pGh9:ISS1 transpositional mutations in Streptococcus uberis UT888 causes reduced bacterial adherence to and internalization into bovine mammary epithelial cells

Abstract

Streptococcus uberis is an important mastitis pathogen that affects dairy cows worldwide. In spite of the economic impact caused by the high prevalence of S. uberis intramammary infections (IMI) in many well-managed dairy herds, pathogenic strategies and associated virulence factors of S. uberis are not well understood. It has been shown that S. uberis attaches to and internalizes into mammary epithelial cells and can survive inside cells for extended periods of time. We hypothesize that early attachment to and internalization into mammary epithelial cells is a critical step for the establishment of intramammary infection. The aim of this study is to identify and characterize chromosomally encoded virulence factors of S. uberis that allow early bacterial attachment to and internalization into mammary epithelial cells. A common approach used to identify virulence factors is by generating random insertion mutants that are defective in adherence to and internalization into mammary epithelial cells using pGh9:ISS1 mutagenesis system. A random insertion mutant library of S. uberis strain UT888 was created using a thermo-sensitive plasmid pGh9:ISS1 carrying ISS1 insertion sequence. Integration of the insertion sequence into the chromosome of these mutant clones was confirmed by PCR and Southern blot. Southern blot analysis of mutant clones also showed that insertional integration was random. Of 1000 random chromosomal insertion mutants of S. uberis strain UT888 screened, 32 had significantly reduced ability to adhere to and internalize into mammary epithelial cells. Chromosomal mapping of insertion sequence integration sites in some of these defective mutants showed integration into penicillin binding protein 2A (pbp2A), sensor histidine kinase, tetR family regulatory protein, phosphoribosylaminoimidazole carboxylase catalytic subunit (purE), lactose phosphotransferase, phosphoribosylamine glycine ligase (purD), and other genes involved in metabolic activities. These proteins may have a significant role in early bacterial colonization of the mammary gland during infection.