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

Sehmi Paik,^1,2, Lauren Senty,^1,2 Sankar Das,¹ Jody C. Noe,^1,2, Cindy L. Munro,³ and Todd Kitten^1,2,4*

The Philips Institute of Oral and Craniofacial Molecular Biology, Departments of,¹ Microbiology and Immunology,² Adult Health Nursing,³ the Center for the Study of Biological Complexity, Virginia Commonwealth University, Richmond, Virginia⁴

Received 27 January 2005/ Returned for modification 16 March 2005/ Accepted 13 May 2005

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.

Supplemental material for this article may be found at http://iai.asm.org/.

Editor: J. N. Weiser

Present address: Wayne State University, NIH Perinatology Research Branch, 275 E. Hancock Ave., Detroit, MI 48201.

Present address: 2200 Bergquist Drive, Suite 1, 859 MDTS/MTLLM, Lackland AFB, TX 78236-5300.

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