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Inactivation of thyA in Staphylococcus aureus attenuates virulence and has a strong impact on metabolism and virulence gene expression.

Kriegeskorte A, Block D, Drescher M, Windmüller N, Mellmann A, Baum C, Neumann C, Lorè NI, Bragonzi A, Liebau E, Hertel P, Seggewiss J, Becker K, Proctor RA, Peters G, Kahl BC - MBio (2014)

Bottom Line: The expression of genes involved in pyrimidine and purine metabolism and nucleotide interconversion changed significantly.In the era of community-associated methicillin-resistant S. aureus, the therapeutic use of TMP-SMX is increasing.Our study helps to understand the clinical nature of TD-SCVs, which emerge frequently once patients are treated with TMP-SMX.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, University Hospital of Münster, Münster, Germany.

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Related in: MedlinePlus

Characteristics and phenotypes of S. aureus SH1000 wild type, ΔthyA mutant, and complemented mutant. Phenotypes of the wild-type strain SH1000 (A), the ΔthyA mutant (B), and the complemented mutant (C) are shown on Columbia blood agar (row I). Susceptibility testing for TMP-SMX was performed on Columbia blood agar (row II). Light microscopy (row III) and transmission electron microscopy (row IV) revealed the typical features of the respective strains.
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fig1: Characteristics and phenotypes of S. aureus SH1000 wild type, ΔthyA mutant, and complemented mutant. Phenotypes of the wild-type strain SH1000 (A), the ΔthyA mutant (B), and the complemented mutant (C) are shown on Columbia blood agar (row I). Susceptibility testing for TMP-SMX was performed on Columbia blood agar (row II). Light microscopy (row III) and transmission electron microscopy (row IV) revealed the typical features of the respective strains.

Mentions: To study the impact of thyA inactivation in detail, we constructed site-directed ΔthyA deletion mutants in the well-characterized S. aureus strain SH1000 (32) and in S. aureus JE2 (belonging to the clinically important USA300 background), in which thyA was replaced by the erythromycin resistance cassette ermB. The SH1000 ΔthyA mutant was not able to grow in or on thymidine-free media (Mueller-Hinton [MH] agar and/or thymidine-free chemical-defined medium [CDM]) and displayed small, nonpigmented, nonhemolytic colonies on Columbia blood agar compared to those of the wild type and the complemented mutant (Fig. 1A-C, row I). Also, pleomorphic cocci in Gram staining and enlarged cocci with incomplete or multiple cross walls in transmission electron microscopy (TEM) were visible in the mutant (Fig. 1B, row III-IV), in contrast to homogeneous cocci of the wild type with regular cross walls in TEM (Fig. 1A, row III-IV). In the complemented mutant, which expressed wild-type thyA constitutively on a plasmid, all these features were restored, leading to the wild-type phenotype (Fig. 1C, row I-IV). Standard susceptibility testing (E test, agar diffusion) according to CLSI guidelines (33) could not be performed on MH agar due to the lack of thymidine and consequently no growth of the mutant. Therefore, Columbia blood agar was used, on which TMP-SMX did not inhibit the growth of the ΔthyA mutant, whereas the wild type and the complemented mutant showed clear inhibition zones around the disks, although within the inhibition zone tiny SCV-like colonies appeared (Fig. 1, row II). Applying CLSI guidelines for susceptibility testing, the mutant has to be considered resistant, whereas the wild type and the complemented mutant have to be considered susceptible, although all strains showed similar tiny colonies around the disk on Columbia blood agar. Tested on MH agar, the wild type and complemented mutant were TMP-SMX susceptible (MIC of 0.064 µg/ml for both strains). All the described features of the SH1000 ΔthyA mutant and the complemented mutant were also obtained with the JE2 ΔthyA mutant (data not shown).


Inactivation of thyA in Staphylococcus aureus attenuates virulence and has a strong impact on metabolism and virulence gene expression.

Kriegeskorte A, Block D, Drescher M, Windmüller N, Mellmann A, Baum C, Neumann C, Lorè NI, Bragonzi A, Liebau E, Hertel P, Seggewiss J, Becker K, Proctor RA, Peters G, Kahl BC - MBio (2014)

Characteristics and phenotypes of S. aureus SH1000 wild type, ΔthyA mutant, and complemented mutant. Phenotypes of the wild-type strain SH1000 (A), the ΔthyA mutant (B), and the complemented mutant (C) are shown on Columbia blood agar (row I). Susceptibility testing for TMP-SMX was performed on Columbia blood agar (row II). Light microscopy (row III) and transmission electron microscopy (row IV) revealed the typical features of the respective strains.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4128360&req=5

fig1: Characteristics and phenotypes of S. aureus SH1000 wild type, ΔthyA mutant, and complemented mutant. Phenotypes of the wild-type strain SH1000 (A), the ΔthyA mutant (B), and the complemented mutant (C) are shown on Columbia blood agar (row I). Susceptibility testing for TMP-SMX was performed on Columbia blood agar (row II). Light microscopy (row III) and transmission electron microscopy (row IV) revealed the typical features of the respective strains.
Mentions: To study the impact of thyA inactivation in detail, we constructed site-directed ΔthyA deletion mutants in the well-characterized S. aureus strain SH1000 (32) and in S. aureus JE2 (belonging to the clinically important USA300 background), in which thyA was replaced by the erythromycin resistance cassette ermB. The SH1000 ΔthyA mutant was not able to grow in or on thymidine-free media (Mueller-Hinton [MH] agar and/or thymidine-free chemical-defined medium [CDM]) and displayed small, nonpigmented, nonhemolytic colonies on Columbia blood agar compared to those of the wild type and the complemented mutant (Fig. 1A-C, row I). Also, pleomorphic cocci in Gram staining and enlarged cocci with incomplete or multiple cross walls in transmission electron microscopy (TEM) were visible in the mutant (Fig. 1B, row III-IV), in contrast to homogeneous cocci of the wild type with regular cross walls in TEM (Fig. 1A, row III-IV). In the complemented mutant, which expressed wild-type thyA constitutively on a plasmid, all these features were restored, leading to the wild-type phenotype (Fig. 1C, row I-IV). Standard susceptibility testing (E test, agar diffusion) according to CLSI guidelines (33) could not be performed on MH agar due to the lack of thymidine and consequently no growth of the mutant. Therefore, Columbia blood agar was used, on which TMP-SMX did not inhibit the growth of the ΔthyA mutant, whereas the wild type and the complemented mutant showed clear inhibition zones around the disks, although within the inhibition zone tiny SCV-like colonies appeared (Fig. 1, row II). Applying CLSI guidelines for susceptibility testing, the mutant has to be considered resistant, whereas the wild type and the complemented mutant have to be considered susceptible, although all strains showed similar tiny colonies around the disk on Columbia blood agar. Tested on MH agar, the wild type and complemented mutant were TMP-SMX susceptible (MIC of 0.064 µg/ml for both strains). All the described features of the SH1000 ΔthyA mutant and the complemented mutant were also obtained with the JE2 ΔthyA mutant (data not shown).

Bottom Line: The expression of genes involved in pyrimidine and purine metabolism and nucleotide interconversion changed significantly.In the era of community-associated methicillin-resistant S. aureus, the therapeutic use of TMP-SMX is increasing.Our study helps to understand the clinical nature of TD-SCVs, which emerge frequently once patients are treated with TMP-SMX.

View Article: PubMed Central - PubMed

Affiliation: Institute of Medical Microbiology, University Hospital of Münster, Münster, Germany.

Show MeSH
Related in: MedlinePlus