Schematic organization of the pneumococcal pezAT operon. The scheme depicts the position of the single promoter sequence (line with arrowhead pointing to the direction of transcription), which includes the -35 and -10 regions, as well as the inverted repeated sequence (PS, convergent arrows) that is the binding site of PezA and PezT proteins. Ribosome-binding site sequences are denoted as RBS and the initial start site is also depicted with arrow. The pezA termination codon, and the overlapping pezT initiation codon are also indicated. Substitution of the entire operon by a gene cassette encoding resistance to kanamycin is shown below.

FIGURE 2

The pezAT mutant strain forms shorter chains and is more prone to lysis at stationary phase. (A) Growth patterns for both R6wt and R6ΔPezAT mutant strains were assessed by measuring OD₆₅₀ every 30 min. (B) The number of CFU/ml at OD₆₅₀∼0.2, 0.4, 0.6, and 0.8 were determined. t-test was used to evaluate the statistical differences and the asterisk symbol (^∗) denotes statistical significance (p-value < 0.05) between R6wt and R6ΔPezAT. (C) The morphology of the cells of both strains was examined under phase-contrast microscope. Pictures were taken at 100× magnifications, 200 ms.

FIGURE 3

Oxidative stress affects equally Streptococcus pneumoniae wt and ΔpezAT strains. Pneumococcal cultures at OD₆₅₀∼0.3 were treated with H₂O₂, 20 min, 37°C, and the number of survival cells were counting to determine the number of CFU/ml on solid medium. t-test was used to evaluate the statistical differences and no differences (p-value < 0.05) were observed for both strains under oxidative stress.

FIGURE 4

Deletion of the pezAT operon increases resistance to ampicillin. (A) Pneumococcal strain-resistances detected by MIC, determined by change of color from resazurin (blue) to resorufin (pink). (B) Color changes measured by light absorbance at 600 nm (resazurin) as a function of the time of incubation, at the different concentrations of ampicillin indicated. Controls: no cells and no antibiotic. t-test was used to evaluate the statistical differences (p-value < 0.05).

FIGURE 5

Deletion of the pezAT operon leads to cells with increased transformability. Competent pneumococcal cells of indicated strains were treated or not with CSP-1 and incubated with transforming DNA prepared from total cell lysates (Chr) or from homogeneous DNA amplified by PCR from total cell lysates (Str41). t-test was used to evaluate the statistical differences and the asterisk symbol (^∗) indicates statistical significance (p-value < 0.05) between R6wt and R6ΔPezAT.

FIGURE 6

Deletion of the pezAT operon increases genetic competence. The development of spontaneous competence of both pneumococcal wt and pezAT mutant cells were assessed by measuring, at different pH, the transcriptional level of Photinus pyralis luc gene that was fused downstream of the promoter of ssbB, which is a late competent gene. Gray lines indicate growth curves at OD₄₉₂, whereas black lines depict RLUs/OD₄₉₂, ranging from pH 7.7–7.9. Vertical line joining both panels points the time differences in the onset of the main peaks of competence. Values correspond to individual cultures representative of three independent experiments (Caymaris et al., 2010).