Koichi Kuwano

Antimicrobial Activity and Stability to Proteolysis of Small Linear Cationic Peptides with D-Amino Acid Substitutions

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.

A Dipalmitoylated Lipoprotein from Mycoplasma pneumoniae Activates NF-κB through TLR1, TLR2, and TLR6

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.

Triacylated lipoproteins derived from Mycoplasma pneumoniae activate nuclear factor-κB through toll-like receptors 1 and 2

Summary Pathogenesis of Mycoplasma pneumoniae infection is considered to be in part attributed to excessive immune responses. Recently, a mycoplasma lipoprotein has been shown to induce nuclear factor κB (NF‐κB) activation through toll‐like receptor 1 (TLR1), TLR2 and TLR6. In this study, we examined the ability of lipoproteins from M. pneumoniae to activate NF‐κB through TLR1‐ and TLR2‐dependent, but TLR6‐independent, pathways, and the active components responsible for the NF‐κB activation through the TLR6‐independent pathway were identified. The active lipoproteins were found to be MPN611 and MPN162 of M. pneumoniae (designated N‐ALP1 and N‐ALP2, respectively). Purified N‐ALP1 and N‐ALP2 from M. pneumoniae and triacylated partial synthetic lipopeptides of N‐ALP1 and N‐ALP2 augmented the levels of NF‐κB induction through TLR1‐ and TLR2‐dependent pathways, whereas diacylated partial synthetic lipopeptides of N‐ALP1 and N‐ALP2 activated NF‐κB through TLR1‐, TLR2‐ and TLR6‐dependent pathways. These data suggest that N‐ALP1 and N‐ALP2 would be triacylated lipoproteins. The activity of N‐ALP1 and N‐ALP2 was decreased with a pretreatment of lipoprotein lipase, and partially decreased by protease treatment, indicating that the lipid moiety of N‐ALP1 and N‐ALP2 is critical for the NF‐κB activation. Thus, triacylated lipoproteins derived from M. pneumoniae might activate NF‐κB through TLR1 and TLR2, but not TLR6.

A novel secreted protease from Pseudomonas aeruginosa activates NF-κB through protease-activated receptors

The 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‐κB‐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.

Ureaplasma parvum lipoproteins, including MB antigen, activate NF-κB through TLR1, TLR2 and TLR6

Ureaplasma species (Ureaplasma parvum and Ureaplasma urealyticum) are commonly isolated pathogens from the female reproductive tract and are associated with perinatal diseases in humans. Inappropriate induction of inflammatory responses may be involved in the occurrence of such diseases; however, pathogenic agents that induce the inflammatory response have not been identified in ureaplasmas. In this study, we examined the involvement of Toll-like receptors (TLRs) in the activation of the immune response by U. parvum lipoproteins, as well as the U. parvum components responsible for nuclear factor kappaB (NF-kappaB) activation. The Triton X-114 (TX-114) detergent phase of U. parvum was found to induce NF-kappaB through TLR2. The active components of the TX-114 detergent phase were lipoproteins, such as multiple banded (MB) antigen, UU012 and UU016 of U. parvum. The activation of NF-kappaB by these lipoproteins was inhibited by dominant negative (DN) constructs of TLR1 and DN TLR6. Thus, the lipoproteins from U. parvum were found to activate NF-kappaB through TLR1, TLR2 and TLR6. Furthermore, these lipoproteins possessed an ability to induce tumour necrosis factor-alpha (TNF-alpha) in mouse peritoneal macrophages.

Serratia marcescens serralysin induces inflammatory responses through protease-activated receptor 2

ABSTRACT 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)-, CCAAT/enhancer-binding protein (C/EBP)-, and nuclear factor-κB (NF-κB)-driven promoters in EBC-1 cells. An electrophoretic mobility shift assay showed that serralysin activates the binding of AP-1, C/EBPβ, and NF-κB in the cells. Inactivation of serralysin resulted in the failure of transactivation of AP-1-, C/EBP-, and NF-κB-driven promoters in the cells. Furthermore, serralysin activated AP-1-, C/EBP-, and NF-κB-driven promoters via PAR-2 in HeLa cells. PAR-2 antagonist peptides decreased serralysin-induced transactivation of AP-1-, C/EBP-, and NF-κB-driven promoters in EBC-1 cells. Considered together, these results suggest that serralysin requires PAR-2 to activate the critical transcription factors AP-1, C/EBPβ, and NF-κB for host inflammatory responses.

A Triacylated Lipoprotein from Mycoplasma genitalium Activates NF-κB through Toll-Like Receptor 1 (TLR1) and TLR2

ABSTRACT Mycoplasma genitalium is a sexually transmitted bacterial pathogen that causes nongonococcal chlamydia-negative urethritis, mucopurulent cervicitis, endometritis, pelvic inflammatory disease, and tubal factor infertility in humans. However, pathogenic agents that induce inflammatory responses have not been identified in M. genitalium. In this study, we examined the involvement of Toll-like receptors (TLRs) in activation of the immune response by a lipoprotein from M. genitalium and their active component responsible for NF-κB activation. The Triton X-114 detergent phase of M. genitalium was found to induce NF-κB through TLR2. The active component of the Triton X-114 detergent phase was a lipoprotein precursor, MG149. The activation of NF-κB by MG149 was inhibited by a dominant negative (DN) construct of TLR1 but not by a DN construct of TLR6. These results indicate that the activation of NF-κB by MG149 is dependent on TLR1 and TLR2. A synthetic lipopeptide derived from MG149 containing three acyl chains also induced NF-κB through TLR1 and TLR2. Thus, the results show that MG149, a triacylated lipoprotein from M. genitalium, activates NF-κB through TLR1 and TLR2.

Mycoplasma pneumoniae-derived lipopeptides induce acute inflammatory responses in the lungs of mice.

ABSTRACT The pathogenesis of Mycoplasma pneumoniae infection is considered to be in part attributable to excessive immune responses. In this study, we investigated whether synthetic lipopeptides of subunit b of F0F1-type ATPase (F0F1-ATPase), NF-κB-activating lipoprotein 1 (N-ALP1), and N-ALP2 (named FAM20, sN-ALP1, and sN-ALP2, respectively) derived from M. pneumoniae induce cytokine and chemokine production and leukocyte infiltration in vivo. Intranasal administration of FAM20 and sN-ALP2 induced infiltration of leukocyte cells and production of chemokines and cytokines in bronchoalveolar lavage fluid, but sN-ALP1 failed to do so. The activity of FAM20 was notably higher than that of sN-ALP2. FAM20 and sN-ALP2 induced tumor necrosis factor alpha (TNF-α) through Toll-like receptor 2 in mouse peritoneal macrophages. Moreover, in the range of low concentrations of lipopeptides, FAM20 showed relatively high activity of inducing TNF-α in mouse peritoneal macrophages compared to synthetic lipopeptides such as MALP-2 and FSL-1, derived from Mycoplasma fermentans and Mycoplasma salivarium, respectively. These findings indicate that the F0F1-ATPase might be a key molecule in inducing cytokines and chemokines contributing to inflammatory responses during M. pneumoniae infection in vivo.

Lipid-associated membrane proteins of Mycoplasma fermentans and M. penetrans activate human immunodeficiency virus long-terminal repeats through Toll-like receptors

Mycoplasmas are known to enhance human immunodeficiency virus (HIV) replication, and mycoplasma‐derived lipid extracts have been reported to activate nuclear factor‐κB (NF‐κB) through Toll‐like receptors (TLRs). In this study, we examined the involvement of TLRs in the activation of HIV long‐terminal repeats (LTR) by mycoplasma and their active components responsible for the TLR activation. Lipid‐associated membrane proteins (LAMPs) from two species of mycoplasma (Mycoplasma fermentans and M. penetrans) that are associated with acquired immune‐deficiency syndrome (AIDS), were found to activate HIV LTRs in a human monocytic cell line, THP‐1. NF‐κB deletion from the LTR resulted in inhibition of the activation. The LTR activation by M. fermentans LAMPs was inhibited by a dominant negative (DN) construct of TLR1 and TLR6, whereas HIV LTR activation by M. penetrans LAMPs was inhibited by DN TLR1, but not by DN TLR6. These results indicate that the activation of HIV LTRs by M. fermentans and M. penetrans LAMPs is dependent on NF‐κB, and that the activation of HIV LTR by M. fermentans LAMPs is mediated through TLR1, TLR2 and TLR6. In contrast, the LTR activation by M. penetrans LAMPs is carried out through TLR1 and TLR2, but not TLR6. Subsequently, the active component of M. penetrans and M. fermentans LAMPs was purified by reverse‐phase high‐performance liquid chromatography (HPLC). Interestingly, the purified lipoprotein of M. penetrans LAMPs (LPMp) was able to activate NF‐κB through TLR1 and TLR2. On the other hand, the activation of NF‐κB by purified lipoprotein of M. fermentans LAMPs (LPMf) was mediated through TLR2 and TLR6, but not TLR1.

Novel Bacterial Lipoprotein Structures Conserved in Low-GC Content Gram-positive Bacteria Are Recognized by Toll-like Receptor 2

Background: The lipid-modified structures of bacterial lipoproteins in low-GC Gram-positive bacteria remains elusive. Results: Three novel structures of bacterial lipoproteins were determined and functioned as TLR2 ligands. Conclusion: Identified novel TLR2-stimulating lipoprotein structures are conserved in low-GC Gram-positive bacteria. Significance: Results open further fields of research concerning functions and biosynthesis of bacterial lipoproteins. Bacterial lipoproteins/lipopeptides inducing host innate immune responses are sensed by mammalian Toll-like receptor 2 (TLR2). These bacterial lipoproteins are structurally divided into two groups, diacylated or triacylated lipoproteins, by the absence or presence of an amide-linked fatty acid. The presence of diacylated lipoproteins has been predicted in low-GC content Gram-positive bacteria and mycoplasmas based on the absence of one modification enzyme in their genomes; however, we recently determined triacylated structures in low-GC Gram-positive Staphylococcus aureus, raising questions about the actual lipoprotein structure in other low-GC content Gram-positive bacteria. Here, through intensive MS analyses, we identified a novel and unique bacterial lipoprotein structure containing an N-acyl-S-monoacyl-glyceryl-cysteine (named the lyso structure) from low-GC Gram-positive Enterococcus faecalis, Bacillus cereus, Streptococcus sanguinis, and Lactobacillus bulgaricus. Two of the purified native lyso-form lipoproteins induced proinflammatory cytokine production from mice macrophages in a TLR2-dependent and TLR1-independent manner but with a different dependence on TLR6. Additionally, two other new lipoprotein structures were identified. One is the “N-acetyl” lipoprotein structure containing N-acetyl-S-diacyl-glyceryl-cysteine, which was found in five Gram-positive bacteria, including Bacillus subtilis. The N-acetyl lipoproteins induced the proinflammatory cytokines through the TLR2/6 heterodimer. The other was identified in a mycoplasma strain and is an unusual diacyl lipoprotein structure containing two amino acids before the lipid-modified cysteine residue. Taken together, our results suggest the existence of novel TLR2-stimulating lyso and N-acetyl forms of lipoproteins that are conserved in low-GC content Gram-positive bacteria and provide clear evidence for the presence of yet to be identified key enzymes involved in the bacterial lipoprotein biosynthesis.