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Fine-tuned characterization of Staphylococcus aureus Newbould 305, a strain associated with mild and chronic mastitis in bovines.

Peton V, Bouchard DS, Almeida S, Rault L, Falentin H, Jardin J, Jan G, Hernandez D, François P, Schrenzel J, Azevedo V, Miyoshi A, Berkova N, Even S, Le Loir Y - Vet. Res. (2014)

Bottom Line: The results were compared with data obtained on S. aureus RF122, a strain representative of the major clone involved in severe bovine mastitis worldwide.In particular, the presence and characteristics of surface exposed proteins correlated well with the greater adhesion and internalization capacities of N305 in bovine mammary epithelial cells.N305 also displayed less diversity of toxin genes but secreted larger quantities of these toxins, associated with a higher cytotoxicity potential.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR 1253 STLO, 65 rue de Saint Brieuc, 35042, Rennes Cedex, France. vincent.peton@rennes.inra.fr.

ABSTRACT
S. aureus is a major aetiological agent of ruminant mastitis worldwide. The chronic nature of S. aureus mastitis makes it difficult to cure and prone to resurgence. In order to identify the bacterial factors involved in this chronicity, Newbould 305 (N305), a strain that can reproducibly induce mild and chronic mastitis in an experimental setting, was characterized in depth. We employed genomic and proteomic techniques combined with phenotype characterization, in order to comprehensively analyse N305. The results were compared with data obtained on S. aureus RF122, a strain representative of the major clone involved in severe bovine mastitis worldwide. Five mobile genetic elements were identified in the N305 genome as carrying virulence factors which correlated with phenotypic features such as cytotoxicity, mammary epithelial cell invasion or host-adaptation. In particular, the presence and characteristics of surface exposed proteins correlated well with the greater adhesion and internalization capacities of N305 in bovine mammary epithelial cells. N305 also displayed less diversity of toxin genes but secreted larger quantities of these toxins, associated with a higher cytotoxicity potential. Our data are consistent with the invasiveness and host-adaptation features which contribute to the chronicity of S. aureus mastitis. Mobile genetic elements, exoproteins and surface exposed proteins constitute good targets for further research to explore the underlying mechanisms related to mastitis chronicity.

No MeSH data available.


Related in: MedlinePlus

2D PAGE ofS. aureusRF122 and N305 protein extracts. Supernatants (A and B) and total proteomes (C and D) were harvested during the stationary phase. Supernatant proteins were precipitated with 10% trichlororacetic acid and washed with high-grade ethanol. Samples were purified using the 2D Clean Up kit (GE Healthcare) then separated by isoelectrofocalization on pH4-7 strips (total proteome) or pH3-11NL strips (supernatant) for the first dimension, and by 12% polyacrylamide gels for the second dimension. The gels were stained with Biosafe (Biorad) Coomassie blue, according to the manufacturer’s instructions. Arrows indicate spots that were over-expressed.
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Fig4: 2D PAGE ofS. aureusRF122 and N305 protein extracts. Supernatants (A and B) and total proteomes (C and D) were harvested during the stationary phase. Supernatant proteins were precipitated with 10% trichlororacetic acid and washed with high-grade ethanol. Samples were purified using the 2D Clean Up kit (GE Healthcare) then separated by isoelectrofocalization on pH4-7 strips (total proteome) or pH3-11NL strips (supernatant) for the first dimension, and by 12% polyacrylamide gels for the second dimension. The gels were stained with Biosafe (Biorad) Coomassie blue, according to the manufacturer’s instructions. Arrows indicate spots that were over-expressed.

Mentions: A comprehensive analysis of the N305 proteome (total cell, surface and secreted proteins) was carried out on cultures grown under conditions mimicking the context of mastitis, as described previously [32]. The N305 proteome was compared with that of RF122. A total of 215 proteins were categorized as being differentially produced in N305 or RF122, and were further identified. Sixty-eight proteins (31.6%) were identified as being over-expressed by RF122, and 147 (68.4%) by N305. In more detail, a total of 33 proteins (15.4% of total proteins) were identified in the culture supernatant (Additional file 4), 11 proteins being more abundant in RF122 and 22 in N305. Supernatant gels (Figure 4A and B) displayed the most flagrant proteomic differences between N305 and RF122. In the total cell lysate, 36 proteins (16.7% of total proteins) were identified (Additional file 5), ten proteins being more abundant in RF122 and 26 in N305. Trypsin shaving enabled the identification of up to 409 proteins because of the sensitivity of this method, and included 146 proteins that were relatively more abundant in one strain (67.9% of total proteins) (Additional file 2), with 47 proteins more abundant in RF122 and 99 in N305.Figure 4


Fine-tuned characterization of Staphylococcus aureus Newbould 305, a strain associated with mild and chronic mastitis in bovines.

Peton V, Bouchard DS, Almeida S, Rault L, Falentin H, Jardin J, Jan G, Hernandez D, François P, Schrenzel J, Azevedo V, Miyoshi A, Berkova N, Even S, Le Loir Y - Vet. Res. (2014)

2D PAGE ofS. aureusRF122 and N305 protein extracts. Supernatants (A and B) and total proteomes (C and D) were harvested during the stationary phase. Supernatant proteins were precipitated with 10% trichlororacetic acid and washed with high-grade ethanol. Samples were purified using the 2D Clean Up kit (GE Healthcare) then separated by isoelectrofocalization on pH4-7 strips (total proteome) or pH3-11NL strips (supernatant) for the first dimension, and by 12% polyacrylamide gels for the second dimension. The gels were stained with Biosafe (Biorad) Coomassie blue, according to the manufacturer’s instructions. Arrows indicate spots that were over-expressed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4230361&req=5

Fig4: 2D PAGE ofS. aureusRF122 and N305 protein extracts. Supernatants (A and B) and total proteomes (C and D) were harvested during the stationary phase. Supernatant proteins were precipitated with 10% trichlororacetic acid and washed with high-grade ethanol. Samples were purified using the 2D Clean Up kit (GE Healthcare) then separated by isoelectrofocalization on pH4-7 strips (total proteome) or pH3-11NL strips (supernatant) for the first dimension, and by 12% polyacrylamide gels for the second dimension. The gels were stained with Biosafe (Biorad) Coomassie blue, according to the manufacturer’s instructions. Arrows indicate spots that were over-expressed.
Mentions: A comprehensive analysis of the N305 proteome (total cell, surface and secreted proteins) was carried out on cultures grown under conditions mimicking the context of mastitis, as described previously [32]. The N305 proteome was compared with that of RF122. A total of 215 proteins were categorized as being differentially produced in N305 or RF122, and were further identified. Sixty-eight proteins (31.6%) were identified as being over-expressed by RF122, and 147 (68.4%) by N305. In more detail, a total of 33 proteins (15.4% of total proteins) were identified in the culture supernatant (Additional file 4), 11 proteins being more abundant in RF122 and 22 in N305. Supernatant gels (Figure 4A and B) displayed the most flagrant proteomic differences between N305 and RF122. In the total cell lysate, 36 proteins (16.7% of total proteins) were identified (Additional file 5), ten proteins being more abundant in RF122 and 26 in N305. Trypsin shaving enabled the identification of up to 409 proteins because of the sensitivity of this method, and included 146 proteins that were relatively more abundant in one strain (67.9% of total proteins) (Additional file 2), with 47 proteins more abundant in RF122 and 99 in N305.Figure 4

Bottom Line: The results were compared with data obtained on S. aureus RF122, a strain representative of the major clone involved in severe bovine mastitis worldwide.In particular, the presence and characteristics of surface exposed proteins correlated well with the greater adhesion and internalization capacities of N305 in bovine mammary epithelial cells.N305 also displayed less diversity of toxin genes but secreted larger quantities of these toxins, associated with a higher cytotoxicity potential.

View Article: PubMed Central - PubMed

Affiliation: INRA, UMR 1253 STLO, 65 rue de Saint Brieuc, 35042, Rennes Cedex, France. vincent.peton@rennes.inra.fr.

ABSTRACT
S. aureus is a major aetiological agent of ruminant mastitis worldwide. The chronic nature of S. aureus mastitis makes it difficult to cure and prone to resurgence. In order to identify the bacterial factors involved in this chronicity, Newbould 305 (N305), a strain that can reproducibly induce mild and chronic mastitis in an experimental setting, was characterized in depth. We employed genomic and proteomic techniques combined with phenotype characterization, in order to comprehensively analyse N305. The results were compared with data obtained on S. aureus RF122, a strain representative of the major clone involved in severe bovine mastitis worldwide. Five mobile genetic elements were identified in the N305 genome as carrying virulence factors which correlated with phenotypic features such as cytotoxicity, mammary epithelial cell invasion or host-adaptation. In particular, the presence and characteristics of surface exposed proteins correlated well with the greater adhesion and internalization capacities of N305 in bovine mammary epithelial cells. N305 also displayed less diversity of toxin genes but secreted larger quantities of these toxins, associated with a higher cytotoxicity potential. Our data are consistent with the invasiveness and host-adaptation features which contribute to the chronicity of S. aureus mastitis. Mobile genetic elements, exoproteins and surface exposed proteins constitute good targets for further research to explore the underlying mechanisms related to mastitis chronicity.

No MeSH data available.


Related in: MedlinePlus