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S. gordonii LTA or lipoproteins unaffected bacterial morphology, size, or growth.Δlgt weakly induced proinflammatory cytokines in macrophages.S. gordonii lipoproteins potently induced proinflammatory cytokines via TLR2.Δlgt was less virulent than the wild-type S. gordonii or ΔltaS in vivo.Gram-positive bacteria such as Streptococcus gordonii causing life-threatening infective endocarditis are mainly recognized by Toll-like receptor 2 (TLR2). Lipoteichoic acid (LTA) and lipoproteins are representative TLR2 ligands that play important roles in bacterial infection and in host inflammatory responses. In the present study, we generated an LTA-deficient mutant (ΔltaS) and a lipoprotein-deficient mutant (Δlgt) and investigated the contributions of LTA and lipoproteins to bacterial morphology and their effect on induction of proinflammatory cytokines in THP-1 and mouse bone-marrow derived macrophages (BMDMs). Deletion of ltaS and lgt was confirmed by PCR analysis of genomic DNA from each mutant. The mutants with absence of LTA or lipoproteins were examined by SDS-PAGE followed by Western blotting with anti-LTA antibodies and silver staining, respectively. Interestingly, scanning and transmission electron microscopies showed no difference in the bacterial cell morphology or size between the wild-type and the mutants even though substantial changes in the cell size and/or morphology have been reported in other Gram-positive bacteria such as Staphylococcus aureus, Listeria monocytogenes, and Bacillus subtilis. However, S. gordonii wild-type and ΔltaS potently induced the expression of proinflammatory cytokines including TNF-α, IL-8, and IL-1β at the mRNA and protein levels, while Δlgt did not have these effects. Furthermore, lipoproteins purified from S. gordonii also induced the expression of the aforementioned cytokines more potently than the purified LTA. Neither LTA nor lipoprotein induced TNF-α, KC (IL-8 counterpart in mouse), and IL-1β in TLR2-deficient BMDMs. S. gordonii Δlgt was less virulent than the wild-type or ΔltaS in a mouse intraperitoneal infection model. Collectively, these results suggest that S. gordonii lipoproteins, but not LTA, are mainly responsible for the infection and inflammatory responses.