Surface lipoprotein PpiA of Streptococcus mutans suppresses scavenger receptor MARCO-dependent phagocytosis by macrophages

Abstract

Streptococcus mutans is associated with the initiation and progression of human dental caries and is occasionally isolated from the blood of patients with bacteremia and infective endocarditis. For the pathogen to survive in the infected host, surface lipoproteins of S. mutans are likely to play important roles in interactions with the innate immune system. To clarify the role that a putative lipoprotein, peptidyl-prolyl cis/trans-isomerase (PpiA), of S. mutans plays in the macrophage response, we investigated the response of THP-1-derived macrophages to S. mutans challenge. The deletion of the gene encoding Lgt eliminated PpiA on the cell surface of S. mutans, which implies that PpiA is a lipoprotein that is lipid anchored in the cell membrane by Lgt. Human and murine peritoneal macrophages both showed higher phagocytic activities for the ppiA and lgt mutants than the wild type, which indicates that the presence of PpiA reduces S. mutans phagocytosis. In addition, infection with S. mutans markedly induced mRNAs of macrophage receptor with collagenous structure (MARCO) and scavenger receptor A (SR-A) in human macrophages. In particular, transcriptional and translational levels of MARCO in human macrophages infected with the ppiA mutant were higher than those in macrophages infected with the wild type. Phagocytosis of S. mutans by human macrophages markedly decreased after treatment with anti-MARCO IgG. These results demonstrate that the S. mutans lipoprotein PpiA contributes to suppression of MARCO-mediated phagocytosis of this bacterium by macrophages.

Fig. 1.

Scheme of construction of the S. mutans ppiA mutant. The ppiA mutant was prepared by the vectorless PCR strategy.

Fig. 2.

Detection of PpiA in S. mutans. Cell extracts prepared from S. mutans were separated by SDS-PAGE, and PpiA (27 kDa) was detected by Western blotting with anti-PpiA serum. Equal amounts of proteins were added to each lane. Lane M, size marker; lane 1, wild-type 109c strain; lane 2, lgt mutant; lane 3, ppiA mutant; lane 4, recombinant His-tagged PpiA purified from recombinant E. coli (positive control).

Fig. 3.

Phagocytosis of S. mutans by macrophages. (A) Phagocytosis of the wild-type (WT) 109c strain, ppiA mutant, lgt mutant, msmE mutant, and ppiA mutant complemented with ppiA (ΔppiA-revertant) by THP-1 macrophages (human macrophages). (B) Phagocytosis of the wild-type 109c strain, ppiA mutant, and msmE mutant by murine peritoneal macrophages. Data shown represent means ± standard deviations of results from three independent experiments. Phagocytosis rate is expressed as the ratio of the number of viable S. mutans cells recovered to the total number of applied S. mutans cells. Statistical significance (P < 0.05; Dunnett's test) is depicted by an asterisk.

Fig. 4.

Expression of SRs (MARCO and/or SR-A) in human macrophages. (A) Transcriptional levels of SR-A and MARCO genes in human macrophages stimulated with the wild-type strain and ppiA mutant. The relative expression levels (to unstimulated human macrophages) for SR-A and MARCO, corrected for 18S rRNA, are shown. Data shown represent means ± standard deviations of results from three independent experiments. Statistical significance (P < 0.05; Dunnett's test) is depicted by an asterisk. (B) Analysis of MARCO expression on human macrophages stimulated by the medium (negative control) (lane 1), wild-type 109c strain (lane 2), and ppiA mutant (lane 3). The other lanes contain size markers (lane M), cell extract of S. mutans ppiA mutant (lane 4), and His-tagged MARCO protein purified from recombinant E. coli (lane 5). Equal amounts of proteins were added to lanes 1 to 3. MARCO protein was detected by Western blotting with anti-MARCO antibody.

Fig. 5.

Analysis of MARCO expression and MARCO-mediated phagocytosis by human macrophages. (A) Expression analysis of MARCO protein on human macrophages stimulated by medium (negative control) (lane 1), heat-killed wild-type 109c strain (lane 2), and heat-killed ppiA mutant (lane 3). The other lanes contain size markers (lane M) and His-tagged MARCO protein purified from recombinant E. coli (lane 4). Equal amounts of proteins were added to lanes 1 to 3. MARCO protein was detected by Western blotting with anti-MARCO antibody. (B and C) Phagocytosis of the ppiA mutant (B) and wild-type strain (C) of S. mutans by human macrophages treated with anti-MARCO antibody. Human macrophages stimulated by heat-killed S. mutans ppiA mutant were treated with medium (lane 1), preimmune IgG (lane 2), and anti-MARCO antibody (lane 3). These treated human macrophages were restimulated by the ppiA mutant (B) and wild-type strain (C) of S. mutans, and the number of S. mutans strains phagocytosed was measured. Data shown represent means ± standard deviations of results from three independent experiments. The phagocytosis rate is expressed as the ratio of the number of viable S. mutans cells recovered to the total number of S. mutans cells applied. Statistical significance (P < 0.05; Dunnett's test) is depicted by an asterisk.