Identification of Virulence Determinants for Endocarditis in Streptococcus sanguinis by Signature-Tagged Mutagenesis

The Philips Institute of Oral and Craniofacial Molecular Biology, Virginia Commonwealth University, 521 North 11th Street, Richmond, VA 23298-0566, USA.
Infection and Immunity (Impact Factor: 3.73). 10/2005; 73(9):6064-74. DOI: 10.1128/IAI.73.9.6064-6074.2005
Source: PubMed


Streptococcus sanguinis is a gram-positive, facultative anaerobe and a normal inhabitant of the human oral cavity. It is also one of the most common agents of infective endocarditis, a serious endovascular infection. To identify virulence factors for infective endocarditis, signature-tagged mutagenesis (STM) was applied to the SK36 strain of S. sanguinis, whose genome is being sequenced. STM allows the large-scale creation, in vivo screening, and recovery of a series of mutants with altered virulence. Screening of 800 mutants by STM identified 38 putative avirulent and 5 putative hypervirulent mutants. Subsequent molecular analysis of a subset of these mutants identified genes encoding undecaprenol kinase, homoserine kinase, anaerobic ribonucleotide reductase, adenylosuccinate lyase, and a hypothetical protein. Virulence reductions ranging from 2-to 150-fold were confirmed by competitive index assays. One putatively hypervirulent strain with a transposon insertion in an intergenic region was identified, though increased virulence was not confirmed in competitive index assays. All mutants grew comparably to SK36 in aerobic broth culture except for the homoserine kinase mutant. Growth of this mutant was restored by the addition of threonine to the medium. Mutants containing an insertion or in-frame deletion in the anaerobic ribonucleotide reductase gene failed to grow under strictly anaerobic conditions. The results suggest that housekeeping functions such as cell wall synthesis, amino acid and nucleic acid synthesis, and the ability to survive under anaerobic conditions are important virulence factors in S. sanguinis endocarditis.

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    • "Over 9,000 PCR fragments were amplified for mutant creation and confirmation. To preclude false identification of genes as essential due to low transformation frequencies, we performed experiments to optimize the transformation efficiency of S. sanguinis SK3626. Using our optimized method, up to 2x106 mutant colonies could be obtained from 107 bacterial cells for non-essential gene transformations. "
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    • "Transformation was done essentially as described previously (Paik et al., 2005) with modifications. Briefly, overnight cultures were grown in TH broth containing 2.5% heatinactivated horse serum (PAA Laboratories; Dartmouth, MA). "
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