RESUMO
Pseudomonas aeruginosa persisters are a rare and poorly characterized subpopulation of cells that are responsible for many recurrent infections. The lack of knowledge on the mechanisms that lead to persister cell development is mainly a result of the difficulty in isolating and characterizing this rare population. Flow cytometry is an ideal method for identifying such subpopulations because it allows for high-content single-cell analysis. However, there are fewer established protocols for bacterial flow cytometry compared to mammalian cell work. Herein, we describe and propose a flow cytometry protocol to identify and isolate P. aeruginosa persister cells. Additionally, we show that the percentage of potential persister cells increases with increasing antibiotic concentrations above the MIC.
Assuntos
Antibacterianos , Pseudomonas aeruginosa , Testes de Sensibilidade Microbiana , Citometria de Fluxo , Antibacterianos/farmacologiaRESUMO
Pseudomonas aeruginosa is an opportunistic pathogen that is a common cause of nosocomial infections. The molecular mechanisms governing immune responses to P. aeruginosa infection remain incompletely defined. Early growth response 1 (Egr-1) is a zinc-finger transcription factor that controls inflammatory responses. Here, we characterized the role of Egr-1 in host defense against P. aeruginosa infection in a mouse model of acute bacterial pneumonia. Egr-1 expression was rapidly and transiently induced in response to P. aeruginosa infection. Egr-1-deficient mice displayed decreased mortality, reduced levels of proinflammatory cytokines (tumor necrosis factor [TNF], interleukin-1ß [IL-1ß], IL-6, IL-12, and IL-17), and enhanced bacterial clearance from the lung. Egr-1 deficiency caused diminished NF-κB activation in P. aeruginosa-infected macrophages independently of IκBα phosphorylation. A physical interaction between Egr-1 and NF-κB p65 was found in P. aeruginosa-infected macrophages, suggesting that Egr-1 could be required for assembly of heterodimeric transcription factors that direct synthesis of inflammatory mediators. Interestingly, Egr-1 deficiency had no impact on neutrophil recruitment in vivo due to its differential effects on chemokine production, which included diminished accumulation of KC (CXCL1), MIP2 (CXCL2), and IP-10 (CXCL10) and increased accumulation of LIX (CXCL5). Importantly, Egr-1-deficient macrophages and neutrophils displayed significant increases in nitric oxide production and bacterial killing ability that correlated with enhanced bacterial clearance in Egr-1-deficient mice. Together, these findings suggest that Egr-1 plays a detrimental role in host defense against P. aeruginosa acute lung infection by promoting systemic inflammation and negatively regulating the nitric oxide production that normally assists with bacterial clearance.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Pneumonia Bacteriana/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Animais , Citocinas/análise , Modelos Animais de Doenças , Proteína 1 de Resposta de Crescimento Precoce/deficiência , Fatores Imunológicos/análise , Camundongos , Análise de SobrevidaRESUMO
Pseudomonas aeruginosa biofilms are typically associated with the chronic lung infection of cystic fibrosis (CF) patients and represent a major challenge for treatment. This opportunistic bacterial pathogen secretes alginate, a polysaccharide that is one of the main components of its biofilm. Targeting this major biofilm component has emerged as a tempting therapeutic strategy for tackling biofilm-associated bacterial infections. The enormous potential in genetic diversity of the marine microbial community make it a valuable resource for mining activities responsible for a broad range of metabolic processes, including the alginolytic activity responsible for degrading alginate. A collection of 36 bacterial isolates were purified from marine water based on their alginolytic activity. These isolates were identified based on their 16S rRNA gene sequences. Pseudoalteromonas sp. 1400 showed the highest alginolytic activity and was further confirmed to produce the enzyme alginate lyase. The purified alginate lyase (AlyP1400) produced by Pseudoalteromonas sp. 1400 showed a band of 23 KDa on a protein electrophoresis gel and exhibited a bifunctional lyase activity for both poly-mannuronic acid and poly-glucuronic acid degradation. A tryptic digestion of this gel band analyzed by liquid chromatography-tandem mass spectrometry confirmed high similarity to the alginate lyases in polysaccharide lyase family 18. The purified alginate lyase showed a maximum relative activity at 30 °C at a slightly acidic condition. It decreased the sodium alginate viscosity by over 90% and reduced the P. aeruginosa (strain PA14) biofilms by 69% after 24 h of incubation. The combined activity of AlyP1400 with carbenicillin or ciprofloxacin reduced the P. aeruginosa biofilm thickness, biovolume and surface area in a flow cell system. The present data revealed that AlyP1400 combined with conventional antibiotics helped to disrupt the biofilms produced by P. aeruginosa and can be used as a promising combinational therapeutic strategy.
Assuntos
Biofilmes/efeitos dos fármacos , Polissacarídeo-Liases/farmacologia , Pseudoalteromonas/enzimologia , Pseudomonas aeruginosa/efeitos dos fármacos , Alginatos/metabolismo , Antibacterianos/farmacologia , Organismos Aquáticos/enzimologia , Organismos Aquáticos/genética , Carbenicilina/farmacologia , Ciprofloxacina/farmacologia , Polissacarídeo-Liases/genética , Polissacarídeo-Liases/metabolismo , Pseudoalteromonas/genética , Pseudomonas aeruginosa/fisiologia , RNA Ribossômico 16S/genéticaRESUMO
Shigella flexneri is an intracellular bacterium that hijacks the host actin cytoskeleton to invade and disseminate within the colonic epithelium. Shigella's virulence factors induce actin polymerization, leading to bacterial uptake, actin tail formation, actin-mediated motility, and cell-to-cell spreading. Many host factors involved in the Shigella-prompted actin rearrangements remain elusive. Here, we studied the role of a host protein receptor for activated C kinase 1 (RACK1) in actin cytoskeleton dynamics and Shigella infection. We used time-lapse imaging to demonstrate that RACK1 facilitates Shigella-induced actin cytoskeleton remodeling at multiple levels during infection of epithelial cells. Silencing RACK1 expression impaired Shigella-induced rapid polymerizing structures, reducing host cell invasion, bacterial motility, and cell-to-cell spreading. In uninfected cells, RACK1 silencing reduced jasplakinolide-mediated filamentous actin aggregate formation and negatively affected actin turnover in fast polymerizing structures, such as membrane ruffles. Our findings provide a role of RACK1 in actin cytoskeleton dynamics and Shigella infection.
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Globally, infectious diseases are one of the leading causes of death among people of all ages. The development of antimicrobials to treat infectious diseases has been one of the most significant advances in medical history. Alarmingly, antimicrobial resistance is a widespread phenomenon that will, without intervention, make currently treatable infections once again deadly. In an era of widespread antimicrobial resistance, there is a constant and pressing need to develop new antibacterial drugs. Unraveling the underlying resistance mechanisms is critical to fight this crisis. In this review, we summarize some emerging evidence of the non-canonical intracellular life cycle of two priority antimicrobial-resistant bacterial pathogens: Pseudomonas aeruginosa and Staphylococcus aureus. The bacterial factors that modulate this unique intracellular niche and its implications in contributing to resistance are discussed. We then briefly discuss some recent research that focused on the promises of boosting host immunity as a combination therapy with antimicrobials to eradicate these two particular pathogens. Finally, we summarize the importance of various strategies, including surveillance and vaccines, in mitigating the impacts of antimicrobial resistance in general.
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Biofilm infections are hard to manage using conventional antibiotic treatment regimens because biofilm structures discourage antibiotics from reaching the entire bacterial community and allow pathogen cells to persistently colonize and develop a plethora of tolerance mechanisms towards antibiotics. Moreover, the dispersed cells from biofilms can cause further complications by colonizing different sites and establishing new cycles of biofilms. Previously, we showed that alginate lyase enzyme (AlyP1400), purified from a marine Pseudoalteromonas bacterium, reduced Pseudomonas aeruginosa biofilm biomass and boosted bactericidal activity of tobramycin by degrading alginate within the biofilm extracellular polymeric substances matrix. In this work, we used a flow cytometry-based assay to analyze collected dispersal cells and demonstrated the synergy between tobramycin with AlyP1400 in enhancing the release of both live and dead biofilm cells from a mucoid P. aeruginosa strain CF27, which is a clinical isolate from cystic fibrosis (CF) patients. Interestingly, this enhanced dispersal was only observed when AlyP1400 was combined with tobramycin and administered simultaneously but not when AlyP1400 was added in advance of tobramycin in a sequential manner. Moreover, neither the combined nor sequential treatment altered the dispersal of the biofilms from a non-mucoid P. aeruginosa laboratory strain PAK. We then carried out the gene expression and tobramycin survival analyses to further characterize the impacts of the combined treatment on the CF27 dispersal cells. Gene expression analysis indicated that CF27 dispersal cells had increased expression in virulence- and antibiotic resistance-related genes, including algR, bdlA, lasB, mexF, mexY, and ndvB. In the CF27 dispersal cell population, the combinational treatment of AlyP1400 with tobramycin further induced bdlA, mexF, mexY, and ndvB genes more than non-treated and tobramycin-treated dispersal cells, suggesting an exacerbated bacterial stress response to the combinational treatment. Simultaneous to the gene expression analysis, the survival ability of the same batch of biofilm dispersal cells to a subsequent tobramycin challenge displayed a significantly higher tobramycin tolerant fraction of cells (~60%) upon the combinational treatment of AlyP1400 and tobramycin than non-treated and tobramycin-treated dispersal cells, as well as the planktonic cells (all below 10%). These results generate new knowledge about the gene expression and antibiotic resistance profiles of dispersed cells from biofilm. This information can guide the design of safer and more efficient therapeutic strategies for the combinational use of alginate lyase and tobramycin to treat P. aeruginosa biofilm-related infections in CF lungs.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Polissacarídeo-Liases/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Tobramicina/farmacologia , Citometria de FluxoRESUMO
The rhizosphere microbiome is composed of diverse microbial organisms, including archaea, viruses, fungi, bacteria as well as eukaryotic microorganisms, which occupy a narrow region of soil directly associated with plant roots. The interactions between these microorganisms and the plant can be commensal, beneficial or pathogenic. These microorganisms can also interact with each other, either competitively or synergistically. Promoting plant growth by harnessing the soil microbiome holds tremendous potential for providing an environmentally friendly solution to the increasing food demands of the world's rapidly growing population, while also helping to alleviate the associated environmental and societal issues of large-scale food production. There recently have been many studies on the disease suppression and plant growth promoting abilities of the rhizosphere microbiome; however, these findings largely have not been translated into the field. Therefore, additional research into the dynamic interactions between crop plants, the rhizosphere microbiome and the environment are necessary to better guide the harnessing of the microbiome to increase crop yield and quality. This review explores the biotic and abiotic interactions that occur within the plant's rhizosphere as well as current agricultural practices, and how these biotic and abiotic factors, as well as human practices, impact the plant microbiome. Additionally, some limitations, safety considerations, and future directions to the study of the plant microbiome are discussed.
Assuntos
Agricultura/métodos , Bactérias/metabolismo , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/microbiologia , Fungos/fisiologia , Microbiota , Bactérias/genética , Fungos/genética , Humanos , Raízes de Plantas/microbiologia , Microbiologia do Solo , SimbioseRESUMO
Pseudomonas aeruginosa is an opportunistic pathogen that is a leading cause of morbidity and mortality in cystic fibrosis patients and immunocompromised individuals. Eradication of P. aeruginosa has become increasingly difficult due to its remarkable capacity to resist antibiotics. Strains of Pseudomonas aeruginosa are known to utilize their high levels of intrinsic and acquired resistance mechanisms to counter most antibiotics. In addition, adaptive antibiotic resistance of P. aeruginosa is a recently characterized mechanism, which includes biofilm-mediated resistance and formation of multidrug-tolerant persister cells, and is responsible for recalcitrance and relapse of infections. The discovery and development of alternative therapeutic strategies that present novel avenues against P. aeruginosa infections are increasingly demanded and gaining more and more attention. Although mostly at the preclinical stages, many recent studies have reported several innovative therapeutic technologies that have demonstrated pronounced effectiveness in fighting against drug-resistant P. aeruginosa strains. This review highlights the mechanisms of antibiotic resistance in P. aeruginosa and discusses the current state of some novel therapeutic approaches for treatment of P. aeruginosa infections that can be further explored in clinical practice.
Assuntos
Antibacterianos/farmacologia , Resistência Microbiana a Medicamentos/genética , Infecções por Pseudomonas/tratamento farmacológico , Pseudomonas aeruginosa/efeitos dos fármacos , Antibacterianos/efeitos adversos , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Fibrose Cística/tratamento farmacológico , Fibrose Cística/microbiologia , Humanos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidadeRESUMO
Toll-like receptors (TLRs) recognize the conserved molecular patterns in microorganisms and trigger myeloid differentiation primary response 88 (MyD88) and/or TIR-domain-containing adapter-inducing interferon-ß (TRIF) pathways that are critical for host defense against microbial infection. However, the molecular mechanisms that govern TLR signaling remain incompletely understood. Regulator of calcineurin-1 (RCAN1), a small evolutionarily conserved protein that inhibits calcineurin phosphatase activity, suppresses inflammation during Pseudomonas aeruginosa infection. Here, we define the roles for RCAN1 in P. aeruginosa lipopolysaccharide (LPS)-activated TLR4 signaling. We compared the effects of P. aeruginosa LPS challenge on bone marrow-derived macrophages from both wild-type and RCAN1-deficient mice and found that RCAN1 deficiency increased the MyD88-NF-κB-mediated cytokine production (IL-6, TNF and MIP-2), whereas TRIF-interferon-stimulated response elements (ISRE)-mediated cytokine production (IFNß, RANTES and IP-10) was suppressed. RCAN1 deficiency caused increased IκBα phosphorylation and NF-κB activity in the MyD88-dependent pathway, but impaired ISRE activation and reduced IRF7 expression in the TRIF-dependent pathway. Complementary studies of a mouse model of P. aeruginosa LPS-induced acute pneumonia confirmed that RCAN1-deficient mice displayed greatly enhanced NF-κB activity and MyD88-NF-κB-mediated cytokine production, which correlated with enhanced pulmonary infiltration of neutrophils. By contrast, RCAN1 deficiency had little effect on the TRIF pathway in vivo. These findings demonstrate a novel regulatory role of RCAN1 in TLR signaling, which differentially regulates MyD88 and TRIF pathways.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Musculares/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Transdução de Sinais , Receptores Toll-Like/metabolismo , Animais , Proteínas de Ligação ao Cálcio , Quimiocinas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Musculares/deficiência , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/metabolismo , Infiltração de Neutrófilos/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Elementos de Resposta/genética , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Increasing anthropogenic activities are creating environmental pressures that threaten marine ecosystems. Effective environmental health assessment requires the development of rapid, sensitive, and cost-effective tools to predict negative impacts at the individual and ecosystem levels. To this end, a number of biological assays using a variety of cells and organisms measuring different end points have been developed for biomonitoring programs. The sea urchin fertilization/development test has been useful for evaluating environmental toxicology and it has been proposed that sea urchin coelomocytes represent a novel cellular biosensor of environmental stress. In this study we investigated the sensitivity of coelomocytes from the sea urchin Lytechinus variegatus to a variety of DNA-damaging agents including ultraviolet (UV) radiation, hydrogen peroxide (H(2)O(2)), methylmethane sulfonate (MMS) and benzo[a]pyrene (BaP). LD(50) values determined for coelomocytes after 24h of exposure to these DNA damaging agents indicated a high level of resistance to all treatments. Significant increases in the formation of apurinic/apyrimidinic (AP or abasic) sites in DNA were only detected using high doses of H(2)O(2), MMS and UV radiation. Comparison of sea urchin coelomocytes with hemocytes from the gastropod mollusk Aplysia dactylomela and the decapod crustacean Panulirus argus indicated that sensitivity to different DNA damaging agents varies between species. The high level of resistance to genotoxic agents suggests that DNA damage may not be an informative end point for environmental health assessment using sea urchin coelomocytes however, natural resistance to DNA damaging agents may have implications for the occurrence of neoplastic disease in these animals.