Construction and characterization of three knockout mutants of the fbl gene in Staphylococcus lugdunensis

Abstract

Staphylococcus lugdunensis is an important human pathogen that causes infectious diseases similar to those caused by Staphylococcus aureus. In contrast to S. aureus, only a very few pathogenicity factors of S. lugdunensis have been characterized. Notably, a genetic manipulation of S. lugdunensis has not yet been described. Ours is the first report where transformation of three different plasmids (pBT2, pRB473, and pT181) into S. lugdunensis and a directed genetic manipulation of S. lugdunensis are described. We constructed fbl knockout mutants from three different strains of S. lugdunensis to show that at least in these strains, the fibrinogen binding is exclusively mediated by Fbl.

Multi-Virulence-Locus Sequence Typing of Staphylococcus lugdunensis Generates Results Consistent with a Clonal Population Structure and Is Reliable for Epidemiological Typing

“…As systems to genetically manipulate S. lugdunensis succeeded only very recently (21,22), a few virulent factors are known to date. We selected (27,30) and IsdJ, a protein of an iron acquisition system, which of the CoNS, is only used by S. lugdunensis (30,32).…”

“…So, in contrast to S. aureus, only very few pathogenicity factors have been characterized. Among them, the fibrinogen binding protein Fbl is similar in structure and organization to S. aureus clumping factor A (ClfA) (23)(24)(25) and is probably the only fibrinogen-binding surface protein of S. lugdunensis (21). A von Willebrand factor-binding protein, denoted vWbl, has also been described (26).…”

“…Furthermore, genetic tools for the manipulation of S. lugdunensis were limited until very recently, when efficient protoplast transformation (21) and electroporation (22) were reported. So, in contrast to S. aureus, only very few pathogenicity factors have been characterized.…”

Sortase A promotes virulence in experimental Staphylococcus lugdunensis endocarditis

“…ClfA contributes to S. aureus pathogenesis in mouse models of experimental septic arthritis, kidney abscess formation and also endocarditis in rats (Josefsson et al, 2001;Moreillon et al, 1995). Previously described fbl mutants of S. lugdunensis were unable to adhere to fibrinogen suggesting that it is the only fibrinogen binding surface protein of S. lugdunensis (Marlinghaus et al, 2012). The von Willebrand factor binding protein (vWbl) from S. lugdunensis is a large .200 kDa multi domain surface protein which contains an Arg-Glu-Asp (RGD) motif that is found in many integrin binding proteins.…”

Important Contribution of the Novel Locus comEB to Extracellular DNA-Dependent Staphylococcus lugdunensis Biofilm Formation

“…Both fragments were sequentially ligated with pCU1, excised as a single 4,072-bp fragment, and then cloned into the EcoRI and PstI sites of the vector pBT2 (34), generating plasmid pBT2comEB::ermB. Subsequently, pBT2comEB:: ermB was introduced into the strain w701 by protoplast transformation to disrupt the comEB gene by homologous recombination essentially as described previously (35). For complementation, DNA fragments encoding either comEB (883 bp) or comEB-comEC (3,147 bp), which were previously amplified from the genomic DNA of strain w701 using primer pairs ComAF and ComBR or ComAF and ComER, respectively (Table 1), were cloned into the EcoRI and BamHI sites of the shuttle vector pRB473 (36), generating pRBcomEB or pRBcomEB/EC, respectively.…”