The expression of a putative exotoxin and an ABC transporter during bovine intramammary infection contributes to the virulence of Staphylococcus aureus

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Abstract

Staphylococcus aureus is a leading cause of intramammary infections (IMI) and bovine mastitis is an important disease for the dairy industry. As this bacterium probably expresses specific genes for establishment of IMI, we studied the transcriptional profile of four S. aureus strains recovered from experimentally infected cows. Microbial RNA was extracted from bacteria isolated from milk, reverse-transcribed and labeled for hybridization to sub-genomic microarrays to detect candidate genes for further investigations. Several S. aureus genes were expressed during IMI; some were detected in samples from more than one strain, more than one cow and at more than one time point during infection. A selection of four genes showing strong expression and with putative functions in pathogenesis was further studied by qPCR. By comparing the expression in different media in vitro, we found that gene SACOL2171 was induced by iron restriction whereas the expression of the transcriptional regulator SACOL2325 and the ABC transporter SACOL0718-720 (vraFG) were induced by milk. In addition, the putative exotoxin SACOL0442 seemed to require the intramammary environment for expression. Gene-disrupted mutants for SACOL0720 and SACOL0442 showed no growth defect in vitro but were attenuated during bovine IMI, causing infections with significant reductions in bacterial and somatic cell counts. The milk from the mammary quarters infected with these mutants also showed better appearance and composition than milk from quarters infected with the wild type. In conclusion, we have identified genes that are most likely important for S. aureus IMI. These represent novel candidates to include in a vaccine.

Introduction

Bovine mastitis, an inflammation of the udder which often results from intramammary infection (IMI), is the most frequent (Olde Riekerink et al., 2008) and costly disease of dairy cows (Sears and McCarthy, 2003). A recent study shows that Staphylococcus aureus is the transmittable bacterium that is responsible for most of the clinical cases of mastitis (Reyher et al., 2011). Staphylococcal IMI commonly leads to a chronic mastitis disease due to the persistence of the original infective organism despite antibiotic treatment (Myllys et al., 1997) Novel approaches to prevent or treat staphylococcal IMI are urgently needed by the dairy industry. The analysis of S. aureus gene expression during infection should help to identify important elements which may be useful as drug targets or as components of a composite vaccine.

It is well known that the in vivo environment (mammalian host) can modulate gene expression of bacteria, including S. aureus (Allard et al., 2006). Identification of genes expressed by S. aureus during IMI was previously attempted by analyzing the protein profile (Taverna et al., 2007) or the gene expression profile of the bacterium cultured in vitro in conditions which mimic some aspects of the mammary gland environment (Lammers et al., 2000). However, the use of fresh milk as cultivation medium in vitro is significantly different from the altered milk of infected mammary glands (Hogarth et al., 2004).

The aim of this work was to identify a selection of genes expressed by S. aureus isolates during bovine IMI and to demonstrate the importance of some of them in this bacterium virulence since they may be interesting targets for immunotherapy or drug development. This was carried out using the most pertinent model system: the cow. To do so, relevant clinical isolates were used for experimental IMI, a rapid method to isolate bacteria from mastitic milk of infected cows was developed, transcriptomic analysis by DNA array and quantitative PCR were performed and the contribution to virulence of two identified genes was evaluated using a mutagenesis approach.

Section snippets

Bacterial isolates

Four different S. aureus strains were selected for this study. Strain SHY97-3906 was isolated from a case of bovine mastitis that occurred during lactation (Diarra et al., 2002) and was previously studied for its in vitro transcriptome (Allard et al., 2006). Isolates CLJ08-3, CLJ08-557 and CLJ08-1290 (herein named 3, 557 and 1290) were originally isolated from cows with persistent mastitis at dry-off. To demonstrate “persistence” we verified that these strains were isolated from cows that shed

Experimental IMI for gene expression studies

The udders of eight cows were inoculated in each quarter with approximately 50 CFU of either one of the four S. aureus strains used in this study. The inoculation of a minimum of eight animals was necessary in order to compare, with a statistical significance, the four tested strains in their ability to infect the mammary gland. However, no significant difference was observed in the ability of the four bacterial strains to generate an IMI considering the mean value of bacterial counts measured

Discussion

This study represents the first attempt to identify some genes expressed by S. aureus during bovine IMI by using the most relevant experimental model system; the cow. As most bacterial genes are regulated at the transcriptional level, we have used a DNA array technology as well as quantitative PCR to explore and pick-up insights on S. aureus genes that are expressed in the mammary gland environment. Many studies have demonstrated that in vitro transcriptomes of pathogenic bacteria were

Conclusion

In summary, using the cow as the experimental model, we have developed a method which allowed the identification of some genes expressed by S. aureus during mastitis. Among those genes were SACOL0442 and SACOL0718-720 for which their importance for S. aureus virulence was validated through mutagenesis and experimental bovine IMI. Our short list of S. aureus genes expressed during IMI now offers a selection of relevant candidate genes/proteins for additional investigations and could be

Acknowledgments

This research was financed by the Natural Science and Engineering Research Council of Canada (NSERC), Alberta Milk, Dairy Farmers of New Brunswick, Nova Scotia, Ontario and Prince Edward Island, Novalait Inc., Dairy Farmers of Canada, Canadian Dairy Network, AAFC, PHAC, Technology PEI Inc., Université de Montréal and University of Prince Edward Island through the Canadian Bovine Mastitis Research Network. We also acknowledge funding support from NSERC (discovery grant 89758-2010) to F.M.

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Present address: South Dakota State University, Brookings, SD 57007, USA.

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