The pathogenesis of Mycoplasma pneumoniae infection is considered to be in part attributed to excessive immune responses. Recently, lipoproteins from mycoplasmas have been reported to induce NF-κB activation. In this study, we examined the ability of lipoproteins from M. pneumoniae to activate NF-κB, and the active component responsible for the NF-κB activation was identified. Lipid-associated membrane proteins from M. pneumoniae were found to induce NF-κB through TLR 2 in a human monocytic cell line, THP-1. The active component of the Lipid-associated membrane proteins was a subunit b of F0F1-type ATPase (F0F1-ATPase). The F0F1-ATPase is assumed to contain two palmitic acids. The activation of NF-κB by the F0F1-ATPase was inhibited by a dominant negative construct of TLR1 and TLR6. These results indicate that the activation of NF-κB by F0F1-ATPase is dependent on TLR1, TLR2, and TLR6. The activity of the F0F1-ATPase was decreased with pretreatment of lipoprotein lipase but not protease, indicating that the lipid moiety of the F0F1-ATPase was important for the NF-κB activation. Thus, a dipalmitoylated lipoprotein from M. pneumoniae was found to activate NF-κB through TLR1, TLR2, and TLR6.

Antimicrobial peptides contribute to innate host defense against a number of bacteria and fungal pathogens. Some of antimicrobial synthetic peptides were systemically administered in vivo; however, effective protection has so far not been obtained because the effective dose of peptides in vivo seems to be very high, often close to the toxic level against the host. Alternatively, peptides administered in vivo may be degraded by certain proteases present in serum. In this study, D‐amino acids were substituted for the L‐amino acids of antimicrobial peptides to circumvent these problems. Initially a peptide (L‐peptide) rich in five arginine residues and consisting of an 11‐amino acid peptide (residues 32–42) of human granulysin was synthesized. Subsequently, the L‐amino acids of the 11‐amino acid peptide were replaced partially (D‐peptide) or wholly (AD‐peptide) with D‐amino acids. Activity and stability to proteolysis, in particular, in the serum of antimicrobial peptides with D‐amino acid substitutions were examined. Peptides with D‐amino acid substitutions were found to lyse bacteria as efficiently as their all‐L‐amino acid parent, L‐peptide. In addition, the peptide composed of L‐amino acids was susceptible to trypsin, whereas peptides containing D‐amino acid substitutions were highly stable to trypsin treatment. Similarly, the peptide consisting of L‐amino acids alone was also susceptible to fetal calf serum (FCS), however, protease inhibitors restored the lowered antimicrobial activity of the FCS‐incubated peptide. Thus, D‐amino acid substitutions can make antimicrobial peptides resistant to proteolysis, suggesting that the antimicrobial peptides consisting of D‐amino acids are potential candidates for clinical therapeutic use.

SummaryThe Pseudomonas aeruginosa-derived alkaline protease (AprA), elastase A (LasA), elastase B (LasB) and protease IV are considered to play an important role in pathogenesis of this organism. Although the sequence analysis of P. aeruginosa genome predicts the presence of several genes encoding other potential proteases in the genome, little has been known about the proteases involving in pathogenesis. Recently, Porphyromonas gingivalis gingipains and Serratia marcescens serralysin have been shown to activate protease-activated receptor 2 (PAR-2), thereby modulating host inflammatory and immune responses. Accordingly, we hypothesized that unknown protease(s) from P. aeruginosa would also modulate such responses through PARs. In this study, we found that P. aeruginosa produces a novel large exoprotease (LepA) distinct from known proteases such as AprA, LasA, LasB and protease IV. Sequence analysis of LepA showed a molecular feature of the proteins transported by the two-partner secretion pathway. Our results indicated that LepA activates NF-kB-driven promoter through human PAR-1, -2 or -4 and cleaves the peptides corresponding to the tethered ligand region of human PAR-1, -2 and -4 at a specific site with exposure of their tethered ligands. Considered together, these results suggest that LepA would require PARs to modulate various host responses against bacterial infection.

Triacylated lipoproteins derived from Mycoplasma pneumoniae activate nuclear factor-jB through toll-like receptors 1 and 2 IntroductionMycoplasmas are wall-less parasitic bacteria, and the smallest organisms capable of self-replication.1 Mycoplasma pneumoniae causes primary atypical pneumonia, tracheobronchitis and pharyngitis in humans.2 However, virulence factors such as endotoxins and exotoxins, which cause such diseases, have not been identified in M. pneumoniae. Adherence of invading mycoplasma to the respiratory epithelium, localized host cell injury and overaggressive inappropriate immune responses seem to contribute to the pathogenesis of M. pneumoniae infection. 3 Recently, it has been reported that toll-like receptors (TLRs), which function as pattern-recognition receptors, play critical roles in early innate recognition and in the inflammatory responses of the host against invading microbes.4,5 Among the 10 TLR family members reported, TLR2, TLR4, TLR5 and TLR9 have been implicated in the recognition of different bacterial components. Peptidoglycan, lipoarabinomannan, zymosan and lipoproteins from various micro-organisms are recognized by TLR2, [6][7][8][9][10][11][12] while lipopolysaccharide, bacterial flagellin and bacterial DNA are recognized by TLR4, TLR5 and TLR9, respectively.13-16 These TLR family members are known to activate nuclear factor jB (NF-jB) via interleukin-1 receptor (IL-1R)-associated signal molecules, including myeloid differentiation protein (MyD88), IL-1R-activated kinase (IRAK), tumour necrosis factor receptor-associated factor 6 (TRAF6) and NF-jB-inducing kinase (NIK). 17 We previously reported that lipid-associated membrane proteins (LAMPs) from M. pneumoniae can induce

The Serratia marcescens-derived protease serralysin is considered to play an important role in the pathogenesis of infection. Protease-activated receptor 2 (PAR-2) is activated by trypsin and also several other trypsin-like serine proteases, leading to the modulation of inflammatory and immune responses. However, little is known about the activation of PAR-2 by bacterial proteases and its roles in bacterial infection. In this study, we investigated whether S. marcescens serralysin activates host inflammatory responses through PAR-2. Our results demonstrated that serralysin induces interleukin-6 (IL-6) and IL-8 mRNA expression in a human lung squamous cell carcinoma, EBC-l cells. In addition, serralysin activated activator protein 1 (AP-1)