Deletion of sua gene reduces the ability of Streptococcus uberis to adhere to and internalize into bovine mammary epithelial cells.

To elucidate the role of Streptococcus uberis adhesion molecule (SUAM) in the pathogenesis of S. uberis mastitis, sua deletion in S. uberis UT888 was achieved by homologous recombination using a thermosensitive plasmid. The deletion mutant was analyzed for sua deletion by PCR, southern blot and DNA sequencing, and was designated Δsua S. uberis UT888. As compared to the isogenic parent strain, Δsua S. uberis UT888 did not produce SUAM based on SDS-PAGE gel and western blot. Deletion of sua and lack of expression of SUAM by Δsua S. uberis UT888 markedly reduced the ability of the sua gene deletion mutant of S. uberis to adhere to and internalize into mammary epithelial cells. These results confirm the central role of SUAM in adherence to and internalization of S. uberis into host cells.

Elucidation of the DNA sequence of Streptococcus uberis adhesion molecule gene (sua) and detection of sua in strains of Streptococcus uberis isolated from geographically diverse locations.

Streptococcus uberis is an important environmental pathogen that causes subclinical and clinical mastitis in lactating and nonlactating cows throughout the world. S. uberis adhesion molecule (SUAM) was identified recently by our laboratory and we hypothesize that SUAM is a potential virulence factor involved in the pathogenesis of S. uberis mastitis. The first objective of the present study was to clone and sequence the SUAM gene (sua) from S. uberis UT888. The second objective was to determine the prevalence of sua in strains of S. uberis isolated from geographically diverse locations. The 20 amino acid N-terminal sequence of purified SUAM was utilized to identify a single open reading frame (ORF) in the S. uberis O140J (ATCC BAA-854) genome database. Three sets of primers were identified from this sequence for amplification of sub-fragments and the complete gene encoding SUAM. Restriction fragment analysis of the largest polymerase chain reaction (PCR) product confirmed the desired fragment had been amplified. This 2970bp PCR fragment was cloned into plasmid pCR-XL-TOPO and sequenced. The S. uberis UT888 sua sequence (NCBI Accession no. DQ232760) was 99% similar to the S. uberis O140J database sequence. The three pairs of PCR primers were used in a subsequent experiment to identify sua in 12 strains of S. uberis isolated in milk from dairy cows with mastitis in Tennessee (n=6), Colorado (n=1), Washington (n=1), New Zealand (n=1) and from the American Type Culture Collection (n=3). Primer pairs yielded the expected 2970, 2639 and 2362bp PCR fragments in all strains evaluated. In conclusion, we cloned and sequenced sua, which codes for the first described S. uberis adhesin, SUAM. sua was detected in all strains of S. uberis evaluated suggesting that it is conserved.

Identification, isolation, and partial characterization of a novel Streptococcus uberis adhesion molecule (SUAM).

The ability to attach to the host cell surface has been considered an important virulence strategy in many bovine mammary gland pathogens, including Streptococcus uberis. Research conducted in our laboratory lead to the identification of an S. uberis adhesion molecule (SUAM) with affinity for bovine lactoferrin (LF) and delineation of its role in adherence of S. uberis to bovine mammary epithelial cells. Using a selected bacterial surface protein extraction protocol and affinity chromatography, a 112-kDa protein that had a similar molecular mass and the LF affinity as one of the identified S. uberis LBP described by Fang and Oliver in 1999 was found. To further characterize SUAM, the N-terminal amino acid sequence of this protein was elucidated. A protein query versus translated database TBLASTN search of the National Center for Biotechnology (NCBI), non-redundant database, nr, with the LBP N-terminal amino acid sequence showed no significant similarity with previous entries. Antibodies directed against SUAM and a 17 amino acid long N-terminal sequence (pep-SUAM) inhibited adherence to and internalization of S. uberis UT888 into bovine mammary epithelial cells. Data presented suggests that we have discovered a novel bacterial protein involved in the pathogenesis of this economically important mastitis pathogen.

Binding of host glycosaminoglycans and milk proteins: possible role in the pathogenesis of Streptococcus uberis mastitis.

The role of indirect binding of host proteins through glycosaminoglycans (GAGs) on adherence and internalization of Streptococcus uberis to bovine mammary epithelial cells was evaluated. Preincubation of S. uberis with GAGs followed by incubation with fetal bovine serum (FBS), bovine milk or milk proteins resulted in greater adherence to and internalization of S. uberis into mammary epithelial cells than observed in untreated controls. Highest values were detected, when final incubation was done with milk. Greater adherence to and internalization into mammary epithelial cells were observed when heparin sulfate (HEP) and milk were used compared with any other GAG and FBS. When individual milk proteins were used, greatest adherence and internalization were observed when S. uberis strains were pretreated with HEP followed by treatment with beta-casein. The findings of this study illustrate a pathogenic strategy of S. uberis that may occur during the very early stages of infection.