RESUMEN
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.
Asunto(s)
Proteínas Bacterianas/inmunología , Mucina 3/metabolismo , Infecciones por Pseudomonas/inmunología , Pseudomonas aeruginosa/enzimología , Sulfatasas/inmunología , Factores de Virulencia/inmunología , Animales , Masculino , Ratones , Ratones Endogámicos ICR , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/patogenicidad , Porcinos , VirulenciaRESUMEN
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.
Asunto(s)
Células Epiteliales/microbiología , Peróxido de Hidrógeno/toxicidad , Mycoplasma pneumoniae/metabolismo , Parthanatos/efectos de los fármacos , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Apoptosis/efectos de los fármacos , Adhesión Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/patogenicidad , NAD/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
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.
Asunto(s)
Traslocación Bacteriana/genética , Genes Bacterianos , Pseudomonas aeruginosa/genética , Sistemas de Secreción Bacterianos/genética , Células CACO-2 , Flagelos/efectos de los fármacos , Flagelos/metabolismo , Humanos , Movimiento , Mutación/genética , Fosfoglicerato-Deshidrogenasa/metabolismo , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/patogenicidad , Serina/farmacología , VirulenciaRESUMEN
Growth factor receptor-bound protein 14 (Grb14) is a negative regulator of insulin receptor (IR) and is involved in a negative feedback mechanism of insulin signaling. Grb14 associates with IR and inhibits its tyrosine kinase activity through the between pleckstrin homology and Src homology-2 (BPS) domain. We previously reported that the pharmacological inhibition and knockdown of glycogen synthase kinase-3 (GSK-3) facilitates the insulin-induced complex formation of human Grb14 (hGrb14) and IR, suggesting that GSK-3 suppresses hGrb14 recruitment to IR. This study further investigated a functional phosphorylation of the serine residues in hGrb14 BPS domain, identified as putative GSK-3 targets to verify an effect of GSK-3 on the hGrb14-IR complex formation. In vitro kinase assay using the motif-derived peptides showed that the serine residues located in N-terminal (Ser358, Ser362 and Ser366) and C-terminal (Ser419 and Ser423) regions of the BPS domain were phosphorylated by GSK-3. Co-immunoprecipitation and yeast two-hybrid (Y2H) experiments suggested that the negative charges genetically introduced on the Ser358, Ser362 and Ser366 suppressed the association of hGrb14 to IR. Surface plasmon resonance experiment gave Kd values of 8 nM for recombinant hGrb14 with respect to the interaction with IR ß-subunit, and this affinity was lost after the replacements of the Ser358, Ser362 and Ser366 with glutamic acid residues. Y2H experiment with the BPS domain alone; however, did not show any difference owing to the same mutations. It is therefore evident that the N-terminus of the BPS domain plays an important role in the regulation of hGrb14-IR complex formation through phosphorylation, in addition to other domains.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Receptor de Insulina/metabolismo , Serina/metabolismo , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Humanos , Fosforilación , Dominios Homologos srcRESUMEN
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.
Asunto(s)
Desoxirribonucleasas/metabolismo , Trampas Extracelulares/metabolismo , Interacciones Huésped-Patógeno , Evasión Inmune , Mycoplasma pneumoniae/enzimología , Mycoplasma pneumoniae/patogenicidad , Neutrófilos/inmunología , Hidrólisis , Magnesio/metabolismo , Mycoplasma pneumoniae/inmunologíaRESUMEN
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.
Asunto(s)
Autofagia/fisiología , Adhesión Bacteriana/fisiología , Infecciones por Mycoplasma/inmunología , Mycoplasma pneumoniae/fisiología , Receptor Toll-Like 4/metabolismo , Animales , Regulación de la Expresión Génica/inmunología , Inflamación/metabolismo , Macrófagos , Ratones , Ratones Noqueados , Mutación , Infecciones por Mycoplasma/microbiología , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/genéticaRESUMEN
To evaluate the biofilm formation of non-typeable Haemophilus influenzae (NTHi) and H. influenzae type b (Hib) clinical isolates, we conducted the following study. Serotyping and polymerase chain reaction were performed to identify ß-lactamase-negative ampicillin (ABPC)-susceptible (BLNAS), ß-lactamase-negative ABPC-resistant (BLNAR), TEM-1 type ß-lactamase-producing ABPC-resistant (BLPAR)-NTHi, and Hib. Biofilm formation was investigated by microtiter biofilm assay, as well as visually observation with a scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) in a continuous-flow chamber. As a result, totally 99 strains were investigated, and were classified into 4 groups which were 26 gBLNAS, 22 gBLNAR, 28 gBLPAR-NTHi and 23 Hib strains. The mean OD600 in the microtiter biofilm assay of gBLNAS, gBLNAR, gBLPAR-NTHi, and Hib strains were 0.57, 0.50, 0.34, and 0.08, respectively. NTHi strains were similar in terms of biofilm formations, which were observed by SEM and CLSM. Five Hib strains with the alternated type b cap loci showed significantly increased biofilm production than the other Hib strains. In conclusion, gBLNAS, gBLNAR, and gBLPAR-NTHi strains were more capable to produce biofilms compared to Hib strains. Our data suggested that resistant status may not be a key factor but capsule seemed to play an important role in H. influenzae biofilm formation.
Asunto(s)
Cápsulas Bacterianas/química , Biopelículas , Haemophilus influenzae/química , Haemophilus influenzae/fisiología , Antibacterianos/farmacología , Infecciones por Haemophilus/microbiología , Haemophilus influenzae/efectos de los fármacos , Humanos , Pruebas de Sensibilidad Microbiana , Nasofaringe/microbiología , Esputo/microbiologíaRESUMEN
The major virulence factors produced by Pseudomonas aeruginosa include secreted proteases that damage host tissues. Of the proteases analyzed, alkaline protease (AprA) and elastase B (LasB) have been characterized extensively. Although P. aeruginosa protein database predicts the presence of several other potential proteases, little has been known about the proteases involving in the pathogenicity of this organism. In this study, we found that P. aeruginosa produces a novel large extracellular protease (LepA) distinct from known proteases such as AprA and LasB. Sequence analysis of LepA showed a molecular future of the proteins transported by the two-partner secretion pathway. We demonstrated that LepA can activate NF-kB-driven promoter through protease-activated receptor-1, -2 or -4. On the other hand, one of the functions of proteases is to hydrolyze proteins and peptides for nutrient acquisition either by degrading host enzymes or even by causing tissue damage to further the survival of the bacterium. Therefore, to investigate the role of LepA in in vivo virulence and growth of P. aeruginosa, we compared the virulence and growth of a wild-type strain and its mutant using a mouse model of acute systemic infection by P. aeruginosa. Our results suggest that LepA contributes to the in vivo virulence and growth of P. aeruginosa.
Asunto(s)
Péptido Hidrolasas/fisiología , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/patogenicidad , Virulencia/genética , Animales , Humanos , FN-kappa B/metabolismo , Péptido Hidrolasas/química , Péptido Hidrolasas/inmunología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crecimiento & desarrollo , Receptores Proteinasa-Activados/inmunología , Receptores Proteinasa-Activados/fisiologíaRESUMEN
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.
Asunto(s)
Transporte Activo de Núcleo Celular/genética , Regulación de la Expresión Génica , Proteína HMGB1/genética , Macrófagos/metabolismo , Fosfoserina/metabolismo , Proteína Fosfatasa 2/genética , Animales , Células COS , Línea Celular , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citoplasma/metabolismo , Proteína HMGB1/metabolismo , Hidrólisis , Macrófagos/citología , Ratones , Datos de Secuencia Molecular , Proteína Fosfatasa 2/metabolismo , Señales de Clasificación de Proteína , Transducción de SeñalRESUMEN
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.
Asunto(s)
Inflamación/patología , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/patogenicidad , Receptores Proteinasa-Activados/metabolismo , Serina Proteasas/metabolismo , Factores de Virulencia/metabolismo , Línea Celular , Células Epiteliales/microbiología , Escherichia coli/genética , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Biopelículas , Polisacáridos Bacterianos/fisiología , Streptococcus pneumoniae/fisiología , Virulencia/genética , Línea Celular , Ingeniería Genética , Interacciones Huésped-Patógeno , Humanos , Microscopía Confocal , Mutación , Infecciones Neumocócicas/microbiología , Polisacáridos Bacterianos/genética , Polisacáridos Bacterianos/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/patogenicidadRESUMEN
Heme oxygenase (HO) catalyzes the O(2)-dependent degradation of heme to biliverdin IXα, carbon monoxide (CO), and free ferrous iron through a multistep mechanism. Electrons required for HO catalysis in mammals are provided by NADPH-cytochrome P450 reductase. Recently, Kim et al. reported for the first time that HO, especially inducible HO-1, appears in caveolae and showed that caveolin-1, a principal isoform of the caveolin family, physically interacts with HO-1 [ Jung , N. H. et al. ( 2003 ) IUBMB Life 55 , 525 - 532 ; Kim , H. P. et al. ( 2004 ) FASEB J. 18 , 1080 - 1089 ]. In the present study, we confirmed by immunoprecipitation experiments that rat HO-1 and rat caveolin-1 (residues 1-101) directly interact with each other and that the HO-1 activity is inhibited by caveolin-1 (1-101). The 82-101 residues of caveolin-1 (CAV(82-101)), called the caveolin scaffolding domain, play essential roles in caveolin-related protein-protein interactions. The HO-1 activity is also inhibited by CAV(82-101) in a competitive manner with hemin, and a hemin titration experiment showed that CAV(82-101) interferes with hemin binding to HO-1. The enzyme kinetics and surface plasmon resonance experiments gave comparable K(i) and K(D) values of 5.2 and 1.0 µM for CAV(82-101), respectively, with respect to the interaction with HO-1. These observations indicated that CAV(82-101) and hemin share a common binding site within the HO-1 protein. The identified caveolin binding motif (FLLNIELF) of rat HO-1 is incomplete compared to the proposed consensus sequence. The affinity between HO-1 and CAV(82-101), however, was almost completely or remarkably eliminated by replacement of Phe(207) and/or Phe(214) with Ala, indicating that HO-1 binds to caveolin-1 via this motif. Among the peptide fragments derived from CAV(82-101), i.e., CAV(82-91), CAV(87-96), CAV(92-101), and CAV(97-101), CAV(92-101) and CAV(97-101) are able to inhibit the HO-1 activity to a similar extent; thus, the five-amino acid sequence (residues 97-101) is considered to be a minimum sequence for binding to HO-1.
Asunto(s)
Caveolina 1/fisiología , Hemo Oxigenasa (Desciclizante)/antagonistas & inhibidores , Animales , Sitios de Unión , Caveolina 1/química , Caveolina 1/metabolismo , Hemo Oxigenasa (Desciclizante)/metabolismo , Reacción en Cadena de la Polimerasa , Unión Proteica , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
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 20â¼25 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.
Asunto(s)
Catelicidinas/metabolismo , Mycoplasma pneumoniae/inmunología , Neutrófilos/inmunología , Neumonía por Mycoplasma/inmunología , Animales , Péptidos Catiónicos Antimicrobianos , Western Blotting , Líquido del Lavado Bronquioalveolar/química , Citoplasma/química , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Ratones , Ratones Endogámicos BALB C , Microscopía Fluorescente , Mycoplasma pneumoniae/crecimiento & desarrollo , Mycoplasma pneumoniae/patogenicidad , Enfermedades de los Roedores/inmunologíaRESUMEN
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.
Asunto(s)
Adhesión Bacteriana/inmunología , Citocinas/biosíntesis , Inflamación/inmunología , Monocitos/inmunología , Mycoplasma pneumoniae/patogenicidad , Western Blotting , Línea Celular , Citocinas/inmunología , Endocitosis/inmunología , Ensayo de Inmunoadsorción Enzimática , Humanos , Inflamasomas/inmunología , Inflamasomas/metabolismo , Inflamación/metabolismo , Inflamación/microbiología , Monocitos/microbiología , Mycoplasma pneumoniae/inmunología , Transducción de Señal/inmunologíaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/enzimología , Fosfolipasas de Tipo C/metabolismo , Albúminas , Animales , Proteínas Bacterianas/genética , Línea Celular , Medios de Cultivo/química , Eritrocitos , Humanos , Hierro , Masculino , Ratones , Mutación , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Ovinos/sangre , Fosfolipasas de Tipo C/clasificación , Fosfolipasas de Tipo C/genética , VirulenciaRESUMEN
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.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Tensoactivos/química , Ácidos Aminoisobutíricos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Cationes , Permeabilidad de la Membrana Celular/efectos de los fármacos , Diseño de Fármacos , Escherichia coli/efectos de los fármacos , Membrana Dobles de Lípidos/química , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Pseudomonas aeruginosa/efectos de los fármacos , Serratia marcescens/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Tensoactivos/farmacologíaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/inmunología , Lipoproteínas/inmunología , FN-kappa B/metabolismo , Receptor Toll-Like 1/inmunología , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 6/inmunología , Ureaplasma/inmunología , Secuencia de Aminoácidos , Animales , Fraccionamiento Celular , Línea Celular , Células Cultivadas , Femenino , Humanos , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/microbiología , Ratones , Datos de Secuencia Molecular , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
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.
Asunto(s)
Lipoproteínas/inmunología , Mycoplasma genitalium/inmunología , FN-kappa B/metabolismo , Receptor Toll-Like 1/inmunología , Receptor Toll-Like 2/inmunología , Secuencia de Aminoácidos , Línea Celular , Humanos , Lipoproteínas/aislamiento & purificación , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/inmunologíaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Endopeptidasas/metabolismo , FN-kappa B/metabolismo , Pseudomonas aeruginosa/enzimología , Receptores Proteinasa-Activados/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Células COS , Bovinos , Línea Celular , Chlorocebus aethiops , Endopeptidasas/genética , Regulación de la Expresión Génica , Humanos , Interleucina-8/metabolismo , Datos de Secuencia Molecular , FN-kappa B/genética , Péptidos/genética , Péptidos/metabolismo , Regiones Promotoras Genéticas , Receptores Proteinasa-Activados/genética , Trombina/metabolismo , Tripsina/metabolismoRESUMEN
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-kappaB-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-alpha) 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-alpha 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.