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Biofilms are microbial communities that inhabit various surfaces and are surrounded by extracellular matrices (ECMs). Clinical microbiologists have shown that the majority of chronic infections are caused by biofilms, following the introduction of the first biofilm infection model by J. W. Costerton and colleagues (J. Lam, R. Chan, K. Lam, and J. W. Costerton, Infect Immun 28:546-556, 1980). However, treatments for chronic biofilm infections are still limited to surgical removal of the infected sites. Pseudomonas aeruginosa and Enterococcus faecalis are two frequently identified bacterial species in biofilm infections; nevertheless, the interactions between these two species, especially during biofilm growth, are not clearly understood. In this study, we observed phenotypic changes in a dual-species biofilm of P. aeruginosa and E. faecalis, including a dramatic increase in biofilm matrix thickness. For clear elucidation of the spatial distribution of the dual-species biofilm, P. aeruginosa and E. faecalis were labeled with red and green fluorescence, respectively. E. faecalis was located at the lower part of the dual-species biofilm, while P. aeruginosa developed a structured biofilm on the upper part. Mutants with altered exopolysaccharide (EPS) productions were constructed in order to determine the molecular basis for the synergistic effect of the dual-species biofilm. Increased biofilm matrix thickness was associated with EPSs, not extracellular DNA. In particular, Pel and Psl contributed to interspecies and intraspecies interactions, respectively, in the dual-species P. aeruginosa and E. faecalis biofilm. Accordingly, targeting Pel and Psl might be an effective part of eradicating P. aeruginosa polymicrobial biofilms.IMPORTANCE Chronic infection is a serious problem in the medical field. Scientists have observed that chronic infections are closely associated with biofilms, and the vast majority of infection-causing biofilms are polymicrobial. Many studies have reported that microbes in polymicrobial biofilms interact with each other and that the bacterial interactions result in elevated virulence, in terms of factors, such as infectivity and antibiotic resistance. Pseudomonas aeruginosa and Enterococcus faecalis are frequently isolated pathogens in chronic biofilm infections. Nevertheless, while both bacteria are known to be agents of numerous nosocomial infections and can cause serious diseases, interactions between the bacteria in biofilms have rarely been examined. In this investigation, we aimed to characterize P. aeruginosa and E. faecalis dual-species biofilms and to determine the molecular factors that cause synergistic effects, especially on the matrix thickening of the biofilm. We suspect that our findings will contribute to the development of more efficient methods for eradicating polymicrobial biofilm infections.
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Biofilmes/crescimento & desenvolvimento , Enterococcus faecalis/fisiologia , Polissacarídeos Bacterianos/metabolismo , Pseudomonas aeruginosa/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Enterococcus faecalis/química , Enterococcus faecalis/genética , Enterococcus faecalis/crescimento & desenvolvimento , Mutação , Polissacarídeos Bacterianos/química , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimentoRESUMO
Pseudomonas aeruginosa, a Gram-negative bacterium of clinical significance, produces elastase as a predominant exoprotease. Here, we screened a library of chemical compounds currently used for human medication and identified diethylene triamine penta-acetic acid (DTPA, pentetic acid) as an agent that suppresses the production of elastase. Elastase activity found in the prototype P. aeruginosa strain PAO1 was significantly decreased when grown with a concentration as low as 20 µM DTPA. Supplementation with Zn(2+) or Mn(2+) ions restored the suppressive effect of DTPA, suggesting that the DTPA-mediated decrease in elastase activity is associated with ion-chelating activity. In DTPA-treated PAO1 cells, transcription of the elastase-encoding lasB gene and levels of the Pseudomonas quinolone signal (PQS), a molecule that mediates P. aeruginosa quorum sensing (QS), were significantly downregulated, reflecting the potential involvement of the PQS QS system in DTPA-mediated elastase suppression. Biofilm formation was also decreased by DTPA treatment. When A549 alveolar type II-like adenocarcinoma cells were infected with PAO1 cells in the presence of DTPA, A549 cell viability was substantially increased. Furthermore, the intranasal delivery of DTPA to PAO1-infected mice alleviated the pathogenic effects of PAO1 cells in the animals. Together, our results revealed a novel function for a known molecule that may help treat P. aeruginosa airway infection.
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Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Metaloendopeptidases/antagonistas & inibidores , Ácido Pentético/farmacologia , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Administração Intranasal , Animais , Antibacterianos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Cátions Bivalentes , Linhagem Celular Tumoral , Reposicionamento de Medicamentos , Inibidores Enzimáticos/metabolismo , Humanos , Quelantes de Ferro/metabolismo , Quelantes de Ferro/farmacologia , Masculino , Manganês/metabolismo , Manganês/farmacologia , Metaloendopeptidases/química , Metaloendopeptidases/metabolismo , Camundongos , Ácido Pentético/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/enzimologia , Pseudomonas aeruginosa/patogenicidade , Quinolonas/metabolismo , Percepção de Quorum/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Virulência , Zinco/metabolismo , Zinco/farmacologiaRESUMO
Supplementation with CBM588, a bifidogenic live bacterial product, has been associated with improved clinical outcomes in persons with metastatic renal cell carcinoma (mRCC) receiving nivolumab and ipilimumab. However, its effect on those receiving tyrosine kinase inhibitor-based combinations is unknown. In this open-label, randomized, investigator-initiated, phase 1 study, 30 participants with locally advanced or mRCC with histological confirmation of clear cell, papillary or sarcomatoid component were randomized in a 2:1 fashion to receive cabozantinib (an inhibitor of vascular endothelial growth factor receptor, MET and AXL) and nivolumab (anti-programmed cell death protein 1) with or without CBM588 as first-line treatment. Metagenomic sequencing was performed on stool samples to characterize their gut microbiome at baseline and 13 weeks into treatment. The primary endpoint was a change in the relative abundance of Bifidobacterium spp.; secondary endpoints included objective response rate (ORR), progression-free survival (PFS) and toxicity profile. The primary endpoint of the study was not met and the addition of CBM588 to cabozantinib and nivolumab did not result in a difference in the relative abundance of Bifidobacterium spp. or alpha diversity (as measured by the Shannon index). However, ORR was significantly higher in participants treated with CBM588 compared to those in the control arm (14 of 19, 74% versus 2 of 10, 20%; P = 0.01). PFS at 6 months was 84% (16 of 19) and 60% (6 of 10) in the experimental and control arms, respectively. No significant difference in toxicity profile was seen between the study arms. Our results provide a preliminary signal of improved clinical activity with CBM588 in treatment-naive participants with mRCC receiving cabozantinib and nivolumab. Further investigation is needed to confirm these findings and better characterize the underlying mechanism driving this effect.ClinicalTrials.gov identifier: NCT05122546.
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Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne disease in animals and MAP involvement in human Crohn disease has been recently emphasized. Evidence from M. tuberculosis studies suggests mycobacterial proteins activate dendritic cells (DCs) via Toll-like receptor (TLR) 4, eventually determining the fate of immune responses. Here, we investigated whether MAP CobT contributes to the development of T cell immunity through the activation of DCs. MAP CobT recognizes TLR4, and induces DC maturation and activation via the MyD88 and TRIF signaling cascades, which are followed by MAP kinases and NF-κB. We further found that MAP CobT-treated DCs activated naive T cells, effectively polarized CD4(+) and CD8(+) T cells to secrete IFN-γ and IL-2, but not IL-4 and IL-10, and induced T cell proliferation. These data indicate that MAP CobT contributes to T helper (Th) 1 polarization of the immune response. MAP CobT-treated DCs specifically induced the expansion of CD4(+)/CD8(+)CD44(high)CD62L(low) memory T cells in the mesenteric lymph node of MAP-infected mice in a TLR4-dependent manner. Our results indicate that MAP CobT is a novel DC maturation-inducing antigen that drives Th1 polarized-naive/memory T cell expansion in a TLR4-dependent cascade, suggesting that MAP CobT potentially links innate and adaptive immunity against MAP.
Assuntos
Complexos Multienzimáticos/genética , Mycobacterium avium subsp. paratuberculosis/metabolismo , Nucleotidiltransferases/genética , Pentosiltransferases/genética , Animais , Proteínas de Bactérias/metabolismo , Células Dendríticas/citologia , Feminino , Sistema Imunitário , Memória Imunológica , Interferon gama/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Complexos Multienzimáticos/fisiologia , Nucleotidiltransferases/fisiologia , Pentosiltransferases/fisiologia , Proteínas Recombinantes/química , Linfócitos T/imunologia , Células Th1/citologia , Receptor 4 Toll-Like/metabolismoRESUMO
RATIONALE: Macrolides, such as clarithromycin (CLR) and azithromycin (AZM), are frequently the only oral antibiotics that are active against Mycobacterium abscessus and M. massiliense infections. OBJECTIVES: To compare the activity of CLR and AZM in experimental models. METHODS: We compared the treatment efficacies of CLR and AZM and determined the correlation between efficacy and induced erythromycin ribosome methyltransferase gene (erm)(41) expression in experimental models of M. abscessus and M. massiliense infections. MEASUREMENTS AND MAIN RESULTS: In all tested M. abscessus isolates, a high level of inducible CLR resistance developed (minimal inhibitory concentration [MIC] on Day 3 versus Day 14; P < 0.001). Whereas the AZM MIC increased on Day 14 (P < 0.01 versus Day 3), the level was significantly lower than the CLR MIC on Day 14 (P < 0.001). However, the MICs of CLR and AZM for the M. massiliense isolates did not change. Compared with CLR, AZM presented greater antibiotic activity against M. abscessus in vitro, ex vivo, and in vivo (P < 0.05), whereas both macrolides were comparably effective against M. massiliense. In M. abscessus infection, the level of erm(41) expression was higher after exposure to CLR than after exposure to AZM (P < 0.001). Experiments using an erm(41)-knockout M. abscessus mutant and an M. massiliense transformant expressing M. abscessus erm(41) confirmed that erm(41) was responsible for inducible CLR resistance. CONCLUSIONS: CLR induces greater erm(41) expression and thus higher macrolide resistance than AZM in M. abscessus infection. AZM may be more effective against M. abscessus, whereas both macrolides appear to be equally effective against M. massiliense.
Assuntos
Azitromicina/uso terapêutico , Claritromicina/uso terapêutico , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Mycobacterium/efeitos dos fármacos , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Azitromicina/farmacologia , Células da Medula Óssea , Claritromicina/farmacologia , Modelos Animais de Doenças , Farmacorresistência Bacteriana Múltipla , Feminino , Humanos , Técnicas In Vitro , Macrolídeos/farmacologia , Macrolídeos/uso terapêutico , Camundongos , Camundongos Endogâmicos C57BL , Mycobacterium/classificação , Mycobacterium/isolamento & purificação , Infecções por Mycobacterium não Tuberculosas/genética , Infecções por Mycobacterium não Tuberculosas/microbiologia , República da CoreiaRESUMO
The gut microbiota-brain axis is suspected to contribute to the development of Alzheimer's disease (AD), a neurodegenerative disease characterized by amyloid-ß plaque deposition, neurofibrillary tangles, and neuroinflammation. To evaluate the role of the gut microbiota-brain axis in AD, we characterized the gut microbiota of female 3xTg-AD mice modeling amyloidosis and tauopathy and wild-type (WT) genetic controls. Fecal samples were collected fortnightly from 4 to 52 weeks, and the V4 region of the 16S rRNA gene was amplified and sequenced on an Illumina MiSeq. RNA was extracted from the colon and hippocampus, converted to cDNA, and used to measure immune gene expression using reverse transcriptase quantitative PCR (RT-qPCR). Diversity metrics were calculated using QIIME2, and a random forest classifier was applied to predict bacterial features that are important in predicting mouse genotype. Gene expression of glial fibrillary acidic protein (GFAP; indicating astrocytosis) was elevated in the colon at 24 weeks. Markers of Th1 inflammation (il6) and microgliosis (mrc1) were elevated in the hippocampus. Gut microbiota were compositionally distinct early in life between 3xTg-AD mice and WT mice (permutational multivariate analysis of variance [PERMANOVA], 8 weeks, P = 0.001, 24 weeks, P = 0.039, and 52 weeks, P = 0.058). Mouse genotypes were correctly predicted 90 to 100% of the time using fecal microbiome composition. Finally, we show that the relative abundance of Bacteroides species increased over time in 3xTg-AD mice. Taken together, we demonstrate that changes in bacterial gut microbiota composition at prepathology time points are predictive of the development of AD pathologies. IMPORTANCE Recent studies have demonstrated alterations in the gut microbiota composition in mice modeling Alzheimer's disease (AD) pathologies; however, these studies have only included up to 4 time points. Our study is the first of its kind to characterize the gut microbiota of a transgenic AD mouse model, fortnightly, from 4 weeks of age to 52 weeks of age, to quantify the temporal dynamics in the microbial composition that correlate with the development of disease pathologies and host immune gene expression. In this study, we observed temporal changes in the relative abundances of specific microbial taxa, including the genus Bacteroides, that may play a central role in disease progression and the severity of pathologies. The ability to use features of the microbiota to discriminate between mice modeling AD and wild-type mice at prepathology time points indicates a potential role of the gut microbiota as a risk or protective factor in AD.
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Chronic type 2 (T2) inflammatory diseases of the respiratory tract are characterized by mucus overproduction and disordered mucociliary function, which are largely attributed to the effects of IL-13 on common epithelial cell types (mucus secretory and ciliated cells). The role of rare cells in airway T2 inflammation is less clear, though tuft cells have been shown to be critical in the initiation of T2 immunity in the intestine. Using bulk and single-cell RNA sequencing of airway epithelium and mouse modeling, we found that IL-13 expanded and programmed airway tuft cells toward eicosanoid metabolism and that tuft cell deficiency led to a reduction in airway prostaglandin E2 (PGE2) concentration. Allergic airway epithelia bore a signature of PGE2 activation, and PGE2 activation led to cystic fibrosis transmembrane receptor-dependent ion and fluid secretion and accelerated mucociliary transport. These data reveal a role for tuft cells in regulating epithelial mucociliary function in the allergic airway.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Fibrose Cística , Animais , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Dinoprostona , Interleucina-13/metabolismo , Camundongos , Sistema RespiratórioRESUMO
The human microbiota is composed of trillions of microbial cells inhabiting the oral cavity, skin, gastrointestinal (GI) tract, airways, and reproductive organs. The gut microbiota is composed of dynamic communities of microorganisms that communicate bidirectionally with the brain via cytokines, neurotransmitters, hormones, and secondary metabolites, known as the gut microbiota-brain axis. The gut microbiota-brain axis is suspected to be involved in the development of neurological diseases, including Alzheimer's disease (AD), Parkinson's disease, and Autism Spectrum Disorder. AD is an irreversible, neurodegenerative disease of the central nervous system (CNS), characterized by amyloid-ß plaques, neurofibrillary tangles, and neuroinflammation. Microglia and astrocytes, the resident immune cells of the CNS, play an integral role in AD development, as neuroinflammation is a driving factor of disease severity. The gut microbiota-brain axis is a novel target for Alzheimer's disease therapeutics to modulate critical neuroimmune and metabolic pathways. Potential therapeutics include probiotics, prebiotics, fecal microbiota transplantation, and dietary intervention. This review summarizes our current understanding of the role of the gut microbiota-brain axis and neuroinflammation in the onset and development of Alzheimer's disease, limitations of current research, and potential for gut microbiota-brain axis targeted therapies.
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Host-associated bacteria and fungi, comprising the microbiota, are critical to host health. In the airways, the composition and diversity of the mucosal microbiota of patients are associated with airway health status. However, the relationship between airway microbiota and respiratory inflammation is not well-understood. Chronic rhinosinusitis (CRS) is a complex disease that affects up to 14% of the US population. Previous studies have shown decreased microbial diversity in CRS patients and enrichment of either Staphylococcus aureus or Pseudomonas aeruginosa. Although bacterial community composition is variable across CRS patients, Malassezia is a dominant fungal genus in the upper airways of the majority of healthy and CRS subjects. We hypothesize that distinct bacterial-fungal interactions differentially influence host mucosal immune response. Thus, we investigated in vitro and in vivo interactions between Malassezia sympodialis, P. aeruginosa, and S. aureus. The in vitro interactions were evaluated using the modified Kirby-Bauer Assay, Crystal Violet assay for biofilm, and FISH. A pilot murine model of acute sinusitis was used to investigate relationships with the host immune response. S. aureus and P. aeruginosa were intranasally instilled in the presence or absence of M. sympodialis (n = 66 total mice; 3-5/group). Changes in the microbiota were determined using 16S rRNA gene sequencing and host immune response was measured using quantitative real-time PCR (qRT-PCR). In vitro, only late stage planktonic P. aeruginosa and its biofilms inhibited M. sympodialis. Co-infection of mice with M. sympodialis and P. aeruginosa or S. aureus differently influenced the immune response. In co-infected mice, we demonstrate different expression of fungal sensing (Dectin-1), allergic responses (IL-5, and IL-13) and inflammation (IL-10, and IL-17) in murine sinus depending on the bacterial species that co-infected with M. sympodialis (p < 0.05). The pilot results suggest that species-specific interactions in airway-associated microbiota may be implicated driving immune responses. The understanding of the role of bacterial-fungal interactions in CRS will contribute to development of novel therapies toward manipulation of the airway microbiota.
Assuntos
Coinfecção , Malassezia , Sinusite , Animais , Bactérias , Biofilmes , Doença Crônica , Humanos , Camundongos , Pseudomonas aeruginosa/genética , RNA Ribossômico 16S/genética , Staphylococcus aureusRESUMO
Staphylococcus aureus is a gram-positive bacterium that causes a wide range of infections. Recently, the spread of methicillin-resistant S. aureus (MRSA) strains has seriously reduced antibiotic treatment options. Anti-virulence strategies, the objective of which is to target the virulence instead of the viability of the pathogen, have become widely accepted as a means of avoiding the emergence of new antibiotic-resistant strains. To increase the number of anti-virulence therapeutic options, it is necessary to identify as many novel virulence-associated genes as possible in MRSA. Co-functional networks have proved useful for mapping gene-to-phenotype associations in various organisms. Herein, we present StaphNet (www.inetbio.org/staphnet), a genome-scale co-functional network for an MRSA strain, S. aureus subsp. USA300_FPR3757. StaphNet, which was constructed by the integration of seven distinct types of genomics data within a Bayesian statistics framework, covers approximately 94% of the coding genome with a high degree of accuracy. We implemented a companion web server for network-based gene prioritization of the phenotypes of 31 different S. aureus strains. We demonstrated that StaphNet can effectively identify genes for virulence-associated phenotypes in MRSA. These results suggest that StaphNet can facilitate target discovery for the development of anti-virulence drugs to treat MRSA infection.
Assuntos
Staphylococcus aureus Resistente à Meticilina/genética , Virulência/genética , Algoritmos , Biofilmes , Biologia Computacional/métodos , Redes Reguladoras de Genes , Hemólise/genética , Internet , Staphylococcus aureus Resistente à Meticilina/patogenicidade , SoftwareRESUMO
Recent studies leveraging next-generation sequencing and functional approaches to understand the human microbiota have demonstrated the presence of diverse, niche-specific microbial communities at nearly every mucosal surface. These microbes contribute to the development and function of physiologic and immunological features that are key to host health status. Not surprisingly, several chronic inflammatory diseases have been attributed to dysbiosis of microbiota composition or function, including chronic rhinosinusitis (CRS). CRS is a heterogeneous disease characterized by inflammation of the sinonasal cavity and mucosal microbiota dysbiosis. Inflammatory phenotypes and bacterial community compositions vary considerably across individuals with CRS, complicating current studies that seek to address causality of a dysbiotic microbiome as a driver or initiator of persistent sinonasal inflammation. Murine models have provided some experimental evidence that alterations in local microbial communities and microbially-produced metabolites influence health status. In this perspective, we will discuss the clinical implications of distinct microbial compositions and community-level functions in CRS and how mucosal microbiota relate to the diverse inflammatory endotypes that are frequently observed. We will also describe specific microbial interactions that can deterministically shape the pattern of co-colonizers and the resulting metabolic products that drive or exacerbate host inflammation. These findings are discussed in the context of CRS-associated inflammation and in other chronic inflammatory diseases that share features observed in CRS. An improved understanding of CRS patient stratification offers the opportunity to personalize therapeutic regimens and to design novel treatments aimed at manipulation of the disease-associated microbiota to restore sinus health.
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Disbiose/microbiologia , Inflamação/microbiologia , Microbiota/genética , Rinite/microbiologia , Sinusite/microbiologia , Animais , Doença Crônica , Modelos Animais de Doenças , Interações entre Hospedeiro e Microrganismos , Humanos , Interações Microbianas , Mucosa Nasal/microbiologia , Mucosa Nasal/patologia , Nariz/microbiologia , Nariz/patologia , RNA Ribossômico 16S/genéticaRESUMO
A biofilm is a community of microbes that typically inhabit on surfaces and are encased in an extracellular matrix. Biofilms display very dissimilar characteristics to their planktonic counterparts. Biofilms are ubiquitous in the environment and influence our lives tremendously in both positive and negative ways. Pseudomonas aeruginosa is a bacterium known to produce robust biofilms. P. aeruginosa biofilms cause severe problems in immunocompromised patients, including those with cystic fibrosis or wound infection. Moreover, the unique biofilm properties further complicate the eradication of the biofilm infection, leading to the development of chronic infections. In this review, we discuss the history of biofilm research and general characteristics of bacterial biofilms. Then, distinct features pertaining to each stage of P. aeruginosa biofilm development are highlighted. Furthermore, infections caused by biofilms on their own or in association with other bacterial species (i.e., multispecies biofilms) are discussed in detail.
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Biofilmes/crescimento & desenvolvimento , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/fisiologia , Coinfecção , Fibrose Cística/microbiologia , Aptidão Genética , Humanos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Percepção de QuorumRESUMO
Pseudomonas aeruginosa is capable of establishing airway infections. Human airway mucus contains a large amount of lysozyme, which hydrolyzes bacterial cell walls. P. aeruginosa, however, is known to be resistant to lysozyme. Here, we performed a genetic screen using a mutant library of PAO1, a prototype P. aeruginosa strain, and identified two mutants (ΔbamB and ΔfabY) that exhibited decrease in survival after lysozyme treatment. The bamB and fabY genes encode an outer membrane assembly protein and a fatty acid synthesis enzyme, respectively. These two mutants displayed retarded growth in the airway mucus secretion (AMS). In addition, these mutants exhibited reduced virulence and compromised survival fitness in two different in vivo infection models. The mutants also showed susceptibility to several antibiotics. Especially, ΔbamB mutant was very sensitive to vancomycin, ampicillin, and ceftazidime that target cell wall synthesis. The ΔfabY displayed compromised membrane integrity. In conclusion, this study uncovered a common aspect of two different P. aeruginosa mutants with pleiotropic phenotypes, and suggests that BamB and FabY could be novel potential drug targets for the treatment of P. aeruginosa infection.
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Antibacterianos/farmacologia , Parede Celular/efeitos dos fármacos , Muramidase/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Animais , Caenorhabditis elegans , Elementos de DNA Transponíveis , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Teste de Complementação Genética , Testes Genéticos , Camundongos Endogâmicos C57BL , Viabilidade Microbiana/efeitos dos fármacos , Mutagênese Insercional , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidade , Vancomicina/farmacologia , Virulência , beta-Lactamas/farmacologiaRESUMO
Pseudomonas aeruginosa is an opportunistic pathogen, known to develop robust biofilms. Its biofilm development increases when antibiotics are presented at subminimal inhibitory concentrations (MICs) for reasons that remain unclear. In order to identify genes that affect biofilm development under such a sublethal antibiotic stress condition, we screened a transposon (Tn) mutant library of PAO1, a prototype P. aeruginosa strain. Among â¼5000 mutants, a fiuA gene mutant was verified to form very defective biofilms in the presence of sub-MIC carbenicillin. The fiuA gene encodes ferrichrome receptor A, involved in the iron acquisition process. Of note, biofilm formation was not decreased in the ΔpchΔpvd mutant defective in the production of pyochelin and pyoverdine, two well-characterized P. aeruginosa siderophore molecules. Moreover, ΔfiuA, a non-polar fiuA deletion mutant, produced a significantly decreased level of elastase, a major virulence determinant. Mouse airway infection experiments revealed that the mutant expressed significantly less pathogenicity. Our results suggest that the fiuA gene has pleiotropic functions that affect P. aeruginosa biofilm development and virulence. The targeting of FiuA could enable the attenuation of P. aeruginosa virulence and may be suitable for the development of a drug that specifically controls the virulence of this important pathogen.
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Proteínas da Membrana Bacteriana Externa/genética , Ferricromo/metabolismo , Pseudomonas aeruginosa/patogenicidade , Fatores de Virulência/genética , Animais , Proteínas da Membrana Bacteriana Externa/metabolismo , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Carbenicilina/farmacologia , Elementos de DNA Transponíveis , Biblioteca Gênica , Ferro/metabolismo , Pulmão/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Oligopeptídeos/biossíntese , Elastase Pancreática/biossíntese , Fenóis/metabolismo , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/genética , Deleção de Sequência , Tiazóis/metabolismo , Fatores de Virulência/metabolismoRESUMO
Indigenous microbes inside the host intestine maintain a complex self-regulating community. The mechanisms by which gut microbes interact with intestinal pathogens remain largely unknown. Here we identify a commensal Escherichia coli strain whose expansion predisposes mice to infection by Vibrio cholerae, a human pathogen. We refer to this strain as 'atypical' E. coli (atEc) because of its inability to ferment lactose. The atEc strain is resistant to reactive oxygen species (ROS) and proliferates extensively in antibiotic-treated adult mice. V. cholerae infection is more severe in neonatal mice transplanted with atEc compared with those transplanted with a typical E. coli strain. Intestinal ROS levels are decreased in atEc-transplanted mice, favouring proliferation of ROS-sensitive V. cholerae. An atEc mutant defective in ROS degradation fails to facilitate V. cholerae infection when transplanted, suggesting that host infection susceptibility can be regulated by a single gene product of one particular commensal species.
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Suscetibilidade a Doenças/microbiologia , Escherichia coli/genética , Gastroenterite/microbiologia , Microbioma Gastrointestinal/genética , Simbiose/genética , Vibrio cholerae/patogenicidade , Animais , Antibacterianos/farmacologia , Catalase/genética , Modelos Animais de Doenças , Enterocolite , Escherichia coli/metabolismo , Transplante de Microbiota Fecal/métodos , Feminino , Microbioma Gastrointestinal/efeitos dos fármacos , Técnicas de Inativação de Genes , Humanos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/microbiologia , Intestinos/microbiologia , Lactose/metabolismo , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The human gastrointestinal tract is colonized by multitudes of microorganisms that exert beneficial effects on human health. Mounting evidence suggests that intestinal microbiota contributes to host resistance against enteropathogenic bacterial infection. However, molecular details that account for such an important role has just begun to be understood. The commensal microbes in the intestine regulate gut homeostasis through activating the development of host innate immunity and producing molecules with antimicrobial activities that directly inhibit propagation of pathogenic bacteria. Understanding the protective roles of gut microbiota will provide a better insight into the molecular basis that underlies complicated interaction among host-pathogen-symbiont. In this review, we highlighted recent findings that help us broaden our knowledge of the intestinal ecosystem and thereby come up with a better strategy for combating enteropathogenic infection.
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Infecções Bacterianas/imunologia , Trato Gastrointestinal/microbiologia , Enteropatias/imunologia , Enteropatias/microbiologia , Microbiota , Simbiose , Animais , Bactérias/classificação , Bactérias/imunologia , Bactérias/metabolismo , Trato Gastrointestinal/fisiopatologia , Homeostase , Humanos , Imunidade Inata , Simbiose/imunologia , Simbiose/fisiologiaRESUMO
Two closely related bacterial species, Segniliparus rotundus and Segniliparus rugosus, have emerged as important human pathogens, but little is known about the immune responses they elicit or their comparative pathophysiologies. To determine the virulence and immune responses of the two species, we compared their abilities to grow in phagocytic and non-phagocytic cells. Both species maintained non-replicating states within A549 epithelial cells. S. rugosus persisted longer and multiplied more rapidly inside murine bone marrow-derived macrophages (BMDMs), induced more pro-inflammatory cytokines, and induced higher levels of macrophage necrosis. Activation of BMDMs by both species was mediated by toll-like receptor 2 (TLR2), followed by mitogen-activated protein kinases (MAPK) and nuclear factor κB (NF-κB) signaling pathways, indicating a critical role for TLR2 in Segniliparus-induced macrophage activation. S. rugosus triggered faster and stronger activation of MAPK signaling and IκB degradation, indicating that S. rugosus induces more pro-inflammatory cytokines than S. rotundus. Multifocal granulomatous inflammations in the liver and lung were observed in mice infected with S. rugosus, but S. rotundus was rapidly cleared from all organs tested within 15 days post-infection. Furthermore, S. rugosus induced faster infiltration of innate immune cells such as neutrophils and macrophages to the lung than S. rotundus. Our results suggest that S. rugosus is more virulent and induces a stronger immune response than S. rotundus.
Assuntos
Infecções por Actinomycetales/imunologia , Actinomycetales/patogenicidade , Actinomycetales/crescimento & desenvolvimento , Actinomycetales/fisiologia , Infecções por Actinomycetales/metabolismo , Infecções por Actinomycetales/patologia , Animais , Células da Medula Óssea/citologia , Morte Celular/imunologia , Citocinas/biossíntese , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Imunidade Celular , Imunidade Humoral , Espaço Intracelular/microbiologia , Cinética , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/microbiologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Fenótipo , Fosforilação/imunologia , Transdução de Sinais/imunologia , Especificidade da Espécie , Receptor 2 Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The failure of Mycobacterium bovis BCG as a TB vaccine against TB reactivation suggests that latency-associated proteins should be included in alternative TB vaccine development. Further, antigens known to generate protective immunity against the strong Th1 stimulatory response to reactivated TB should be included in novel vaccine design. Recent studies have emphasized the importance of Rpfs from Mycobacterium tuberculosis in the reactivation process and cellular immunity. However, little is known about how RpfB mediates protective immunity against M. tuberculosis. Here, we investigated the functional roles and signaling mechanisms of RpfB in DCs and its implications in the development of T cell immunity. DCs treated with RpfB displayed features of mature and functional status, with elevated expression of cell surface molecules (CD80, CD86, and MHC class I and II) and proinflammatory cytokine production (TNF-α, IL-1ß, IL-6, and IL-12p70). Activation of DCs was mediated by direct binding of RpfB to TLR4, followed by MyD88/TRIF-dependent signaling to MAPKs and NF-κB signaling pathways. Specifically, we found that the RpfB G5 domain is the most important part in RpfB binding to TLR4. RpfB-treated DCs effectively polarized naïve CD4(+) and CD8(+) T cells to secrete IFN-γ and IL-2. Importantly, RpfB induced the expansion of memory CD4(+)/CD8(+)CD44(high)CD62L(low) T cells in the spleen of M. tuberculosis-infected mice. Our data suggest that RpfB regulates innate immunity and activates adaptive immunity through TLR4, a finding that may help in the design of more effective vaccines.
Assuntos
Proteínas de Bactérias/metabolismo , Células Dendríticas/imunologia , Imunidade Celular/imunologia , Mycobacterium tuberculosis/imunologia , Células Th1/imunologia , Receptor 4 Toll-Like/metabolismo , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/metabolismo , Morte Celular , Diferenciação Celular/imunologia , Citocinas/metabolismo , Células Dendríticas/citologia , Endotoxinas/metabolismo , Ativação Enzimática , Feminino , Memória Imunológica , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Testes de Neutralização , Fenótipo , Ligação Proteica , Reprodutibilidade dos Testes , Deleção de Sequência/genética , Transdução de Sinais/imunologia , Células Th1/citologia , Receptor 2 Toll-Like/metabolismoRESUMO
Biofilms, communities of micro-organisms attached to a surface, are responsible for many chronic diseases and are often associated with environmental reservoirs or lifestyles. Bacillus anthracis is a Gram-positive, endospore-forming bacterium and is the aetiological agent of pulmonary, gastrointestinal and cutaneous anthrax. Anthrax infections are part of the natural lifecycle of many ruminants in North America, including cattle and bison, and B. anthracis is thought to be a central part of this ecosystem. However, in endemic areas in which humans and livestock interact, chronic cases of cutaneous anthrax are commonly reported. This suggests that biofilms of B. anthracis exist in the environment and are part of the ecology associated with its lifecycle. Currently, there are few data that account for the importance of the biofilm mode of life in B. anthracis, yet biofilms have been characterized in other pathogenic and non-pathogenic Bacillus species, including Bacillus cereus and Bacillus subtilis, respectively. This study investigated the phenotypic and functional role of biofilms in B. anthracis. The results demonstrate that B. anthracis readily forms biofilms which are inherently resistant to commonly prescribed antibiotics, and that antibiotic resistance is not solely the function of sporulation.