The present study is the first to report that PC expression was found in S. pyogenes, and PC expression was different even for the same emm type. There was a significant positive correlation between PC expression and the number of bacteria adhering to Detroit 562 cells. Significant negative correlations were observed between PC expression and the number of adhered and invading bacteria after pretreatment with TEPC-15 and ABT-491.
The presence of PC was first discovered in Spn by Tomasz in 1967 . The molecule PC is a small haptenic moiety that is bound to teichoic acid in the cell wall of several Gram-positive bacteria, including S. pneumoniae . PC is a structural component of a wide variety of pathogens and possesses immunomodulatory properties, and the amount of PC on the bacterial surface is modulated by phase variation . The present study demonstrates that the level of PC expressed on the cell surface of S. pyogenes differs in each strain, as assessed by fluorescence intensity in flow cytometry. Furthermore, PC expression was different, even for the same emm type.
In contrary, typing based on the M protein, a cell surface protein that is the major virulence and immunological determinant of S. pyogenes, is the most widely used method . The M protein, which is encoded by the emm gene, possesses a hypervariable region of the amino-terminal with 40–50 amino acid residues . Kuhn et al. have reported that the distribution of emm genotypes among patients with recurrent pharyngitis was in the order of emm12 (24.2%), emm3 (18.2%), emm1 (15.2%), and emm4 (12.1%), and emm12 was the most frequently detected genotype in recurrent streptococcal pharyngitis cases . In this study, emm75 strains were 50% (4/8), emm89, emm28, emm12, and emm11 strains were 12.5% (1/8). Moreover, emm4, emm6, and emm75 strains showed significantly higher levels of invasion capacity into Detroit 562 cells than strains with other genotypes . Since the bacteria used in this study were collected from patients with recurrent tonsillitis, it is possible that many emm75 strains that invaded the cells were detected.
High PC expression is considered to increase S. pneumoniae and H. influenzae virulence . For instance, it has been reported that S. pneumoniae with high PC expression causes more invasive infection than that with low PC expression . Andersson et al. have found that the adhesive capacity of S. pneumoniae was highest among the strains isolated from patients with acute otitis media (AOM) and suggested that the capacity to attach to the pharyngeal mucosa is a virulence factor in S. pneumoniae causing AOM . Furthermore, it has also been reported that isolates recovered from the blood of patients with severe S. pyogenes infections adhere more readily to human epithelial cells than isolates from patients with uncomplicated infections . In this study, there was a significant positive correlation between PC expression and the number of bacteria adhering to Detroit 562 cells. Additionally, the adhesiveness of the emm75 strain was high. These findings suggest that PC expression in S. pyogenes is associated with their adhesive activity to mucosal surfaces, which might be one of the reasons that PC-expressing strains exhibit increased virulence.
This study showed that there was a significant negative correlation between TEPC-15 inhibitory effects and PC expression in S. pyogenes. The antibody response to PC has been thoroughly described in mice , and the cells involved in regulating anti-PC antibody production have been well characterized . Kurono et al. have reported that the adherence of Spn and NTHi was remarkably reduced by treating bacteria with nasopharyngeal secretions with secretory IgA antibody activity against these bacteria . Furthermore, intranasal immunization of mice with PC induced PC-specific IgA in mucosal secretions such as saliva, which reacted with most Spn and NTHi strains and enhanced the clearance of these bacteria from the nasal cavity . These findings indicate the inhibitory effect of PC-specific IgA on the adherence of these bacteria.
Furthermore, intracellular invasion assay experiments showed that there was a significant negative correlation between TEPC-15 inhibitory effects and PC expression. In this study, there was a significant positive correlation between PC expression and the number of bacteria invading Detroit 562 cells. A number of bacterial species are able to enter host cells by internalization via phagosomes or endosomes , including S. pyogenes . In general, immediate bacterial degradation with lysosomes follows phagocytosis or endocytosis. However, some bacteria have evolved strategies to avoid the host defense system by escaping from the endocytic compartment into the cytoplasm or modifying the lysosomal acidic environment . Streptococcus pyogenes enters human epithelial cells via engulfment by early endosomes, after which endosomes containing bacteria disappear within a few hours, indicating escape into the cytoplasm . Considering these facts, it has been considered that the vaccine targeting PC is also effective against bacteria that invade cells. Additionally, more than half of the emm75 strains temporarily escaped killing by penicillin alone by internalization into epithelial cells, even when the antibiotic concentration used was greater than the 10-fold minimum inhibitory concentration for planktonic S. pyogenes . In this study, emm75 strains had high PC expression and also had a large number of intracellular invasions. Vaccines targeting PC may also be effective against emm75 strains, which avoid the effects of antibacterial agents.
To confirm the previously reported association between PC and PAF-R in bacterial adherence, the presence of PAF-R on Detroit 562 cell surfaces and the effects of blocking PAF-R by pretreatment with ABT-491 on the adherence of S. pyogenes were examined. PAF-R expression in Detroit 562 cells was confirmed by FACS, as previously reported . Cundell et al. have investigated the attachment of bacterial PC to PAF-R and found that binding between PC and PAF-R enhanced the adherence of Spn and that only virulent Spn engaged with PAF-R . Moreover, the enhanced expression of PAF-R induced by viral antigens is associated with susceptibility of host epithelial cells to Spn infection and contributes to the incidence of recurrent and persistent bacterial upper airway infections . In this study, significant negative correlations were observed between PC expression and the number of adhered and invaded bacteria after pretreatment with ABT-491. These results indicate that PAF-R and its binding to PC play an important role in the adherence of virulent strains of PC-high bacteria.
Our study has some limitations. First, the number of bacterial strains used was small. However, we believe that the reliability was also obtained by examining the emm type. Second, in this study, epithelial cells were used. Although the use of normal human epithelial cells may be more clinically relevant, we used a pharyngeal cancer-derived cell line. Because these cells were of human origin, we consider that the results of this study were similar to those that would have been obtained with the use of normal cells.