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1.
Microbiol Immunol ; 64(4): 280-295, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31907968

RESUMO

Mucin is a glycoprotein that is the primary component of the mucus overlaying the epithelial tissues. Because mucin functions as a first line of the innate immune system, Pseudomonas aeruginosa appears to require interaction with mucin to establish infection in the host. However, the interactions between P. aeruginosa and mucin have been poorly understood. In this study, using in vivo expression technology (IVET), we attempted to identify mucin-inducible promoters that are likely to be involved in the establishment of P. aeruginosa infection. The IVET analysis revealed that the genes encoding glycosidases, sulfatases, and peptidases that are thought to be required for the utilization of mucin as a nutrient are present in 13 genes downstream of the identified promoters. Our results indicated that, among them, sdsA1 encoding a secreted sulfatase plays a central role in the degradation of mucin. It was then demonstrated that disruption of sdsA1 leads to a decreased release of sulfate from mucin and sulfated sugars. Furthermore, the sdsA1 mutant showed a reduction in the ability of mucin gel penetration and an attenuation of virulence in leukopenic mice compared with the wild-type strain. Collectively, these results suggest that SdsA1 plays an important role as a virulence factor of P. aeruginosa.


Assuntos
Proteínas de Bactérias/imunologia , Mucina-3/metabolismo , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/enzimologia , Sulfatases/imunologia , Fatores de Virulência/imunologia , Animais , Masculino , Camundongos , Camundongos Endogâmicos ICR , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/patogenicidade , Suínos , Virulência
2.
Cell Microbiol ; 21(6): e13015, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30702185

RESUMO

Epithelial cell shedding is a defence mechanism against infectious microbes that use these cells as an infection foothold and that eliminate microbes from infection foci by removing infected cells. Mycoplasma pneumoniae, a causative agent of respiratory infections, is known to adhere to and colonise the surface of ciliated airway epithelial cells; it produces a large amount of hydrogen peroxide, indicating its capability of regulating hydrogen peroxide-induced infected cell detachment. In this study, we found that M. pneumoniae reduces exogenous hydrogen peroxide-induced detachment of the infected cells from culture plates. This cell detachment occurred dependently of DNA damage-initiated, poly (ADP-ribose) polymerase 1 (PARP1)-mediated cell death, or parthanatos. In cells infected with M. pneumoniae, exogenous hydrogen peroxide failed to induce DNA damage-initiated poly (ADP-ribose) (PAR) synthesis and concomitant increased cytoplasmic membrane rupture, both of which are biochemical hallmarks of parthanatos. The impairment of PAR synthesis was attributed to a reduction in the amount of cytosolic nicotinamide adenine dinucleotide (NAD), a substrate of PARP1, caused by M. pneumoniae. On the other hand, nonadherent mutant strains of M. pneumoniae showed a lower ability to reduce cell detachment than wild-type strains, but the extent to which NAD was decreased in infected cells was comparable to that seen in the wild-type strain. We found that NAD depletion could induce PARP1-independent cell detachment pathways following stimulation with hydrogen peroxide and that M. pneumoniae could also regulate PARP1-independent cell detachment in a cytoadhesion-dependent manner. These results suggest that M. pneumoniae might regulate infected cell detachment induced by hydrogen peroxide that it produces itself, and such a mechanism may contribute to sustaining the bacterial infection.


Assuntos
Células Epiteliais/microbiologia , Peróxido de Hidrogênio/toxicidade , Mycoplasma pneumoniae/metabolismo , Parthanatos/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Dano ao DNA/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/patogenicidade , NAD/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Espécies Reativas de Oxigênio/metabolismo
3.
J Vet Med Sci ; 79(9): 1472-1479, 2017 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-28757508

RESUMO

First-generation cephalosporins such as cefazolin (CEZ) have been widely used for mastitis treatment in dairy cattle. However, the use of antibiotics results in the presence of antibiotic residues in milk, which is used for human consumption. Nisin A, a bacteriocin produced by Lactococcus lactis, has been used as a broad-spectrum food preservative for over 50 years. Therefore, a combination of CEZ and nisin A might provide an extended activity spectrum against mastitis pathogens and reduce the antibiotic dose for mastitis treatment. This study aimed to evaluate the combined effect of CEZ and nisin A against mastitis pathogens using the checkerboard and time-kill assays. In the checkerboard assay, the CEZ-nisin A combination exhibited a synergistic effect against Staphylococcus aureus (n=20/20) and Enterococcus faecalis (n=13/18), and meanwhile exhibited a mostly additive effect against Staphylococcus intermedius (n=12/20), Streptococcus agalactiae (n=10/10), Streptococcus dysgalactiae (n=18/18), and Escherichia coli (n=14/18). There were no indifferent or antagonistic effects between CEZ and nisin A. In the time-kill assay, the CEZ-nisin A combination at 0.5 × or 1 × minimum inhibitory concentration exhibited synergistic reduction of bacterial growth by over 3 log10 colony forming units per ml relative to that observed with either antimicrobial substance alone. These results suggest that the CEZ-nisin A combination can be used for developing an intramammary infusion for mastitis treatment, with lower antibiotic concentrations than normal.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Cefazolina/farmacologia , Mastite Bovina/microbiologia , Nisina/farmacologia , Animais , Bovinos , Sinergismo Farmacológico , Enterococcus/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Mastite Bovina/tratamento farmacológico , Testes de Sensibilidade Microbiana , Staphylococcus/efeitos dos fármacos , Streptococcus/efeitos dos fármacos , Fatores de Tempo
4.
PLoS One ; 12(1): e0169367, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28046014

RESUMO

To specify critical factors responsible for Pseudomonas aeruginosa penetration through the Caco-2 cell epithelial barrier, we analyzed transposon insertion mutants that demonstrated a dramatic reduction in penetration activity relative to P. aeruginosa PAO1 strain. From these strains, mutations could be grouped into five classes, specifically flagellin-associated genes, pili-associated genes, heat-shock protein genes, genes related to the glycolytic pathway, and biosynthesis-related genes. Of these mutants, we here focused on the serA mutant, as the association between this gene and penetration activity is yet unknown. Inactivation of the serA gene caused significant repression of bacterial penetration through Caco-2 cell monolayers with decreased swimming and swarming motilities, bacterial adherence, and fly mortality rate, as well as repression of ExoS secretion; however, twitching motility was not affected. Furthermore, L-serine, which is known to inhibit the D-3-phosphoglycerate dehydrogenase activity of the SerA protein, caused significant reductions in penetration through Caco-2 cell monolayers, swarming and swimming motilities, bacterial adherence to Caco-2 cells, and virulence in flies in the wild-type P. aeruginosa PAO1 strain. Together, these results suggest that serA is associated with bacterial motility and adherence, which are mediated by flagella that play a key role in the penetration of P. aeruginosa through Caco-2 cell monolayers. Oral administration of L-serine to compromised hosts might have the potential to interfere with bacterial translocation and prevent septicemia caused by P. aeruginosa through inhibition of serA function.


Assuntos
Translocação Bacteriana/genética , Genes Bacterianos , Pseudomonas aeruginosa/genética , Sistemas de Secreção Bacterianos/genética , Células CACO-2 , Flagelos/efeitos dos fármacos , Flagelos/metabolismo , Humanos , Movimento , Mutação/genética , Fosfoglicerato Desidrogenase/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/patogenicidade , Serina/farmacologia , Virulência
5.
Cell Microbiol ; 19(3)2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27603754

RESUMO

Neutrophils play an important role in antimicrobial defense as the first line of innate immune system. Recently, the release of neutrophil extracellular traps (NETs) has been identified as a killing mechanism of neutrophils against invading microbes. Mycoplasma pneumoniae, a causative agent of respiratory infection, has been shown to be resistant to in vitro killing by neutrophils, suggesting that the bacterium might circumvent bactericidal activity of NETs. In this study, we investigated whether M. pneumoniae possesses resistance mechanisms against the NETs-mediated killing of neutrophils and found that the bacterium degrades the NETs induced upon M. pneumoniae infection. The NETs-degrading ability of M. pneumoniae required the production of a secreted nuclease, Mpn491, capable of using Mg2+ as a cofactor for its hydrolytic activity. Moreover, the inactivation of the nuclease resulted in increased susceptibility of M. pneumoniae to the NETs-mediated killing of neutrophils. The results suggest that M. pneumoniae employs Mpn491 as a means for evading the killing mechanism of neutrophils.


Assuntos
Desoxirribonucleases/metabolismo , Armadilhas Extracelulares/metabolismo , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Mycoplasma pneumoniae/enzimologia , Mycoplasma pneumoniae/patogenicidade , Neutrófilos/imunologia , Hidrólise , Magnésio/metabolismo , Mycoplasma pneumoniae/imunologia
6.
Infect Immun ; 82(7): 3076-86, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24799628

RESUMO

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 ε 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.


Assuntos
Autofagia/fisiologia , Aderência Bacteriana/fisiologia , Infecções por Mycoplasma/imunologia , Mycoplasma pneumoniae/fisiologia , Receptor 4 Toll-Like/metabolismo , Animais , Regulação da Expressão Gênica/imunologia , Inflamação/metabolismo , Macrófagos , Camundongos , Camundongos Knockout , Mutação , Infecções por Mycoplasma/microbiologia , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/genética
7.
J Reprod Immunol ; 100(2): 118-27, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24238827

RESUMO

Ureaplasma spp. are members of the family Mycoplasmataceae and have been considered to be associated with chorioamnionitis and preterm delivery. However, it is unclear whether Ureaplasma spp. have virulence factors related to these manifestations. The purpose of the present study was to determine whether the immunogenic protein multiple-banded antigen (MBA) from Ureaplasma parvum is a virulence factor for preterm delivery. We partially purified MBA from a type strain and clinical isolates of U. parvum, and also synthesized a diacylated lipopeptide derived from U. parvum, UPM-1. Using luciferase assays, both MBA-rich fraction MRF and UPM-1 activated the NF-κB pathway via TLR2. UPM-1 upregulated IL-1ß, IL-6, IL-12p35, TNF-α, MIP2, LIX, and iNOS in mouse peritoneal macrophage. MRF or UPM-1 was injected into uteri on day 15 of gestation on pregnant C3H/HeN mice. The intrauterine MRF injection group had a significantly higher incidence of intrauterine fetal death (IUFD; 38.5%) than the control group (14.0%). Interestingly, intrauterine injection of UPM-1 caused preterm deliveries at high concentration (80.0%). In contrast, a low concentration of UPM-1 induced a significantly higher rate of fetal deaths (55.2%) than the control group (14.0%). The placentas of the UPM-1 injection group showed neutrophil infiltration and increased iNOS protein expression. Our data indicate that MBA from the clinical isolate of U. parvum is a potential virulence factor for IUFD and preterm delivery in mice and that the N-terminal diacylated lipopeptide is essential for the initiation of inflammation.


Assuntos
Proteínas de Bactérias/administração & dosagem , Corioamnionite/imunologia , Morte Fetal/imunologia , Macrófagos Peritoneais/imunologia , Fragmentos de Peptídeos/administração & dosagem , Nascimento Prematuro/imunologia , Infecções por Ureaplasma/imunologia , Ureaplasma/imunologia , Animais , Proteínas de Bactérias/síntese química , Proteínas de Bactérias/isolamento & purificação , Linhagem Celular Tumoral , Feminino , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Neutrófilos/patologia , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/isolamento & purificação , Placenta/metabolismo , Placenta/patologia , Gravidez , Transdução de Sinais , Receptor 2 Toll-Like/metabolismo , Ureaplasma/patogenicidade , Fatores de Virulência
8.
J Biochem ; 154(3): 299-308, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23782844

RESUMO

High mobility group box 1 (HMGB1), a non-histone chromosomal protein, is a proinflammatory cytokine. There are two known pathways for the release of HMGB1 into the extracellular milieu-passive and active. The passive pathway is attributable to cell death from damage or necrosis, and the active pathway is secretion from immunocompetent cells activated by proinflammatory stimuli. Recent studies have shown that post-translational modifications of HMGB1, including phosphorylation, are involved in the relocation of HMGB1 to the cytoplasm and subsequent secretion. With regard to the HMGB1 phosphorylation, Youn and Shin [Nucleocytoplasmic shuttling of HMGB1 is regulated by phosphorylation that redirects it toward secretion. J Immunol 2006;177:7889-97] reported that treatment of the murine macrophage RAW264.7 with okadaic acid resulted in nucleocytoplasmic translocation and secretion of HMGB1. Herein, we demonstrate the physical interaction between HMGB1 and protein phosphatase 2A (PP2A) in the RAW264.7. The results of in vitro phosphatase assay further indicate that PP2A dephosphorylates specific phosphoserine residues within one of the two nuclear localization signals (NLSs) of HMGB1. The cytoplasmic relocation of HMGB1 through PP2A inhibition was markedly suppressed by replacement of the Ser residues within the NLS with Ala. These consequences imply that PP2A correlates in the nucleocytoplasmic shuttling of HMGB1.


Assuntos
Transporte Ativo do Núcleo Celular/genética , Regulação da Expressão Gênica , Proteína HMGB1/genética , Macrófagos/metabolismo , Fosfosserina/metabolismo , Proteína Fosfatase 2/genética , Animais , Células COS , Linhagem Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citoplasma/metabolismo , Proteína HMGB1/metabolismo , Hidrólise , Macrófagos/citologia , Camundongos , Dados de Sequência Molecular , Proteína Fosfatase 2/metabolismo , Sinais Direcionadores de Proteínas , Transdução de Sinais
9.
Cell Microbiol ; 15(7): 1168-81, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23311922

RESUMO

PA3535 (EprS), an autotransporter (AT) protein of Pseudomonas aeruginosa, is predicted to contain a serine protease motif. The eprS encodes a 104.5 kDa protein with a 30-amino-acid-long signal peptide, a 51.2 kDa amino-terminal secreted passenger domain and a 50.1 kDa carboxyl-terminal outer membrane channel formed translocator. Although the majority of AT proteins have been reported to be virulence factors, little is known about the functions of EprS in the pathogenicity of P. aeruginosa. In this study, we performed functional analyses of recombinant EprS secreted by Escherichia coli. The proteolytic activity of EprS was markedly decreased by changing Ser to Ala at position 308 or by serine protease inhibitors. EprS preferred to cleave substrates that terminated with arginine or lysine residues. Thus, these results indicate that EprS, a serine protease, displays the substrate specificity, cleaving after basic residues. We demonstrated that EprS activates NF-κB-driven promoters through protease-activated receptor (PAR)-1, -2 or -4 and induces IL-8 production through PAR-2 in a human bronchiole epithelial cell line. Moreover, EprS cleaved the peptides corresponding to the tethered ligand region of PAR-1, -2 and -4 at a specific site with exposure oftheir tethered ligands. Collectively, these results suggest that EprS activates host inflammatory responses through PARs.


Assuntos
Inflamação/patologia , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/patogenicidade , Receptores Ativados por Proteinase/metabolismo , Serina Proteases/metabolismo , Fatores de Virulência/metabolismo , Linhagem Celular , Células Epiteliais/microbiologia , Escherichia coli/genética , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
10.
J Infect Chemother ; 19(2): 261-71, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23229613

RESUMO

Streptococcus pneumoniae has been reported to form biofilms. Many different surface molecules, including capsular polysaccharide (CPS), may play a fundamental role in pneumococcal biofilm development. We designed a CPS mutant, TIGR4cps4D(-), from the TIGR4 strain and detected enhanced biofilm formation. The pathogenic diversities of the mutant were also investigated with the in vitro expression levels of pavA, lytA, IgA1, piaA, psaA, ply, and spxB. The mean OD595 of TIGR4cps4D(-) biofilm was 1.77 and 1.74, whereas that of TIGR4 was 0.76 and 0.33 on day 1 and day 2, respectively. Scanning electron microscopy and confocal laser scanning microscopy showed TIGR4cps4D(-) formed a biofilm that was significantly thicker than that formed by TIGR4 (~12.22 vs. ~6.29 µm). Compared to TIGR4, the gene expression of lytA, IgA1, and, psaA in TIGR4cps4D(-) was 1.9 × 10(-5)-, 2.4 × 10(-5)-, and 3.2 × 10(-3) fold lower under the planktonic condition, and 1.9 × 10(-5)- and 9.7 × 10(-5) fold lower in biofilms, respectively. Furthermore, TIGR4cps4D(-) seemed to induce less cell death, compared to the results of TIGR4 (21.38 vs. 33.47 %, after a 5-h exposure; P < 0.05). Our data indicate that impaired pneumococcal CPS may increase biofilm formation and be involved in inhibition of virulence, possibly by influencing the gene expression.


Assuntos
Biofilmes , Polissacarídeos Bacterianos/fisiologia , Streptococcus pneumoniae/fisiologia , Virulência/genética , Linhagem Celular , Engenharia Genética , Interações Hospedeiro-Patógeno , Humanos , Microscopia Confocal , Mutação , Infecções Pneumocócicas/microbiologia , Polissacarídeos Bacterianos/genética , Polissacarídeos Bacterianos/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/patogenicidade
11.
J Biol Chem ; 287(16): 13170-81, 2012 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-22303020

RESUMO

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.


Assuntos
Bactérias Gram-Positivas/imunologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/microbiologia , Receptor 2 Toll-Like/imunologia , Receptor 2 Toll-Like/metabolismo , Animais , Bacillus cereus/imunologia , Bacillus cereus/metabolismo , Bacillus subtilis/imunologia , Bacillus subtilis/metabolismo , Enterococcus faecalis/imunologia , Enterococcus faecalis/metabolismo , Geobacillus/imunologia , Geobacillus/metabolismo , Bactérias Gram-Positivas/metabolismo , Lactobacillus/imunologia , Lactobacillus/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia por Mycoplasma/imunologia , Pneumonia por Mycoplasma/metabolismo , Streptococcus sanguis/imunologia , Streptococcus sanguis/metabolismo , Receptor 1 Toll-Like/genética , Receptor 1 Toll-Like/imunologia , Receptor 1 Toll-Like/metabolismo , Receptor 2 Toll-Like/genética , Receptor 6 Toll-Like/genética , Receptor 6 Toll-Like/imunologia , Receptor 6 Toll-Like/metabolismo
12.
Inflamm Res ; 61(5): 493-501, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22270622

RESUMO

OBJECTIVE: To investigate the involvement of reactive oxygen species (ROS) in the activation of Janus kinase2 (Jak2)/signal transducers and activators of transcription3 (Stat3), and IL-8 expression in pulmonary epithelial cells stimulated with lipid-associated membrane proteins (LAMP) from Mycoplasma pneumoniae using a known antioxidant, N-acetylcysteine (NAC). METHODS: Pulmonary epithelial A549 cells were treated with or without NAC in the presence or absence of LAMP. Intracellular ROS levels were detected by fluorescent analysis for fluorescent dichlorofluorescein. mRNA expression of IL-8 was analyzed by reverse transcription-polymerase chain reaction. IL-8 protein in the medium was determined by enzyme-linked immunosorbent assay. Activation of Jak2/Stat3 was determined by the increases in phospho-specific forms of Jak2/Stat3 compared to total forms of Jak2/Stat3 by western blotting. Stat3-DNA binding activity was assessed by electrophoretic mobility shift assay. RESULTS: LAMP increased the level of ROS, phosphorylation of Jak2/Stat3, Stat3-DNA binding activity, and IL-8 expression in A549 cells, which were inhibited by NAC dose-dependently. CONCLUSION: LAMP of M. pneumoniae induces the production of ROS, Jak2/Stat3 activation, and IL-8 induction in A549 cells. Antioxidants such as NAC may be beneficial for preventing pulmonary inflammation caused by M. pneumoniae.


Assuntos
Proteínas de Bactérias/farmacologia , Interleucina-8/genética , Janus Quinase 2/fisiologia , Pulmão/metabolismo , Proteínas de Membrana/farmacologia , Mycoplasma pneumoniae/patogenicidade , Espécies Reativas de Oxigênio/metabolismo , Fator de Transcrição STAT3/fisiologia , Acetilcisteína/farmacologia , Linhagem Celular , DNA/metabolismo , Ativação Enzimática , Células Epiteliais/metabolismo , Humanos , NF-kappa B/fisiologia , Receptores Toll-Like/fisiologia
13.
Microbiol Immunol ; 55(8): 582-8, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21605160

RESUMO

In innate immunity, cationic antimicrobial peptides including cathelin-related antimicrobial peptide (CRAMP) are known to play critical roles in protecting the host from infection by invasive microbes, including Gram-positive and -negative bacteria. However, little is known about the interactions between CRAMP and mycoplasmas. In the present study, the antimicrobial activity of CRAMP against M. pneumoniae and the expression of CRAMP in bronchoalveolar lavage fluid (BALF) of M. pneumoniae-infected mice was examined. CRAMP at 10-20 µg/mL reduced the growth of two strains of M. pneumoniae by 100 to 1000-fold. The amount of CRAMP in the BALF of M. pneumoniae-infected mice was 20∼25 ng/mL by ELISA. The presence of mature CRAMP in BALF was observed by Western blotting. Neutrophils in BALF showed a fair amount of CRAMP in their cytoplasm by immunofluorescence. Furthermore, the addition of M. pneumoniae resulted in the release of a large amount of CRAMP from neutrophils induced by thioglycolate. These results suggest that CRAMP from neutrophils may play an important role in protection against M. pneumoniae infection.


Assuntos
Catelicidinas/metabolismo , Mycoplasma pneumoniae/imunologia , Neutrófilos/imunologia , Pneumonia por Mycoplasma/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos , Western Blotting , Líquido da Lavagem Broncoalveolar/química , Citoplasma/química , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Mycoplasma pneumoniae/crescimento & desenvolvimento , Mycoplasma pneumoniae/patogenicidade , Doenças dos Roedores/imunologia
14.
Immunology ; 133(1): 51-61, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21320122

RESUMO

Pathogenesis of Mycoplasma pneumoniae infection is considered to be in part attributed to excessive immune responses. Mycoplasma pneumoniae shows strong cytoadherence to host cells and this cytoadherence is thought to be involved in the progression of pneumonia. However, the interaction between the cytoadherence and the immune responses is not known in detail. In this study, we demonstrated that the induction of pro-inflammatory cytokines in the human monocyte cell line THP-1 is dependent on the property of cytoadherence of M. pneumoniae. A wild-type strain of M. pneumoniae with cytoadherence ability induced pro-inflammatory cytokines such as tumour necrosis factor-α and interleukin-1ß (IL-1ß). Whereas, heat-killed M. pneumoniae and cytoadherence-deficient mutants of M. pneumoniae caused significantly less production of pro-inflammatory cytokines than the wild-type strain. The wild-type strain induced pro-inflammatory cytokines in an endocytosis-independent manners, but the induction by heat-killed M. pneumoniae and cytoadherence-deficient mutants was dependent on endocytosis. Moreover, the wild-type strain induced caspase-1 production and ATP efflux, promoting the maturation of IL-1ß and release of the pro-IL-1ß precursor, whereas heat-killed M. pneumoniae and the cytoadherence-deficient mutants failed to induce them. These data suggest that the cytoadherence ability of M. pneumoniae activates immune responses and is involved in the pathogenesis of M. pneumoniae infection.


Assuntos
Aderência Bacteriana/imunologia , Citocinas/biossíntese , Inflamação/imunologia , Monócitos/imunologia , Mycoplasma pneumoniae/patogenicidade , Western Blotting , Linhagem Celular , Citocinas/imunologia , Endocitose/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Inflamassomos/imunologia , Inflamassomos/metabolismo , Inflamação/metabolismo , Inflamação/microbiologia , Monócitos/microbiologia , Mycoplasma pneumoniae/imunologia , Transdução de Sinais/imunologia
15.
Infect Immun ; 79(1): 211-9, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21041488

RESUMO

Pseudomonas aeruginosa-derived large extracellular protease (LepA) and hemolytic phospholipase C (PlcH) are considered to play an important role in the pathogenicity of this organism. Although bacterial growth appears to be closely related to virulence, little is known about whether LepA and PlcH participate in the growth and virulence of P. aeruginosa. In this study, we investigated whether LepA and PlcH contribute to the virulence and growth of P. aeruginosa using a wild-type strain and mutants. The growth rate of the isogenic lepA single mutant was lower than that of the wild-type strain in a minimal medium containing serum albumin or hemoglobin as the sole carbon and nitrogen source. Furthermore, the growth rate of the lepA plcH double mutant decreased greatly compared with that of the wild-type strain in a minimal medium containing erythrocytes as a sole nutrient source for growth. Thus, these results indicate that cooperation between LepA and PlcH would contribute to the utilization of erythrocytes as a sole nutrient source for the growth of P. aeruginosa. In addition, mouse infection experiments demonstrated that the virulence of the lepA and plcH single mutants was attenuated, and the numbers of the mutants were lower than the numbers of the wild-type strain in peritoneal lavage fluid and whole-blood specimens. In particular, the virulence and growth rate of the lepA plcH double mutant were markedly lower than those of the wild-type strain. Collectively, these results suggest that LepA and PlcH contribute to the in vivo virulence and growth of P. aeruginosa.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/enzimologia , Fosfolipases Tipo C/metabolismo , Albuminas , Animais , Proteínas de Bactérias/genética , Linhagem Celular , Meios de Cultura/química , Eritrócitos , Humanos , Ferro , Masculino , Camundongos , Mutação , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Ovinos/sangue , Fosfolipases Tipo C/classificação , Fosfolipases Tipo C/genética , Virulência
16.
J Pept Sci ; 16(11): 607-12, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20648478

RESUMO

The widespread natural sources-derived cationic peptides have been reported to reveal bacterial killing and/or growth-inhibiting properties. Correspondingly, a number of artificial peptides have been designed to understand antibacterial mechanism of the cationic peptides. These peptides are expected to be an alternative antibiotic against drug-resistant pathogenic bacteria because major antimicrobial mechanism of cationic peptides involves bacterial membrane disorder, although those availabilities have not been well evaluated. In this study, cationic peptides containing Aib were prepared to evaluate the availability as an antimicrobial agent, especially against representative pathogenic bacteria. Among them, BRBA20, consisting of five repeated Aib-Arg-Aib-Ala sequences, showed strong antibacterial activity against both Gram-negative and Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. Additionally, growth of Serratia marcescens and multidrug-resistant Pseudomonas aeruginosa, known as proteases-secreting pathogenic bacteria, were also completely inhibited by BRBA20 under 20 µg/ml peptide concentrations. Our results suggested availabilities of Aib-derived amphiphilicity and protease resistance in the design of artificial antimicrobial peptides. Comparing BRBA20 with BKBA20, it was also concluded that Arg residue is the preferred cationic source than Lys for antimicrobial action of amphiphilic helices.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Tensoativos/química , Ácidos Aminoisobutíricos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Cátions , Permeabilidade da Membrana Celular/efeitos dos fármacos , Desenho de Fármacos , Escherichia coli/efeitos dos fármacos , Bicamadas Lipídicas/química , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/efeitos dos fármacos , Serratia marcescens/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Tensoativos/farmacologia
17.
Arch Gerontol Geriatr ; 49(2): 278-283, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19095315

RESUMO

Urinary incontinence (UI) is one of the most common and distressing conditions among nursing home residents. Although scheduled care is usually provided for them, incontinence care should be individualized regarding going to the toilet, changing diapers, and taking food and water. We have developed an individualized and comprehensive care strategy to address the problem. We conducted an intervention study that involved training chiefs of staffs, who in turn trained other staffs, and encouraging residents. A total of 153 elderly subjects selected from 1290 residents in 17 nursing homes were eligible to receive our individualized and comprehensive care. The goals of the care strategy were (i) to complete meal intake; (ii) to take fluids up to 1500 ml/day; (iii) to urinate in a toilet; (iv) to spend over 6h out of bed; and (v) to reduce time spent in wet diapers. We explained the aims of our strategy to the chiefs of staff of each nursing home and instructed them to encourage residents to take an active part in our individualized and comprehensive care strategy for 12 weeks. For 3 days before and after that period, we assessed the changes in fluid volume intake, time spent in wet diapers, size of diaper pads, and urination habits. The result was that fluid volume intake significantly increased (p<0.001) while time spent in wet diapers decreased (p<0.001). The number of residents wearing diapers decreased as did the size of pads during the day (p=0.0017). The proportion of residents using diapers at night was reduced and those using toilets at night increased (p=0.007). This study suggests that such an individualized and comprehensive care strategy can offer a measurable improvement in UI care.


Assuntos
Instituição de Longa Permanência para Idosos , Casas de Saúde , Planejamento de Assistência ao Paciente , Incontinência Urinária/terapia , Fraldas para Adultos/estatística & dados numéricos , Feminino , Hidratação , Humanos , Capacitação em Serviço , Masculino , Estudos de Casos Organizacionais , Treinamento no Uso de Toaletes
18.
Microbiology (Reading) ; 154(Pt 5): 1318-1325, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18451040

RESUMO

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.


Assuntos
Proteínas de Bactérias/imunologia , Lipoproteínas/imunologia , NF-kappa B/metabolismo , Receptor 1 Toll-Like/imunologia , Receptor 2 Toll-Like/imunologia , Receptor 6 Toll-Like/imunologia , Ureaplasma/imunologia , Sequência de Aminoácidos , Animais , Fracionamento Celular , Linhagem Celular , Células Cultivadas , Feminino , Humanos , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/microbiologia , Camundongos , Dados de Sequência Molecular , Fator de Necrose Tumoral alfa/biossíntese
19.
Infect Immun ; 76(8): 3672-8, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18474641

RESUMO

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-kappaB activation. The Triton X-114 detergent phase of M. genitalium was found to induce NF-kappaB through TLR2. The active component of the Triton X-114 detergent phase was a lipoprotein precursor, MG149. The activation of NF-kappaB 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-kappaB by MG149 is dependent on TLR1 and TLR2. A synthetic lipopeptide derived from MG149 containing three acyl chains also induced NF-kappaB through TLR1 and TLR2. Thus, the results show that MG149, a triacylated lipoprotein from M. genitalium, activates NF-kappaB through TLR1 and TLR2.


Assuntos
Lipoproteínas/imunologia , Mycoplasma genitalium/imunologia , NF-kappa B/metabolismo , Receptor 1 Toll-Like/imunologia , Receptor 2 Toll-Like/imunologia , Sequência de Aminoácidos , Linhagem Celular , Humanos , Lipoproteínas/isolamento & purificação , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/imunologia
20.
Cell Microbiol ; 10(7): 1491-504, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18331590

RESUMO

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-kappaB-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.


Assuntos
Proteínas de Bactérias/metabolismo , Endopeptidases/metabolismo , NF-kappa B/metabolismo , Pseudomonas aeruginosa/enzimologia , Receptores Ativados por Proteinase/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Células COS , Bovinos , Linhagem Celular , Chlorocebus aethiops , Endopeptidases/genética , Regulação da Expressão Gênica , Humanos , Interleucina-8/metabolismo , Dados de Sequência Molecular , NF-kappa B/genética , Peptídeos/genética , Peptídeos/metabolismo , Regiões Promotoras Genéticas , Receptores Ativados por Proteinase/genética , Trombina/metabolismo , Tripsina/metabolismo
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