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Cytadherence of Mycoplasma pneumoniae induces inflammatory responses through autophagy and toll-like receptor 4.

ABSTRACT: Mycoplasma pneumoniae causes pneumonia, tracheobronchitis, pharyngitis, and asthma in humans. The pathogenesis of M. pneumoniae infection is attributed to excessive immune responses. We previously demonstrated that M. pneumoniae lipoproteins induced inflammatory responses through Toll-like receptor 2 (TLR2). In the present study, we demonstrated that M. pneumoniae induced strong inflammatory responses in macrophages derived from TLR2 knockout (KO) mice. Cytokine production in TLR2 KO macrophages was increased compared with that in the macrophages of wild-type (WT) mice. Heat-killed, antibiotic-treated, and overgrown M. pneumoniae failed to induce inflammatory responses in TLR2 KO macrophages. 3-Methyladenine and chloroquine, inhibitors of autophagy, decreased the induction of inflammatory responses in TLR2 KO macrophages. These inflammatory responses were also inhibited in macrophages treated with the TLR4 inhibitor VIPER and those obtained from TLR2 and TLR4 (TLR2/4) double-KO mice. Two mutants that lacked the ability to induce inflammatory responses in TLR2 KO macrophages were obtained by transposon mutagenesis. The transposons were inserted in atpC encoding an ATP synthase F0F1 ? subunit and F10_orf750 encoding hypothetical protein MPN333. These mutants showed deficiencies in cytadherence. These results suggest that cytadherence of M. pneumoniae induces inflammatory responses through TLR4 and autophagy.

SUBMITTER: Shimizu T

PROVIDER: S-EPMC4097642 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Cytadherence of Mycoplasma pneumoniae induces inflammatory responses through autophagy and toll-like receptor 4.

Shimizu Takashi T Kimura Yui Y Kida Yutaka Y Kuwano Koichi K Tachibana Masato M Hashino Masanori M Watarai Masahisa M

Infection and immunity 20140505 7

Mycoplasma pneumoniae causes pneumonia, tracheobronchitis, pharyngitis, and asthma in humans. The pathogenesis of M. pneumoniae infection is attributed to excessive immune responses. We previously demonstrated that M. pneumoniae lipoproteins induced inflammatory responses through Toll-like receptor 2 (TLR2). In the present study, we demonstrated that M. pneumoniae induced strong inflammatory responses in macrophages derived from TLR2 knockout (KO) mice. Cytokine production in TLR2 KO macrophages ...[more]

PMID: 24799628

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Project description:Toll-like receptor 4 (TLR4) is a critical pattern recognition receptor that plays a critical role in the host innate immune system's recognition of Gram-negative bacteria. Since it is the lipopolysaccharide (LPS) receptor, it links the activated inflammatory response with autophagy and oxidative stress. Autophagy, or type II programmed cell death, was reported to have defensive functions in response to the production of inflammatory cytokines and oxidative stress. To explore the relationship between autophagy, inflammation, and oxidative stress, a TLR4-enriched transgenic (Tg) animal model (sheep) was generated. Autophagy activity in the Tg blood monocytes was significantly higher than in the wild-type animal under LPS stress, and it returned to normal after transfection of TLR4 siRNA. Pretreatment with 3-methyladenine (3-MA) inhibited autophagy and enhanced oxidative stress and the production of TNF-?. The LPS-induced reactive oxygen species (ROS) level was markedly increased in the Tg group at an early stage before quickly returning to normal values. In addition, suppressing ROS production by N-acetyl-L-cysteine down-regulated the number of intracellular autophagosomes and the expression of Beclin-1, ATG5, and cytokines IL-1?, IL-6, and TNF-?. Further mechanistic investigation suggested that the TLR4-associated p38 mitogen-activated protein kinase (MAPK) signaling pathway was involved in autophagy and oxidative stress. P38 MAPK promotes intracellular autophagy, ROS production, and inflammatory response. Moreover, TLR4 over-expression suppressed oxidative stress and the production of inflammatory cytokines and increased autophagy activity in vivo. Taken together, our results showed that LPS induced autophagy, which was related to TLR4-mediated ROS production through the p38 MAPK signaling pathway. In addition, our study also provided a novel transgenic animal model to analyze the effects of TLR4 on autophagy, oxidative stress, and inflammatory responses.

Project description:The Mycoplasma pneumoniae hmw3 gene was sequenced and its gene product was characterized with the goal of elucidating the functional role of HMW3 as an accessory component in cytadherence. A total of 2,016 bp of the hmw3 locus was sequenced, revealing an open reading frame large enough to encode a 672-amino-acid protein with a molecular weight of 73,725. No consensuslike ribosome binding or promoter sequences were identified. However, hmw3 was flanked by upstream and downstream open reading frames. Gene identity was confirmed by comparing the deduced amino acid sequence with the amino acid sequence obtained directly from N-terminal amino acid sequencing of HMW3. Analysis of the deduced amino acid sequence indicated an acidic pI (4.4), an unusual distribution of charged residues, a high degree of hydrophilicity, and a high proline content for HMW3. M. pneumoniae protein profiles obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) following culturing in the presence of [3H]Pro were consistent with the high Pro content predicted for HMW3 and indicated the same for cytadherence accessory protein HMW1. A discrepancy existed between the predicted size of HMW3 (Mr, 73,725) and the size of HMW3 obtained by SDS-PAGE (Mr, 140,000). Variable electrophoretic mobility in SDS-PAGE was observed at different acrylamide concentrations for HMW3, indicating that anomalous migration might account for the size discrepancy. The relative mobility of HMW3 was enhanced only slightly in the presence of magnesium acetate, suggesting that the unusual charge distribution might only be partially responsible for the anomalous migration. Secondary structure predictions were dominated by beta-sheets and nonrepetitive turns or coils, suggesting a probable extended, rigid conformation for HMW3. Finally, an unusual, highly acidic domain (residues 180 to 280) which might have particular functional significance relative to the role of HMW3 as a cytadherence accessory protein was identified.

Dataset Information

Cytadherence of Mycoplasma pneumoniae induces inflammatory responses through autophagy and toll-like receptor 4.

Publications

Cytadherence of Mycoplasma pneumoniae induces inflammatory responses through autophagy and toll-like receptor 4.

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