Expression of Staphylococcus aureus Clumping Factor A in Lactococcus lactis subsp. cremoris Using a New Shuttle Vector

Abstract

Staphylococcus aureus harbors redundant adhesins mediating tissue colonization and infection. To evaluate their intrinsic role outside of the staphylococcal background, a system was designed to express them in Lactococcus lactis subsp. cremoris 1363. This bacterium is devoid of virulence factors and has a known genetic background. A new Escherichia coli-L. lactis shuttle and expression vector was constructed for this purpose. First, the high-copy-number lactococcal plasmid pIL253 was equipped with the oriColE1 origin, generating pOri253 that could replicate in E. coli. Second, the lactococcal promoters P23 or P59 were inserted at one end of the pOri253 multicloning site. Gene expression was assessed by a luciferase reporter system. The plasmid carrying P23 (named pOri23) expressed luciferase constitutively at a level 10,000 times greater than did the P59-containing plasmid. Transcription was absent in E. coli. The staphylococcal clumping factor A (clfA) gene was cloned into pOri23 and used as a model system. Lactococci carrying pOri23-clfA produced an unaltered and functional 130-kDa ClfA protein attached to their cell walls. This was indicated both by the presence of the protein in Western blots of solubilized cell walls and by the ability of ClfA-positive lactococci to clump in the presence of plasma. ClfA-positive lactococci had clumping titers (titer of 4,112) similar to those of S. aureus Newman in soluble fibrinogen and bound equally well to solid-phase fibrinogen. These experiments provide a new way to study individual staphylococcal pathogenic factors and might complement both classical knockout mutagenesis and modern in vivo expression technology and signature tag mutagenesis.Staphylococcus aureus is a major pathogen responsible for a wide range of both acute and chronic infections (37). A key step in S. aureus infection is its ability to attach to various surfaces and colonize host tissues. For this purpose, S. aureus carries several functionally redundant surface adhesins, which have high affinity for either soluble proteins or extracellularmatrix components of the host (26, 38). These include, for instance, prothrombin (28), fibrinogen (18, 25), fibronectin (11, 33), vitronectin (14), and collagen (27), as well as other constituents.Since attachment to host tissues is an essential step of S. aureus disease, it was assumed that interfering with bacterial adhesion could be a means to prevent infection. This possibility was investigated in staphylococcal mutants defective in one or more of these cell-wall-associated ligands (9,18,22). Knockout mutants were tested both in vitro for their decreased adherence to purified ligands and in vivo for their lower pathogenicity in various animal models. When compared to their parent strains, the defective mutants were always significantly less able to attach to surfaces coated with purified ligands (4, 9, 18). Unexpectedly, however, these differences did not translate into major alterations of pathogenicity in vivo. Infectivity was either moderately or not affecte...

RNase Y-mediated regulation of the streptococcal pyrogenic exotoxin B

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The Lactococcus lactis FabF fatty acid synthetic enzyme can functionally replace both the FabB and FabF proteins of Escherichia coli and the FabH protein of Lactococcus lactis

“…Both plasmids were sequenced to verify fidelity (Keck Genomics Center, University of Illinois). pRK49 was digested with the restriction enzymes BamHI and PstI and ligated into the Lactococcus expression-shuttle vector pOri23 (Que et al 2000) cut with the same enzymes to produce pRK51. Plasmid pRK50 was digested with the restriction enzymes EcoR1 and XbaI and the fabF fragment was ligated into E. coli expression vector pBAD24 cut with the same enzymes to produce the plasmid pRK52.…”

Degradation of SsrA-tagged proteins in streptococci

“…Various Cterminal tags (SsrA tag and its derivatives) were added to the gfp gene by fusion PCR. These PCR products were cloned under the P 23 promoter (a strong lactococcal phage promoter) in plasmid pIB184Km for expression of GFPs in S. mutans Que et al, 2000). All the constructs were sequenced.…”