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1.
Int J Mol Sci ; 23(8)2022 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-35457206

RESUMO

Bacterial biofilm represents a multicellular community embedded within an extracellular matrix attached to a surface. This lifestyle confers to bacterial cells protection against hostile environments, such as antibiotic treatment and host immune response in case of infections. The Pseudomonas genus is characterised by species producing strong biofilms difficult to be eradicated and by an extraordinary metabolic versatility which may support energy and carbon/nitrogen assimilation under multiple environmental conditions. Nutrient availability can be perceived by a Pseudomonas biofilm which, in turn, readapts its metabolism to finally tune its own formation and dispersion. A growing number of papers is now focusing on the mechanism of nutrient perception as a possible strategy to weaken the biofilm barrier by environmental cues. One of the most important nutrients is amino acid L-arginine, a crucial metabolite sustaining bacterial growth both as a carbon and a nitrogen source. Under low-oxygen conditions, L-arginine may also serve for ATP production, thus allowing bacteria to survive in anaerobic environments. L-arginine has been associated with biofilms, virulence, and antibiotic resistance. L-arginine is also a key precursor of regulatory molecules such as polyamines, whose involvement in biofilm homeostasis is reported. Given the biomedical and biotechnological relevance of biofilm control, the state of the art on the effects mediated by the L-arginine nutrient on biofilm modulation is presented, with a special focus on the Pseudomonas biofilm. Possible biotechnological and biomedical applications are also discussed.


Assuntos
GMP Cíclico , Pseudomonas aeruginosa , Arginina/metabolismo , Arginina/farmacologia , Proteínas de Bactérias/metabolismo , Biofilmes , Carbono/metabolismo , Carbono/farmacologia , GMP Cíclico/metabolismo , Nitrogênio/metabolismo , Nitrogênio/farmacologia , Nutrientes , Pseudomonas/metabolismo , Pseudomonas aeruginosa/fisiologia
2.
Elife ; 112022 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-35438634

RESUMO

Biofilm formation is one of most important causes leading to persistent infections. Exopolysaccharides are usually a main component of biofilm matrix. Genes encoding glycosyl hydrolases are often found in gene clusters that are involved in the exopolysaccharide synthesis. It remains elusive about the functions of intracellular glycosyl hydrolase and why a polysaccharide synthesis gene cluster requires a glycosyl hydrolase-encoding gene. Here, we systematically studied the physiologically relevant role of intracellular PslG, a glycosyl hydrolase whose encoding gene is co-transcribed with 15 psl genes, which is responsible for the synthesis of exopolysaccharide PSL, a key biofilm matrix polysaccharide in opportunistic pathogen Pseudomonas aeruginosa. We showed that lack of PslG or its hydrolytic activity in this opportunistic pathogen enhances the signaling function of PSL, changes the relative level of cyclic-di-GMP within daughter cells during cell division and shapes the localization of PSL on bacterial periphery, thus results in long chains of bacterial cells, fast-forming biofilm microcolonies. Our results reveal the important roles of intracellular PslG on the cell fate and biofilm development.


Assuntos
Biofilmes , Pseudomonas aeruginosa , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , GMP Cíclico , Regulação Bacteriana da Expressão Gênica , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Polissacarídeos , Pseudomonas aeruginosa/fisiologia
3.
Front Cell Infect Microbiol ; 12: 851784, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35295755

RESUMO

Biofilm-associated bacteria exhibit profound changes in bacterial physiology. They thrive in the environment but also in the human host in protected sessile communities. Antimicrobial therapy usually fails, despite the absence of genotypic resistance, and it is commonly accepted that biofilm-grown bacteria are up to 1,000-fold more resistant than planktonic cells. We are only at the beginning to understand the reasons for biofilm recalcitrance, and systematic approaches to describe biofilm-induced tolerance phenotypes are lacking. In this study, we investigated a large and highly diverse collection of 352 clinical Pseudomonas aeruginosa isolates for their antimicrobial susceptibility profiles under biofilm growth conditions towards the antibiotics ciprofloxacin, tobramycin, and colistin. We discovered characteristic patterns of drug-specific killing activity and detected conditional tolerance levels far lower (in the range of the minimal inhibitory concentration (MIC)), but also far higher (up to 16,000-fold increase compared to planktonic cells) than generally believed. This extremely broad distribution of biofilm-induced tolerance phenotypes across the clinical isolates was greatly influenced by the choice of the antibiotic. We furthermore describe cross-tolerance against ciprofloxacin and tobramycin, but not colistin, and observed an additive activity between biofilm-induced tolerance and genetically determined resistance. This became less evident when the biofilm-grown cells were exposed to very high antibiotic concentrations. Although much more remains to be learned on the molecular mechanisms underlying biofilm-induced tolerance, our data on intra-species variations in tolerance profiles provide valuable new insights. Furthermore, our observation that colistin appears to act independently of the tolerance mechanisms of individual clinical strains could make colistin a valuable therapeutic option in chronic biofilm-associated infections characterized by the presence of particularly tolerant strains.


Assuntos
Infecções por Pseudomonas , Pseudomonas aeruginosa , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Biofilmes , Humanos , Testes de Sensibilidade Microbiana , Plâncton , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/fisiologia , Tobramicina/farmacologia
4.
Immunol Cell Biol ; 100(5): 352-370, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35318736

RESUMO

A population of neutrophils recruited into cystic fibrosis (CF) airways is associated with proteolytic lung damage, exhibiting high expression of primary granule exocytosis marker CD63 and reduced phagocytic receptor CD16. Causative factors for this population are unknown, limiting intervention. Here we present a laboratory model to characterize responses of differentiated airway epithelium and neutrophils following respiratory infection. Pediatric primary airway epithelial cells were cultured at the air-liquid interface, challenged individually or in combination with rhinovirus (RV) and Pseudomonas aeruginosa, then apically washed with medical saline to sample epithelial infection milieus. Cytokine multiplex analysis revealed epithelial antiviral signals, including IP-10 and RANTES, increased with exclusive RV infection but were diminished if P. aeruginosa was also present. Proinflammatory signals interleukin-1α and ß were dominant in P. aeruginosa infection milieus. Infection washes were also applied to a published model of neutrophil transmigration into the airways. Neutrophils migrating into bacterial and viral-bacterial co-infection milieus exhibited the in vivo CF phenotype of increased CD63 expression and reduced CD16 expression, while neutrophils migrating into milieus of RV-infected or uninfected cultures did not. Individually, bacterial products lipopolysaccharide and N-formylmethionyl-leucyl-phenylalanine and isolated cytokine signals only partially activated this phenotype, suggesting that additional soluble factors in the infection microenvironment trigger primary granule release. Findings identify P. aeruginosa as a trigger of acute airway inflammation and neutrophil primary granule exocytosis, underscoring potential roles of airway microbes in prompting this neutrophil subset. Further studies are required to characterize microbes implicated in primary granule release, and identify potential therapeutic targets.


Assuntos
Fibrose Cística , Infecções por Pseudomonas , Citocinas/metabolismo , Exocitose , Humanos , Neutrófilos/metabolismo , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/fisiologia
5.
Front Immunol ; 13: 840272, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35273616

RESUMO

Mitochondria are central players in host immunometabolism as they function not only as metabolic hubs but also as signaling platforms regulating innate immunity. Environmental exposures to mitochondrial toxicants occur widely and are increasingly frequent. Exposures to these mitotoxicants may pose a serious threat to organismal health and the onset of diseases by disrupting immunometabolic pathways. In this study, we investigated whether the Complex I inhibitor rotenone could alter C. elegans immunometabolism and disease susceptibility. C. elegans embryos were exposed to rotenone (0.5 µM) or DMSO (0.125%) until they reached the L4 larval stage. Inhibition of mitochondrial respiration by rotenone and disruption of mitochondrial metabolism were evidenced by rotenone-induced detrimental effects on mitochondrial efficiency and nematode growth and development. Next, through transcriptomic analysis, we investigated if this specific but mild mitochondrial stress that we detected would lead to the modulation of immunometabolic pathways. We found 179 differentially expressed genes (DEG), which were mostly involved in detoxification, energy metabolism, and pathogen defense. Interestingly, among the down-regulated DEG, most of the known genes were involved in immune defense, and most of these were identified as commonly upregulated during P. aeruginosa infection. Furthermore, rotenone increased susceptibility to the pathogen Pseudomonas aeruginosa (PA14). However, it increased resistance to Salmonella enterica (SL1344). To shed light on potential mechanisms related to these divergent effects on pathogen resistance, we assessed the activation of the mitochondrial unfolded protein response (UPRmt), a well-known immunometabolic pathway in C. elegans which links mitochondria and immunity and provides resistance to pathogen infection. The UPRmt pathway was activated in rotenone-treated nematodes further exposed for 24 h to the pathogenic bacteria P. aeruginosa and S. enterica or the common bacterial food source Escherichia coli (OP50). However, P. aeruginosa alone suppressed UPRmt activation and rotenone treatment rescued its activation only to the level of DMSO-exposed nematodes fed with E. coli. Module-weighted annotation bioinformatics analysis was also consistent with UPRmt activation in rotenone-exposed nematodes consistent with the UPR being involved in the increased resistance to S. enterica. Together, our results demonstrate that the mitotoxicant rotenone can disrupt C. elegans immunometabolism in ways likely protective against some pathogen species but sensitizing against others.


Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/metabolismo , Dimetil Sulfóxido/metabolismo , Escherichia coli/metabolismo , Pseudomonas aeruginosa/fisiologia , Rotenona/toxicidade
6.
Molecules ; 27(5)2022 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-35268822

RESUMO

Natural products derived from marine sponges have exhibited bioactivity and, in some cases, serve as potent quorum sensing inhibitory agents that prevent biofilm formation and attenuate virulence factor expression by pathogenic microorganisms. In this study, the inhibitory activity of the psammaplin-type compounds, psammaplin A (1) and bisaprasin (2), isolated from the marine sponge, Aplysinellarhax, are evaluated in quorum sensing inhibitory assays based on the Pseudomonas aeruginosa PAO1 lasB-gfp(ASV) and rhlA-gfp(ASV) biosensor strains. The results indicate that psammaplin A (1) showed moderate inhibition on lasB-gfp expression, but significantly inhibited the QS-gene promoter, rhlA-gfp, with IC50 values at 14.02 µM and 4.99 µM, respectively. In contrast, bisaprasin (2) displayed significant florescence inhibition in both biosensors, PAO1 lasB-gfp and rhlA-gfp, with IC50 values at 3.53 µM and 2.41 µM, respectively. Preliminary analysis suggested the importance of the bromotyrosine and oxime functionalities for QSI activity in these molecules. In addition, psammaplin A and bisaprasin downregulated elastase expression as determined by the standard enzymatic elastase assay, although greater reduction in elastase production was observed with 1 at 50 µM and 100 µM. Furthermore, the study revealed that bisaprasin (2) reduced biofilm formation in P. aeruginosa.


Assuntos
Pseudomonas aeruginosa , Percepção de Quorum , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Biofilmes , Dissulfetos , Elastase Pancreática , Pseudomonas aeruginosa/fisiologia , Tirosina/análogos & derivados , Fatores de Virulência/metabolismo
7.
Cells ; 11(5)2022 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-35269409

RESUMO

DNA methyltransferase 3b (Dnmt3b) has been suggested to play a role in the host immune response during bacterial infection. Neutrophils and other myeloid cells are crucial for lung defense against Pseudomonas (P.) aeruginosa infection. This study aimed to investigate the role of Dnmt3b in neutrophils and myeloid cells during acute pneumonia caused by P. aeruginosa. Neutrophil-specific (Dnmt3bfl/flMrp8Cre) or myeloid cell-specific (Dnmt3bfl/flLysMCre) Dnmt3b-deficient mice and littermate control mice were infected with P. aeruginosa PAK via the airways. Bacteria burdens, neutrophil recruitment, and activation (CD11b expression, myeloperoxidase, and elastase levels), interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF) were measured in bronchoalveolar lavage fluid (BALF) at 6 and 24 h after infection. Our data showed that the bacterial loads and neutrophil recruitment and activation did not differ in BALF obtained from neutrophil-specific Dnmt3b-deficient and control mice, whilst BALF IL-6 and TNF levels were lower in the former group at 24 but not at 6 h after infection. None of the host response parameters measured differed between myeloid cell-specific Dnmt3b-deficient and control mice. In conclusion, dnmt3b deficiency in neutrophils or myeloid cells does not affect acute immune responses in the airways during Pseudomonas pneumonia.


Assuntos
Pneumonia , Infecções por Pseudomonas , Animais , DNA (Citosina-5-)-Metiltransferases , Imunidade , Interleucina-6/metabolismo , Camundongos , Neutrófilos/metabolismo , Pneumonia/patologia , Pseudomonas , Pseudomonas aeruginosa/fisiologia
8.
Infect Immun ; 90(3): e0047021, 2022 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-35130452

RESUMO

Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that causes nosocomial pneumonia, urinary tract infections, and bacteremia. A hallmark of P. aeruginosa pathogenesis is disruption of host cell function by the type III secretion system (T3SS) and its cognate exoenzyme effectors. The T3SS effector ExoU is phospholipase A2 (PLA2) that targets the host cell plasmalemmal membrane to induce cytolysis and is an important virulence factor that mediates immune avoidance. In addition, ExoU has been shown to subvert the host inflammatory response in a noncytolytic manner. In primary bone marrow-derived macrophages (BMDMs), P. aeruginosa infection is sensed by the nucleotide-binding domain containing leucine-rich repeats-like receptor 4 (NLRC4) inflammasome, which triggers caspase-1 activation and inflammation. ExoU transiently inhibits NLRC4 inflammasome-mediated activation of caspase-1 and its downstream target, interleukin 1ß (IL-1ß), to suppress activation of inflammation. In the present study, we sought to identify additional noncytolytic virulence functions for ExoU and discovered an unexpected association between ExoU, host mitochondria, and NLRC4. We show that infection of BMDMs with P. aeruginosa strains expressing ExoU elicited mitochondrial oxidative stress. In addition, mitochondria and mitochondrion-associated membrane fractions enriched from infected cells exhibited evidence of autophagy activation, indicative of damage. The observation that ExoU elicited mitochondrial stress and damage suggested that ExoU may also associate with mitochondria during infection. Indeed, ExoU phospholipase A2 enzymatic activity was present in enriched mitochondria and mitochondrion-associated membrane fractions isolated from P. aeruginosa-infected BMDMs. Intriguingly, enriched mitochondria and mitochondrion-associated membrane fractions isolated from infected Nlrc4 homozygous knockout BMDMs displayed significantly lower levels of ExoU enzyme activity, suggesting that NLRC4 plays a role in the ExoU-mitochondrion association. These observations prompted us to assay enriched mitochondria and mitochondrion-associated membrane fractions for NLRC4, caspase-1, and IL-1ß. NLRC4 and pro-caspase-1 were detected in enriched mitochondria and mitochondrion-associated membrane fractions isolated from noninfected BMDMs, and active caspase-1 and active IL-1ß were detected in response to P. aeruginosa infection. Interestingly, ExoU inhibited mitochondrion-associated caspase-1 and IL-1ß activation. The implications of ExoU-mediated effects on mitochondria and the NLRC4 inflammasome during P. aeruginosa infection are discussed.


Assuntos
Infecções por Pseudomonas , Pseudomonas aeruginosa , Animais , Caspase 1/metabolismo , Inflamassomos/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Camundongos , Fosfolipases/metabolismo , Pseudomonas aeruginosa/fisiologia , Sistemas de Secreção Tipo III/metabolismo
9.
Nat Commun ; 13(1): 721, 2022 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-35132084

RESUMO

Much of our understanding of bacterial behavior stems from studies in liquid culture. In nature, however, bacteria frequently live in densely packed spatially-structured communities. How does spatial structure affect bacterial cooperative behaviors? In this work, we examine rhamnolipid production-a cooperative and virulent behavior of Pseudomonas aeruginosa. Here we show that, in striking contrast to well-mixed liquid culture, rhamnolipid gene expression in spatially-structured colonies is strongly associated with colony specific growth rate, and is impacted by perturbation with diffusible quorum signals. To interpret these findings, we construct a data-driven statistical inference model which captures a length-scale of bacterial interaction that develops over time. Finally, we find that perturbation of P. aeruginosa swarms with quorum signals preserves the cooperating genotype in competition, rather than creating opportunities for cheaters. Overall, our data demonstrate that the complex response to spatial localization is key to preserving bacterial cooperative behaviors.


Assuntos
Interações Microbianas/fisiologia , Modelos Biológicos , Proteínas de Bactérias/genética , Biomassa , Contagem de Colônia Microbiana , Regulação Bacteriana da Expressão Gênica , Glicolipídeos/genética , Glicolipídeos/metabolismo , Locomoção , Interações Microbianas/genética , Mutação , Imagem Óptica , Regiões Promotoras Genéticas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/fisiologia , Percepção de Quorum , Análise Espaço-Temporal
10.
PLoS One ; 17(2): e0263887, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35196336

RESUMO

Antibiotic resistant bacterial pathogens are increasingly prevalent, driving the need for alternative approaches to chemical antibiotics when treating infections. One such approach is bacteriophage therapy: the use of bacteria-specific viruses that lyse (kill) their host cells. Just as the effect of environmental conditions (e.g. elevated temperature) on antibiotic efficacy is well-studied, the effect of environmental stressors on the potency of phage therapy candidates demands examination. Therapeutic phage OMKO1 infects and kills the opportunistic human pathogen Pseudomonas aeruginosa. Here, we used phage OMKO1 as a model to test how environmental stressors can lead to damage and decay of virus particles. We assessed the effects of elevated temperatures, saline concentrations, and urea concentrations. We observed that OMKO1 particles were highly tolerant to different saline concentrations, but decayed more rapidly at elevated temperatures and under high concentrations of urea. Additionally, we found that exposure to elevated temperature reduced the ability of surviving phage particles to suppress the growth of P. aeruginosa, suggesting a temperature-induced damage. Our findings demonstrate that OMKO1 is highly tolerant to a range of conditions that could be experienced inside and outside the human body, while also showing the need for careful characterization of therapeutic phages to ensure that environmental exposure does not compromise their expected potency, dosing, and pharmacokinetics.


Assuntos
Bacteriófagos/patogenicidade , Pseudomonas aeruginosa/virologia , Estresse Fisiológico , Bacteriófagos/fisiologia , Interações Hospedeiro-Patógeno , Terapia por Fagos , Pseudomonas aeruginosa/fisiologia , Salinidade , Temperatura
11.
J Infect Chemother ; 28(5): 595-601, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35168878

RESUMO

BACKGROUND: Increased expression of efflux pumps is an important mechanism of antibiotic resistance in Pseudomonas aeruginosa, and treatment with inhibitors of active efflux pumps seems an attractive strategy to combat with multidrug resistance. Assays using ethidium bromide (EtBr), which accumulates by binding to nucleic acids, are often employed to assess the efficacy of efflux pump inhibitors (EPIs). However, few studies have reported on assays using other nucleic acid dyes. OBJECTIVE: We used different classes of EPIs for MexAB- or MexXY-OprM to measure the accumulation of various fluorescent dyes, including SYBR Safe, AtlasSight, and GelGreen. METHODS: Escherichia coli MG1655ΔacrBΔtolC strain harboring plasmids carrying the mexAB-oprM (pABM) or mexXY-oprM (pXYM) genes of P. aeruginosa were constructed. Then, the accumulation of the above-mentioned nucleic acid dyes and EtBr was measured to assess the efflux ability in the presence and absence of EPIs (MexAB-OprM-specific inhibitor of pyridopyrimidine derivative [ABI-PP], berberine, non-specific inhibitor of phenylalanine-arginine ß-naphthylamide [PAßN], and protonophore of carbonyl cyanide m-chlorophenyl hydrazone [CCCP]). RESULTS: Decreased accumulations of nucleic acid dyes were observed in strains with pABM or pXYM compared with the parental strain. ABI-PP or berberine addition significantly increased the accumulation of any nucleic acids in the strains with the specific pumps. PAßN or CCCP addition showed increased accumulation of almost all dye in strains with pABM or pXYM. However, the inhibition patterns of EPIs differed according to the nucleic acid dyes used. CONCLUSIONS: Accumulation assays for EPIs were suitable to evaluate EPI candidates using various nucleic acid dyes.


Assuntos
Ácidos Nucleicos , Pseudomonas aeruginosa , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Corantes/metabolismo , Humanos , Proteínas de Membrana Transportadoras/genética , Testes de Sensibilidade Microbiana , Pseudomonas aeruginosa/fisiologia
12.
Proc Natl Acad Sci U S A ; 119(4)2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-35064082

RESUMO

Work on surface sensing in bacterial biofilms has focused on how cells transduce sensory input into cyclic diguanylate (c-di-GMP) signaling, low and high levels of which generally correlate with high-motility planktonic cells and low-motility biofilm cells, respectively. Using Granger causal inference methods, however, we find that single-cell c-di-GMP increases are not sufficient to imply surface commitment. Tracking entire lineages of cells from the progenitor cell onward reveals that c-di-GMP levels can exhibit increases but also undergo oscillations that can propagate across 10 to 20 generations, thereby encoding more complex instructions for community behavior. Principal component and factor analysis of lineage c-di-GMP data shows that surface commitment behavior correlates with three statistically independent composite features, which roughly correspond to mean c-di-GMP levels, c-di-GMP oscillation period, and surface motility. Surface commitment in young biofilms does not correlate to c-di-GMP increases alone but also to the emergence of high-frequency and small-amplitude modulation of elevated c-di-GMP signal along a lineage of cells. Using this framework, we dissect how increasing or decreasing signal transduction from wild-type levels, by varying the interaction strength between PilO, a component of a principal surface sensing appendage system, and SadC, a key hub diguanylate cyclase that synthesizes c-di-GMP, impacts frequency and amplitude modulation of c-di-GMP signals and cooperative surface commitment.


Assuntos
Fenômenos Fisiológicos Bacterianos , GMP Cíclico/análogos & derivados , Transdução de Sinais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , GMP Cíclico/metabolismo , Mutação , Ligação Proteica , Pseudomonas aeruginosa/fisiologia
13.
J Vis Exp ; (179)2022 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-35068473

RESUMO

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of induced swarming motility, specific concentration of an inducer may not be able to report events occurring within the optimal concentration range to elicit the desired responses from a species. Semisolid plates containing multiple concentrations are commonly used to investigate the response within an inducer concentration range. However, separate semisolid plates increase variations in medium viscosity and moisture content within each plate due to nonuniform solidification time. This paper describes a one-step method to simultaneously test surface swarming motility on a single gradient plate, where the isometrically arranged test wells allow the simultaneous acquisition of multiconcentration responses. In the present work, the surface swarming of Escherichia coli K12 and Pseudomonas aeruginosa PAO1 were evaluated in response to a concentration gradient of inducers such as resveratrol and arabinose. Periodically, the swarm morphologies were imaged using an imaging system to capture the entire surface swarming process. The quantitative measurement of the swarm morphologies was acquired using ImageJ software, providing analyzable information of the swarm area. This paper presents a simple gradient swarm plate method that provides qualitative and quantitative information about the inducers' effects on surface swarming, which can be extended to study the effects of other inducers on a broader range of motile bacterial species.


Assuntos
Escherichia coli K12 , Pseudomonas aeruginosa , Bactérias , Proteínas de Bactérias/genética , Meios de Cultura/farmacologia , Pseudomonas aeruginosa/fisiologia
14.
ACS Appl Mater Interfaces ; 14(5): 6307-6319, 2022 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-35099179

RESUMO

Biofilms are central to some of the most urgent global challenges across diverse fields of application, from medicine to industries to the environment, and exert considerable economic and social impact. A fundamental assumption in anti-biofilms has been that the coating on a substrate surface is solid. The invention of slippery liquid-infused porous surfaces─a continuously wet lubricating coating retained on a solid surface by capillary forces─has led to this being challenged. However, in situations where flow occurs, shear stress may deplete the lubricant and affect the anti-biofilm performance. Here, we report on the use of slippery omniphobic covalently attached liquid (SOCAL) surfaces, which provide a surface coating with short (ca. 4 nm) non-cross-linked polydimethylsiloxane (PDMS) chains retaining liquid-surface properties, as an antibiofilm strategy stable under shear stress from flow. This surface reduced biofilm formation of the key biofilm-forming pathogens Staphylococcus epidermidis and Pseudomonas aeruginosa by three-four orders of magnitude compared to the widely used medical implant material PDMS after 7 days under static and dynamic culture conditions. Throughout the entire dynamic culture period of P. aeruginosa, SOCAL significantly outperformed a typical antibiofilm slippery surface [i.e., swollen PDMS in silicone oil (S-PDMS)]. We have revealed that significant oil loss occurred after 2-7 day flow for S-PDMS, which correlated to increased contact angle hysteresis (CAH), indicating a degradation of the slippery surface properties, and biofilm formation, while SOCAL has stable CAH and sustainable antibiofilm performance after 7 day flow. The significance of this correlation is to provide a useful easy-to-measure physical parameter as an indicator for long-term antibiofilm performance. This biofilm-resistant liquid-like solid surface offers a new antibiofilm strategy for applications in medical devices and other areas where biofilm development is problematic.


Assuntos
Biofilmes/crescimento & desenvolvimento , Dimetilpolisiloxanos/química , Óleos de Silicone/química , Biofilmes/efeitos dos fármacos , Biomassa , Dimetilpolisiloxanos/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Porosidade , Pseudomonas aeruginosa/fisiologia , Staphylococcus epidermidis/fisiologia , Propriedades de Superfície , Molhabilidade
15.
Carbohydr Polym ; 279: 118778, 2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-34980345

RESUMO

Eliminating biofilms from infected tissue presents one of the most challenging issues in clinical treatment of chronic wounds. In biofilms, the extracellular polymeric substances (EPS) form gel structures by electrostatic forces between macromolecules. We hypothesized that cationic polymers could induce the gel-to-sol phase transition of the network, leading to biofilms disruptions. We first validated this assumption by using polyethyleneimine (PEI) as a model molecule, and further synthesized two cationic dextrans with high biodegradability for in vitro and in vivo evaluation. All the cationic polymers could destruct Pseudomonas aeruginosa (P. aeruginosa) biofilms. Treating biofilm with cationic dextrans significantly enhanced the bacterial antibiotic sensitivity. When tested in a biofilm-presenting mouse wound healing model, the cationic dextrans efficiently controlled infection, and accelerated the healing process. Our findings suggest that devising cationic polymers to trigger phase transition of biofilm is an effective, straightforward, and perhaps generic strategy for anti-bacterial therapies.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Dextranos/farmacologia , Hidrogéis/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Alginatos/química , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocinas/imunologia , Dextranos/química , Feminino , Hidrogéis/química , Camundongos Endogâmicos BALB C , Transição de Fase , Polietilenoimina/química , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/fisiologia , Pele/efeitos dos fármacos , Pele/imunologia , Infecção dos Ferimentos/tratamento farmacológico , Infecção dos Ferimentos/imunologia
16.
Nano Lett ; 22(3): 1129-1137, 2022 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-35040647

RESUMO

Mechano-bactericidal surfaces deliver lethal effects to contacting bacteria. Until now, cell death has been attributed to the mechanical stress imparted to the bacterial cell envelope by the surface nanostructures; however, the process of bacterial death encountering nanostructured surfaces has not been fully illuminated. Here, we perform an in-depth investigation of the mechano-bactericidal action of black silicon (bSi) surfaces toward Gram-negative bacteria Pseudomonas aeruginosa. We discover that the mechanical injury is not sufficient to kill the bacteria immediately due to the survival of the inner plasma membrane. Instead, such sublethal mechanical injury leads to apoptosis-like death (ALD) in affected bacteria. In addition, when the mechanical stress is removed, the self-accumulated reactive oxygen species (ROS) incur poststress ALD in damaged cells in a nonstressed environment, revealing that the mechano-bactericidal actions have sustained physiological effects on the bacterium. This work creates a new facet and can introduce many new regulation tools to this field.


Assuntos
Nanoestruturas , Pseudomonas aeruginosa , Antibacterianos/química , Antibacterianos/farmacologia , Bactérias Gram-Negativas , Nanoestruturas/química , Pseudomonas aeruginosa/fisiologia , Propriedades de Superfície
17.
Microbiol Spectr ; 10(1): e0146321, 2022 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-35019684

RESUMO

Anthranilate is a diffusible molecule produced by Pseudomonas aeruginosa and accumulates as P. aeruginosa grows. Anthranilate is an important intermediate for the synthesis of tryptophan and the Pseudomonas quinolone signal (PQS), as well as metabolized by the anthranilate dioxygenase complex (antABC operon products). Here we demonstrate that anthranilate is a key factor that modulates the pathogenicity-related phenotypes of P. aeruginosa and other surrounding bacteria in the environment, such as biofilm formation, antibiotic tolerance, and virulence. We found that the anthranilate levels in P. aeruginosa cultures rapidly increased in the stationary phase and then decreased again, forming an anthranilate peak. Biofilm formation, antibiotic susceptibility, and virulence of P. aeruginosa were significantly altered before and after this anthranilate peak. In addition, these phenotypes were all modified by the mutation of antABC and exogenous addition of anthranilate. Anthranilate also increased the antibiotic susceptibility of other species of bacteria, such as Escherichia coli, Salmonella enterica, Bacillus subtilis, and Staphylococcus aureus. Before the anthranilate peak, the low intracellular anthranilate level was maintained through degradation from the antABC function, in which induction of antABC was also limited to a small extent. The premature degradation of anthranilate, due to its high levels, and antABC expression early in the growth phase, appears to be toxic to the cells. From these results, we propose that by generating an anthranilate peak as a signal, P. aeruginosa may induce some sort of physiological change in surrounding cells. IMPORTANCE Pseudomonas aeruginosa is a notorious pathogen with high antibiotic resistance, strong virulence, and ability to cause biofilm-mediated chronic infection. We found that these characteristics change profoundly before and after the time when anthranilate is produced as an "anthranilate peak". This peak acts as a signal that induces physiological changes in surrounding cells, decreasing their antibiotic tolerance and biofilm formation. This study is important in that it provides a new insight into how microbial signaling substances can induce changes in the pathogenicity-related phenotypes of cells in the environment. In addition, this study shows that anthranilate can be used as an adjuvant to antibiotics.


Assuntos
Antibacterianos/farmacologia , Biofilmes , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , ortoaminobenzoatos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/efeitos dos fármacos , Humanos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Salmonella enterica/efeitos dos fármacos , Salmonella enterica/genética , Salmonella enterica/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Virulência
18.
J Bacteriol ; 204(1): e0029721, 2022 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-34723645

RESUMO

Pseudomonas aeruginosa, an opportunistic bacterial pathogen, can synthesize and catabolize several small cationic molecules known as polyamines. In several clades of bacteria, polyamines regulate biofilm formation, a lifestyle-switching process that confers resistance to environmental stress. The polyamine putrescine and its biosynthetic precursors, l-arginine and agmatine, promote biofilm formation in Pseudomonas spp. However, it remains unclear whether the effect is a direct effect of polyamines or occurs through a metabolic derivative. Here, we used a genetic approach to demonstrate that putrescine accumulation, either through disruption of the spermidine biosynthesis pathway or the catabolic putrescine aminotransferase pathway, promoted biofilm formation in P. aeruginosa. Consistent with this observation, exogenous putrescine robustly induced biofilm formation in P. aeruginosa that was dependent on putrescine uptake and biosynthesis pathways. Additionally, we show that l-arginine, the biosynthetic precursor of putrescine, also promoted biofilm formation but did so by a mechanism independent of putrescine or agmatine conversion. We found that both putrescine and l-arginine induced a significant increase in the intracellular level of bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) (c-di-GMP), a bacterial second messenger widely found in Proteobacteria that upregulates biofilm formation. Collectively these data show that putrescine and its metabolic precursor, arginine, promote biofilm and c-di-GMP synthesis in P. aeruginosa. IMPORTANCE Biofilm formation allows bacteria to physically attach to a surface, confer tolerance to antimicrobial agents, and promote resistance to host immune responses. As a result, the regulation of biofilm formation is often crucial for bacterial pathogens to establish chronic infections. A primary mechanism of biofilm promotion in bacteria is the molecule c-di-GMP, which promotes biofilm formation. The level of c-di-GMP is tightly regulated by bacterial enzymes. In this study, we found that putrescine, a small molecule ubiquitously found in eukaryotic cells, robustly enhances P. aeruginosa biofilm and c-di-GMP. We propose that P. aeruginosa may sense putrescine as a host-associated signal that triggers a lifestyle switch that favors chronic infection.


Assuntos
Arginina/farmacologia , Biofilmes/crescimento & desenvolvimento , GMP Cíclico/análogos & derivados , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Putrescina/farmacologia , GMP Cíclico/biossíntese , Regulação Bacteriana da Expressão Gênica/fisiologia , Pseudomonas aeruginosa/efeitos dos fármacos , Regulação para Cima
19.
J Appl Microbiol ; 132(4): 2781-2794, 2022 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-34846774

RESUMO

AIMS: The cost of Microbiologically Influenced Corrosion (MIC) significantly affects a wide range of sectors. This study aims to assess the efficiency of a novel technology based on the use of plasma-activated water (PAW) in inhibiting corrosion caused by bacteria. METHODS AND RESULTS: This study evaluated the effectiveness of PAW, produced by a plasma bubble reactor, in reducing corrosion causing Pseudomonas aeruginosa planktonic cells in tap water and biofilms were grown onto stainless steel (SS) coupons. Planktonic cells and biofilms were treated with PAW at different discharge frequencies (500-1500 Hz) and exposure times (0-20 min). P. aeruginosa cells in tap water were significantly reduced after treatment, with higher exposure times and discharge frequencies achieving higher reductions. Also, PAW treatment led to a gradual reduction for young and mature biofilms, achieving >4-Log reductions after 20 min. Results were also used to develop two predictive inactivation models. CONCLUSIONS: This work presents evidence that PAW can be used to inactivate both planktonic cells and biofilms of P. aeruginosa. Experimental and theoretical results also demonstrate that reduction is dependent on discharge frequency and exposure time. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrates the potential of using PAW as means to control MIC.


Assuntos
Pseudomonas aeruginosa , Água , Biofilmes , Corrosão , Pseudomonas aeruginosa/fisiologia , Aço Inoxidável
20.
Infect Immun ; 90(1): e0033921, 2022 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-34662210

RESUMO

Contact lenses are biomaterials worn on the eye to correct refractive errors. Bacterial adhesion and colonization of these lenses results in adverse events, such as microbial keratitis. The adsorption of tear proteins to contact lens materials enhances bacterial adhesion. Glycoprotein 340 (Gp340), a tear component, is known to promote microbial colonization in the oral cavity; however, it has not been investigated in any contact lens-related adverse event. Therefore, this study examined the adsorption of Gp340 and its recombinantly expressed scavenger receptor cysteine-rich (iSRCR1Gp340) domain on two common contact lens materials, etafilcon A and lotrafilcon B, and the concomitant effects on the adherence of clinical isolates of microbial keratitis causative agents, Pseudomonas aeruginosa (PA6206; PA6294), and Staphylococcus aureus (SA38; USA300). Across all strains and materials, iSRCR1Gp340 enhanced adherence of bacteria in a dose-dependent manner. However, iSRCR1Gp340 did not modulate the lysozyme's or lactoferrin's effects on bacterial adhesion to the contact lens. The Gp340 binding serine-rich surface protein (SraP) significantly enhanced the binding of USA300 to iSRCR1Gp340-coated lenses. In addition, iSRCR1Gp340-coated surfaces had significantly diminished biofilms with the SraP mutant (ΔSraP), and there was a further reduction in biofilms with the sortase A mutant (ΔSrtA), indicating the likely involvement of additional surface proteins. Finally, the binding affinities between iSRCR1Gp340 and SraP were determined using surface plasmon resonance (SPR), where the complete SraP binding region displayed nanomolar affinity, whereas its smaller fragments adhered with micromolar affinities. This study concludes that Gp340 and its SRCR domains play an important role in bacterial adhesion to the contact lens.


Assuntos
Aderência Bacteriana , Lentes de Contato , Polímeros , Domínios e Motivos de Interação entre Proteínas , Pseudomonas aeruginosa/fisiologia , Receptores Imunológicos/metabolismo , Staphylococcus aureus/fisiologia , Adesinas Bacterianas/metabolismo , Biofilmes , Interações Hospedeiro-Patógeno , Humanos , Hidrogéis , Metacrilatos , Muramidase/metabolismo , Ligação Proteica , Receptores Imunológicos/química , Silicones
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