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

View Article: PubMed Central - PubMed

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).

No MeSH data available.


Related in: MedlinePlus

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.
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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.

Mentions: Streptococcus pneumoniae is a facultative anaerobe that colonizes the human nasopharynx of up to 70% of healthy individuals (Chan et al., 2012; Gamez and Hammerschmidt, 2012). As a consequence, the bacteria are customarily exposed to an oxygen-rich environment under colonization conditions, and thus the most frequent stress found by pneumococcal cells in vivo is oxidation. Pneumococci also produce H2O2 in amounts that can exceed 1 mM (which is 1,000-fold higher than the concentration needed to inhibit growth of E. coli cells) under aerobic and rich-nutrient conditions. In this way, resident pneumococci will kill or inhibit growth of other respiratory tract-colonizing flora, but growth of S. pneumoniae was not impaired even at the high levels of endogenously produced H2O2 (Pericone et al., 2003). Further, it was shown that pyruvate oxidase, which is the enzyme responsible for production of endogenous H2O2, also contributed to H2O2 resistance in pneumococci (Pericone et al., 2003). Although the ability of S. pneumoniae to cope with oxidative stress is still not well-understood, we explored whether pezAT could be involved in the response to this stress. This was done by exposing R6wt and R6ΔPezAT mutant to high concentration of exogenously added H2O2. The results (Figure 3) did not show any significant survival differences between the two strains at any of the tested H2O2 concentrations (20 and 40 mM), indicating that the pezAT operon was not involved in protecting the pneumococcal cells from oxidative stress. Another set of experiments were designed to test whether differences in biofilm formation between the wt and the mutant strains could be observed, since it has been shown that some TAs influence biofilm formation (Gonzalez Barrios et al., 2006; Wen et al., 2014).


The Streptococcus pneumoniae pezAT Toxin – Antitoxin System Reduces β -Lactam Resistance and Genetic Competence
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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4997998&req=5

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.
Mentions: Streptococcus pneumoniae is a facultative anaerobe that colonizes the human nasopharynx of up to 70% of healthy individuals (Chan et al., 2012; Gamez and Hammerschmidt, 2012). As a consequence, the bacteria are customarily exposed to an oxygen-rich environment under colonization conditions, and thus the most frequent stress found by pneumococcal cells in vivo is oxidation. Pneumococci also produce H2O2 in amounts that can exceed 1 mM (which is 1,000-fold higher than the concentration needed to inhibit growth of E. coli cells) under aerobic and rich-nutrient conditions. In this way, resident pneumococci will kill or inhibit growth of other respiratory tract-colonizing flora, but growth of S. pneumoniae was not impaired even at the high levels of endogenously produced H2O2 (Pericone et al., 2003). Further, it was shown that pyruvate oxidase, which is the enzyme responsible for production of endogenous H2O2, also contributed to H2O2 resistance in pneumococci (Pericone et al., 2003). Although the ability of S. pneumoniae to cope with oxidative stress is still not well-understood, we explored whether pezAT could be involved in the response to this stress. This was done by exposing R6wt and R6ΔPezAT mutant to high concentration of exogenously added H2O2. The results (Figure 3) did not show any significant survival differences between the two strains at any of the tested H2O2 concentrations (20 and 40 mM), indicating that the pezAT operon was not involved in protecting the pneumococcal cells from oxidative stress. Another set of experiments were designed to test whether differences in biofilm formation between the wt and the mutant strains could be observed, since it has been shown that some TAs influence biofilm formation (Gonzalez Barrios et al., 2006; Wen et al., 2014).

View Article: PubMed Central - PubMed

ABSTRACT

Chromosomally encoded Type II Toxin&ndash;Antitoxin operons are ubiquitous in bacteria and archaea. Antitoxins neutralize the toxic effect of cognate Toxins by protein&ndash;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&ndash;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 (&beta;-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 &beta;-lactams and genetic competence) and gain of other traits (virulence).

No MeSH data available.


Related in: MedlinePlus