Abstract
Streptococcus pyogenes secretes various virulence factors for evasion from complement-mediated bacteriolysis. However, full understanding of the molecules possessed by this organism that interact with complement C1q, an initiator of the classical complement pathway, remains elusive. In this study, we identified an endopeptidase of S. pyogenes, PepO, as an interacting molecule, and investigated its effects on complement immunity and pathogenesis. Enzyme-linked immunosorbent assay and surface plasmon resonance analysis findings revealed that S. pyogenes recombinant PepO bound to human C1q in a concentration-dependent manner under physiological conditions. Sites of inflammation are known to have decreased pH levels, thus the effects of PepO on bacterial evasion from complement immunity was analyzed in a low pH condition. Notably, under low pH conditions, PepO exhibited a higher affinity for C1q as compared with IgG, and PepO inhibited the binding of IgG to C1q. In addition, pepO deletion rendered S. pyogenes more susceptible to the bacteriocidal activity of human serum. Also, observations of the morphological features of the pepO mutant strain (ΔpepO) showed damaged irregular surfaces as compared with the wild-type strain (WT). WT-infected tissues exhibited greater severity and lower complement activity as compared with those infected by ΔpepO in a mouse skin infection model. Furthermore, WT infection resulted in a larger accumulation of C1q than that with ΔpepO. Our results suggest that interaction of S. pyogenes PepO with C1q interferes with the complement pathway, which enables S. pyogenes to evade complement-mediated bacteriolysis under acidic conditions, such as seen in inflammatory sites.
Highlights
Streptococcus pyogenes secretes various virulence factors for evasion from complement-mediated bacteriolysis
The expression level of PepO was not related to disease severity, and there was no obvious correlation between PepO production and the emm type of the S. pyogenes strains (Fig. 1, A and B)
We found that streptococcal pyrogenic exotoxin B (SpeB) has an ability to interrupt the host complement system via degradation of vari
Summary
S. pyogenes Endopeptidase PepO Is Conserved and Expressed in Various Strains—To examine the subcellular localization of PepO, we prepared culture supernatant, cell wall, and cytoplasmic fractions from batch cultures of S. pyogenes clinical isolates obtained from patients with invasive and non-invasive diseases, as well as a laboratory strain (Table 1), subjected them to Western blot analysis using anti-PepO antiserum. 10 mM phosphate buffer at pH 5.0 (data not shown) These findings suggest that human serum-induced morphological changes of the bacterial surface under a low pH condition are inhibited by PepO. To verify the effect of the interaction between PepO and C1q on S. pyogenes virulence, C1q levels in the lesions were semiquantified Those levels were increased in skin lesions of mice infected with WT as compared with those infected with the ⌬pepO strain (Fig. 6D). These findings indicate that the PepO-C1q interaction participates in development of inflammatory lesions via suppression of complement activity
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