RESUMO
Inhibiting quorum sensing (QS), a central communication system, is a promising strategy to combat bacterial pathogens without antibiotics. Here, we designed novel hybrid compounds targeting the PQS (Pseudomonas quinolone signal)-dependent quorum sensing (QS) of Pseudomonas aeruginosa that is one of the multidrug-resistant and highly virulent pathogens with urgent need of new antibacterial strategies. We synthesized 12 compounds using standard procedures to combine halogen-substituted anthranilic acids with 4-(2-aminoethyl/4-aminobuthyl)amino-7-chloroquinoline, linked via 1,3,4-oxadiazole. Their antibiofilm activities were first pre-screened using Gram-negative Chromobacterium violaceum-based reporter, which identified compounds 15-19 and 23 with the highest anti-QS and minimal bactericidal effects in a single experiment. These five compounds were then evaluated against P. aeruginosa PAO1 to assess their ability to prevent biofilm formation, eradicate pre-formed biofilms, and inhibit virulence using pyocyanin as a representative marker. Compound 15 displayed the most potent antibiofilm effect, reducing biofilm formation by nearly 50% and pre-formed biofilm masses by 25%. On the other hand, compound 23 exhibited the most significant antivirulence effect, reducing pyocyanin synthesis by over 70%. Thus, our study highlights the potential of 1,3,4-oxadiazoles 15 and 23 as promising scaffolds to combat P. aeruginosa. Additionally, interactive QS systems should be considered to achieve maximal anti-QS activity against this clinically relevant species.
Assuntos
Quinolinas , Percepção de Quorum , Piocianina/farmacologia , Biofilmes , Virulência , Antibacterianos/farmacologia , Fatores de Virulência , Quinolinas/farmacologia , Pseudomonas aeruginosa , ChromobacteriumRESUMO
In the present study, we report the design and synthesis of novel amide-type hybrid molecules based on anthranilic acid and quinoline or ß-carboline heterocyclic scaffolds. Three types of biological screenings were performed: (i) in vitro antiproliferative screening against a panel of solid tumor and leukemia cell lines, (ii) antiviral screening against several RNA viruses, and (iii) anti-quorum sensing screening using gram-negative Chromobacterium violaceum as the reporter strain. Antiproliferative screening revealed a high activity of several compounds. Anthranilamides 12 and 13 with chloroquine core and halogenated anthranilic acid were the most active agents toward diverse cancer cell lines such as glioblastoma, pancreatic adenocarcinoma, colorectal carcinoma, lung carcinoma, acute lymphoblastic, acute myeloid, chronic myeloid leukemia, and non-Hodgkin lymphoma, but also against noncancerous cell lines. Boc-protected analogs 2 and 3 showed moderate activities against the tested cancer cells without toxic effects against noncancerous cells. A nonhalogenated quinoline derivative 10 with N-benzylanthranilic acid residue was equally active as 12 and 13 and selective toward tumor cells. Chloroquine and quinoline anthranilamides 10-13 exerted pronounced antiviral effect against human coronaviruses 229E and OC43, whereas 12 and 13 against coronavirus OC43 (EC50 values in low micromolar range; selectivity indices from 4.6 to > 10.4). Anthranilamides 14 and 16 with PQ core inhibited HIV-1 with EC50 values of 9.3 and 14.1 µM, respectively. Compound 13 displayed significant anti-quorum/biofilm effect against the quorum sensing reporter strain (IC50 of 3.7 µM) with no apparent bactericidal effect.
Assuntos
Adenocarcinoma , Neoplasias Pancreáticas , Quinolinas , Amidas , Antibacterianos/farmacologia , Antivirais/química , Antivirais/farmacologia , Carbolinas/farmacologia , Cloroquina , Humanos , Quinolinas/química , Quinolinas/farmacologia , ortoaminobenzoatosRESUMO
Antibacterial screenings are most commonly targeted at planktonic bacteria but less effort is dedicated to the exploration of agents acting on biofilms. Here, a natural compounds library was screened against Staphylococcus aureus using a 384-well plate platform to identify compounds preventing biofilm formation. Five structurally diverse hits were selected for follow-up studies: honokiol, tschimganidin, ferutinin, oridonin and deoxyshikonin. The compounds were evaluated against different bacterial species for their capacity to prevent and disrupt biofilms. The development of resistance and cytotoxicity were also investigated. Ferutinin displayed the best antibacterial activity, with a minimum inhibitory, bactericidal and biofilm preventive concentration of 25 µM against S. aureus. It efficiently disrupted pre-formed biofilms (over 5-log reduction of viable cells) and reduced biofilm formation on a catheter in the presence of neutrophils. This work provides new information on the antibacterial activity of five natural compounds and identified ferutinin as a promising candidate against S. aureus biofilms.
Assuntos
Infecções Estafilocócicas , Staphylococcus aureus , Antibacterianos/farmacologia , Benzoatos , Biofilmes , Compostos Bicíclicos com Pontes , Cicloeptanos , Humanos , Testes de Sensibilidade Microbiana , SesquiterpenosRESUMO
Quorum sensing inhibitors (QSIs) that specifically interfere with bacterial cell-to-cell communication are considered as an alternative approach to conventional antibacterial therapy. In our study, a set of twenty-six fumardiamides with a quinoline head-group were evaluated as potential QSIs. Two strains of Gram-negative Chromobacterium violaceum (violacein-producing strain ATCC31532 and violacein-negative, mini-Tn5 mutant derivative CV026) were used as QS reporters for testing anti-QS and bactericidal activity of various quinoline fumardiamides. The initial screening of eighteen fumardiamides with primaquine, mefloquine and chloroquine scaffolds identified chloroquine derivatives as the most promising QSIs. Tail-group optimization of chloroquine fumardiamides led to the most active compounds 27, 29 and 30 bearing aminoethyl or piperidine moieties. At 400 µM concentration, these compounds inhibited the QS of C. violaceum strains in a manner similar to quercetin (the model QSI), while at the 40 µM concentration their inhibitory effect was twice less than that of quercetin. As none of the compounds displayed a bactericidal effect and that the QS inhibition was specific to the CV026 strain, our findings indicate that the structurally optimized chloroquine derivatives could function as quorum quenching (QQ) agents with a potential to block the signaling without entering the cell. In conclusion, our finding provides an important step toward the further design of agents targeting cell-to-cell communication.
Assuntos
Amidas/farmacologia , Antibacterianos/farmacologia , Cloroquina/farmacologia , Chromobacterium/efeitos dos fármacos , Percepção de Quorum/efeitos dos fármacos , Amidas/química , Antibacterianos/química , Cloroquina/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
In recent years, bacterial infections have become a main concern following the spread of antimicrobial resistance. In addition, bacterial biofilms are known for their high tolerance to antimicrobials and they are regarded as a main cause of recalcitrant infections in humans. Many efforts have been deployed in order to find new antibacterial therapeutic options and the high-throughput screening (HTS) of large libraries of compounds is one of the utilized strategies. However, HTS efforts for anti-biofilm discovery remain uncommon. Here, we miniaturized a 96-well plate (96WP) screening platform, into a 384-well plate (384WP) format, based on a sequential viability and biomass measurements for the assessment of anti-biofilm activity. During the assay optimization process, different parameters were evaluated while using Staphylococcus aureus and Pseudomonas aeruginosa as the bacterial models. We compared the performance of the optimized 384WP platform to our previously established 96WP-based platform by carrying out a pilot screening of 100 compounds, followed by the screening of a library of 2000 compounds to identify new repurposed anti-biofilm agents. Our results show that the optimized 384WP platform is well-suited for screening purposes, allowing for the rapid screening of a higher number of compounds in a run in a reliable manner.
Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Ensaios de Triagem em Larga Escala/métodos , Testes de Sensibilidade Microbiana/métodos , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Corantes , Violeta Genciana , Ensaios de Triagem em Larga Escala/instrumentação , Humanos , Testes de Sensibilidade Microbiana/instrumentação , Miniaturização , Oxazinas , Projetos Piloto , Pseudomonas aeruginosa/fisiologia , Bibliotecas de Moléculas Pequenas , Coloração e Rotulagem/métodos , Staphylococcus aureus/fisiologia , XantenosRESUMO
A Gram-stain-positive, catalase-positive and pleomorphic rod organism was isolated from malted barley in Finland, classified initially by partial 16S rRNA gene sequencing and originally deposited in the VTT Culture Collection as a strain of Propionibacterium acidipropionici (currently Acidipropionibacterium acidipropionici). The subsequent comparison of the whole 16S rRNA gene with other representatives of the genus Acidipropionibacterium revealed that the strain belongs to a novel species, most closely related to Acidipropionibacterium microaerophilum and Acidipropionibacterium acidipropionici, with similarity values of 98.46 and 98.31â%, respectively. The whole genome sequencing using PacBio RS II platform allowed further comparison of the genome with all of the other DNA sequences available for the type strains of the Acidipropionibacterium species. Those comparisons revealed the highest similarity of strain JS278T to A. acidipropionici, which was confirmed by the average nucleotide identity analysis. The genome of strain JS278T is intermediate in size compared to the A. acidipropionici and Acidipropionibacterium jensenii at 3â432â872 bp, the G+C content is 68.4 mol%. The strain fermented a wide range of carbon sources, and produced propionic acid as the major fermentation product. Besides its poor ability to grow at 37 °C and positive catalase reaction, the observed phenotype was almost indistinguishable from those of A. acidipropionici and A. jensenii. Based on our findings, we conclude that the organism represents a novel member of the genus Acidipropionibacterium, for which we propose the name Acidipropionibacteriumvirtanenii sp. nov. The type strain is JS278T (=VTT E-113202T=DSM 106790T).
Assuntos
Hordeum/microbiologia , Filogenia , Propionibacterium/classificação , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Fermentação , Finlândia , Propionibacterium/genética , Propionibacterium/isolamento & purificação , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
BACKGROUND: Propionibacterium freudenreichii is an industrially important bacterium granted the Generally Recognized as Safe (the GRAS) status, due to its long safe use in food bioprocesses. Despite the recognized role in the food industry and in the production of vitamin B12, as well as its documented health-promoting potential, P. freudenreichii remained poorly characterised at the genomic level. At present, only three complete genome sequences are available for the species. RESULTS: We used the PacBio RS II sequencing platform to generate complete genomes of 20 P. freudenreichii strains and compared them in detail. Comparative analyses revealed both sequence conservation and genome organisational diversity among the strains. Assembly from long reads resulted in the discovery of additional circular elements: two putative conjugative plasmids and three active, lysogenic bacteriophages. It also permitted characterisation of the CRISPR-Cas systems. The use of the PacBio sequencing platform allowed identification of DNA modifications, which in turn allowed characterisation of the restriction-modification systems together with their recognition motifs. The observed genomic differences suggested strain variation in surface piliation and specific mucus binding, which were validated by experimental studies. The phenotypic characterisation displayed large diversity between the strains in ability to utilise a range of carbohydrates, to grow at unfavourable conditions and to form a biofilm. CONCLUSION: The complete genome sequencing allowed detailed characterisation of the industrially important species, P. freudenreichii by facilitating the discovery of previously unknown features. The results presented here lay a solid foundation for future genetic and functional genomic investigations of this actinobacterial species.
Assuntos
Genômica/métodos , Propionibacterium freudenreichii/genética , Adaptação Fisiológica/genética , Genoma Bacteriano/genética , Ilhas Genômicas/genética , Plasmídeos/genética , Análise de SequênciaRESUMO
The present study reports the effect of Penicillin G (PenG) on the proteome dynamics of the Staphylococcus aureus strain Newman during biofilm mode of growth. The viability of the 18-h-old biofilm cells challenged with PenG at the concentration of 1mgmL(-1) was first assessed by plate counting, resazurin and LIVE/DEAD fluorescence staining, which indicated that the viability was reduced by â¼35% and â¼90% at 2h and 24h, respectively, after the addition of PenG. Subsequent two-dimensional difference gel electrophoresis (2D DIGE) assay of the treated and non-treated biofilm cells at the indicated time points revealed 45 proteins showing time- and treatment-specific change (1.5-fold, p<0.01). The 2D DIGE results suggested that the PenG-induced decrease in viability was accompanied by an increased synthesis of pyruvate oxidase (CidC), a suicidal marker known to potentiate acetate-dependent cell death in S. aureus. Increased abundance was also found for the TCA cycle associated malate-quinone oxidoreductase (Mqo), the ClpC ATPase, the HlgBC toxin and phage-associated proteins, which suggests that surviving cells have induced these activities as a last effort to overcome lethal doses of PenG. Proteomic results also revealed that the surviving cells were likely to strengthen their peptidoglycan due to the increased abundance of cell-wall biogenesis associated proteins, FemA and Pbp2; a phenomenon associated with dormancy in S. aureus.
Assuntos
Antibacterianos/metabolismo , Proteínas de Bactérias/biossíntese , Biofilmes/efeitos dos fármacos , Penicilina G/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Fatores de Virulência/biossíntese , Eletroforese em Gel Bidimensional , Viabilidade Microbiana/efeitos dos fármacos , Proteoma/análise , Staphylococcus aureus/fisiologiaRESUMO
The present study reports the identification and comparison of all expressed cell-surface exposed proteins from the well-known probiotic L. rhamnosus GG and a related dairy strain, Lc705. To obtain this information, the cell-surface bound proteins were released from intact cells by trypsin shaving under hypertonic conditions with and without DTT. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses of the purified peptides identified a total of 102 and 198 individual proteins from GG and Lc705, respectively. Comparison of both data sets suggested that the Msp-type antigens (Msp1, Msp2) and the serine protease HtrA were uniquely exposed at the cell surface of GG, whereas the Lc705-specific proteins included lactocepin and a wider range of different moonlighting proteins. ImmunoEM analyses with the GG and Lc705 antibodies suggested that the whole-cell immunization yielded antibodies toward surface-bound proteins and proteins that were secreted or released from the cell-surface. One of the detected antigens was a pilus-like structure on the surface of GG cells, which was not detected with Lc705 antibodies. Further 2-DE immunoblotting analysis of GG proteins with both L. rhamnosus antisera revealed that majority of the detected antigens were moonlighting proteins with potential roles in adhesion, pathogen exclusion or immune stimulation. The present study provides the first catalog of surface-exposed proteins from lactobacilli and highlights the importance of the specifically exposed moonlighting proteins for adaptation and probiotic functions of L. rhamnosus.
Assuntos
Proteínas de Bactérias/análise , Immunoblotting/métodos , Lacticaseibacillus rhamnosus/química , Proteínas de Membrana/análise , Proteoma/análise , Proteômica/métodos , Anticorpos Antibacterianos , Proteínas de Bactérias/classificação , Proteínas de Bactérias/fisiologia , Proteínas de Membrana/classificação , Proteínas de Membrana/fisiologia , Proteoma/químicaRESUMO
BACKGROUND: Propionibacterium freudenreichii is a food grade bacterium that has gained attention as a producer of appreciable amounts of cobalamin, a cobamide with activity of vitamin B12. Production of active form of vitamin is a prerequisite for attempts to naturally fortify foods with B12 by microbial fermentation. Active vitamin B12 is distinguished from the pseudovitamin by the presence of 5,6-dimethylbenzimidazole (DMBI) as the lower ligand. Genomic data indicate that P. freudenreichii possesses a fusion gene, bluB/cobT2, coding for a predicted phosphoribosyltransferase/nitroreductase, which is presumably involved in production of vitamin B12. Understanding the mechanisms affecting the synthesis of different vitamin forms is useful for rational strain selection and essential for engineering of strains with improved B12 production properties. RESULTS: Here, we investigated the activity of heterologously expressed and purified fusion enzyme BluB/CobT2. Our results show that BluB/CoBT2 is responsible for the biosynthesis of the DMBI base and its activation into α-ribazole phosphate, preparing it for attachment as the lower ligand of cobalamin. The fusion enzyme was found to be efficient in metabolite channeling and the enzymes' inability to react with adenine, a lower ligand present in the pseudovitamin, revealed a mechanism favoring the production of the active form of the vitamin. P. freudenreichii did not produce cobalamin under strictly anaerobic conditions, confirming the requirement of oxygen for DMBI synthesis. In vivo experiments also revealed a clear preference for incorporating DMBI over adenine into cobamide under both microaerobic and anaerobic conditions. CONCLUSIONS: The herein described BluB/CobT2 is responsible for the production and activation of DMBI. Fusing those two activities results in high pressure towards production of the true vitamin B12 by efficiently activating DMBI formed within the same enzymatic complex. This indicates that BluB/CobT2 is the crucial enzyme in the B12 biosynthetic pathway of P. freudenreichii. The GRAS organism status and the preference for synthesizing active vitamin form make P. freudenreichii a unique candidate for the in situ production of vitamin B12 within food products.
Assuntos
Proteínas de Bactérias/metabolismo , Propionibacterium/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Vitamina B 12/metabolismo , Proteínas de Bactérias/genética , Proteínas Recombinantes de Fusão/genéticaRESUMO
The present study reports a comparative proteome cataloging of a bovine mastitis and a human-associated Staphylococcus epidermidis strain with a specific focus on surfome (cell-wall bound and extracellular) proteins. Protein identification by 1DE coupled with LC-MS/MS analyses resulted in 1400 and 1287 proteins from the bovine (PM221) and human (ATCC12228) strains, respectively, covering over 50% of all predicted and more than 30% of all predicted surfome proteins in both strains. Comparison of the identification results suggests elevated levels of proteins involved in adherence, biofilm formation, signal transduction, house-keeping functions, and immune evasion in PM221, whereas ATCC12228 was more effective in expressing host defense evasion proteases, skin adaptation lipases, hemagglutination, and heavy-metal resistance proteins. Phenotypic analyses showed that only PM221 displays protein- and DNA-mediated adherent growth, and that PM221 was more efficient in cleaving tributyrin, a natural compound of milk fat under low CO2 conditions. These findings are in line with the identification data and suggest that distinct expression of lipases and adhesive surfome proteins could lead to the observed phenotypes. This study is the first extensive survey of S. epidermidis proteomes to date, providing several protein candidates to be examined for their roles in adaptation and virulence in vivo. All MS data have been deposited in the ProteomeXchange with identifier PXD000404 (http://proteomecentral.proteomexchange.org/dataset/PXD000404).
Assuntos
Proteínas de Bactérias/metabolismo , Infecções Estafilocócicas/microbiologia , Staphylococcus epidermidis/metabolismo , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/análise , Bovinos , Humanos , Proteoma/análise , Proteoma/metabolismo , Staphylococcus epidermidis/patogenicidade , Espectrometria de Massas em Tandem , Fatores de Virulência/análiseRESUMO
Staphylococcus epidermidis (SE) includes commensal and pathogenic strains capable of infecting humans and animals. This study reports global exoproteome profiling of bovine mastitis strain PM221 and two human strains, commensal-type ATCC12228 and sepsis-associated RP62A. We identified 451, 395, and 518 proteins from culture supernatants of PM221, ATCC12228, and RP62A, respectively. Comparison of the identified exoproteomes revealed several strain-specific differences related to secreted antigens and adhesins, higher virulence capability for RP62A, and similarities between the PM221 and RP62A exoproteomes. The majority of the identified proteins (â¼80%) were predicted to be cytoplasmic, including proteins known to be associated in membrane vesicles (MVs) in Staphylococcus aureus and immunogenic/adhesive moonlighting proteins. Enrichment of MV fractions from culture supernatants and analysis of their protein composition indicated that this nonclassical protein secretion pathway was being exploited under the conditions used and that there are strain-specific differences in nonclassical protein export. In addition, several predicted cell-surface proteins were identified in the culture media. In summary, the present study is the first in-depth exoproteome analysis of SE highlighting strain-specific factors able to contribute to virulence and adaptation.
Assuntos
Proteínas de Bactérias/metabolismo , Staphylococcus epidermidis/metabolismo , Virulência , Animais , Humanos , Staphylococcus epidermidis/patogenicidade , Urease/metabolismo , beta-Lactamases/metabolismoRESUMO
The present study reports comparative genomics and proteomics of Staphylococcus epidermidis (SE) strains isolated from bovine intramammary infection (PM221) and human hosts (ATCC12228 and RP62A). Genome-level profiling and protein expression analyses revealed that the bovine strain and the mildly infectious ATCC12228 strain are highly similar. Their genomes share high sequence identity and synteny, and both were predicted to encode the commensal-associated fdr marker gene. In contrast, PM221 was judged to differ from the sepsis-associated virulent human RP62A strain on the basis of distinct protein expression patterns and overall lack of genome synteny. The 2D DIGE and phenotypic analyses suggest that PM221 and ATCC12228 coordinate the TCA cycle activity and the formation of small colony variants in a way that could result in increased viability. Pilot experimental infection studies indicated that although ATCC12228 was able to infect a bovine host, the PM221 strain caused more severe clinical signs. Further investigation revealed strain- and condition-specific differences among surface bound proteins with likely roles in adhesion, biofilm formation, and immunomodulatory functions. Thus, our findings revealed a close link between the bovine and commensal-type human strains and suggest that humans could act as a reservoir of bovine mastitis-causing SE strains.
RESUMO
Mycobacterium marinum, a slow-growing Actinobacterium, typically induces tuberculosis-like disease in fish. Here, we report a new reference sequence for M. marinum ATCC 927T, along with its DNA methylome. This aims to maximize the research potential of this type strain and facilitates investigations into the pathomechanisms of human tuberculosis.
RESUMO
The coronavirus disease 2019 (COVID-19) pandemic has heavily challenged the global healthcare system. Despite the vaccination programs, the new virus variants are circulating. Further research is required for understanding of the biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and for discovery of therapeutic agents against the virus. Here, we took advantage of drug repurposing to identify if existing drugs could inhibit SARS-CoV-2 infection. We established an open high throughput platform for in vitro screening of drugs against SARS-CoV-2 infection. We screened â¼1000 drugs for their ability to inhibit SARS-CoV-2-induced cell death in the African green monkey kidney cell line (Vero-E6), analyzed how the hit compounds affect the viral N (nucleocapsid) protein expression in human cell lines using high-content microscopic imaging and analysis, determined the hit drug targets in silico, and assessed their ability to cause phospholipidosis, which can interfere with the viral replication. Duvelisib was found by in silico interaction assay as a potential drug targeting virus-host protein interactions. The predicted interaction between PARP1 and S protein, affected by Duvelisib, was further validated by immunoprecipitation. Our results represent a rapidly applicable platform for drug repurposing and evaluation of the new emerging viruses' responses to the drugs. Further in silico studies help us to discover the druggable host pathways involved in the infectious cycle of SARS-CoV-2.
Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Animais , Chlorocebus aethiops , Reposicionamento de Medicamentos , Bioensaio , Morte Celular , Proteínas do NucleocapsídeoRESUMO
Lactobacillus rhamnosus GG (GG) is a widely used and intensively studied probiotic bacterium. Although the health benefits of strain GG are well documented, the systematic exploration of mechanisms by which this strain exerts probiotic effects in the host has only recently been initiated. The ability to survive the harsh conditions of the gastrointestinal tract, including gastric juice containing bile salts, is one of the vital characteristics that enables a probiotic bacterium to transiently colonize the host. Here we used gene expression profiling at the transcriptome and proteome levels to investigate the cellular response of strain GG toward bile under defined bioreactor conditions. The analyses revealed that in response to growth of strain GG in the presence of 0.2% ox gall the transcript levels of 316 genes changed significantly (p < 0.01, t test), and 42 proteins, including both intracellular and surface-exposed proteins (i.e. surfome), were differentially abundant (p < 0.01, t test in total proteome analysis; p < 0.05, t test in surfome analysis). Protein abundance changes correlated with transcriptome level changes for 14 of these proteins. The identified proteins suggest diverse and specific changes in general stress responses as well as in cell envelope-related functions, including in pathways affecting fatty acid composition, cell surface charge, and thickness of the exopolysaccharide layer. These changes are likely to strengthen the cell envelope against bile-induced stress and signal the GG cells of gut entrance. Notably, the surfome analyses demonstrated significant reduction in the abundance of a protein catalyzing the synthesis of exopolysaccharides, whereas a protein dedicated for active removal of bile compounds from the cells was up-regulated. These findings suggest a role for these proteins in facilitating the well founded interaction of strain GG with the host mucus in the presence of sublethal doses of bile. The significance of these findings in terms of the functionality of a probiotic bacterium is discussed.
Assuntos
Bile/metabolismo , Perfilação da Expressão Gênica , Lacticaseibacillus rhamnosus/metabolismo , Proteoma , Proteômica/métodos , Proteínas de Bactérias/genética , Eletroforese em Gel Bidimensional , Fermentação , Humanos , Espectrometria de Massas/métodos , Fases de Leitura Aberta , Polissacarídeos/química , Probióticos/química , Coloração pela PrataRESUMO
A series of quaternary ammonium fluoroquinolones was obtained by exhaustive methylation of the amine groups present at the 7-position of fluoroquinolones, including ciprofloxacin, enoxacin, gatifloxacin, lomefloxacin, and norfloxacin. The synthesized molecules were tested for their antibacterial and antibiofilm activities against Gram-positive and Gram-negative human pathogens, i.e. Staphylococcus aureus and Pseudomonas aeruginosa. The study showed that the synthesized compounds are potent antibacterial agents (MIC values at the lowest 6.25 µM) with low cytotoxicity in vitro as assessed on the BALB 3T3 mouse embryo cell line. Further experiments proved that the tested derivatives are able to bind to the DNA gyrase and topoisomerase IV active sites in a fluoroquinolone-characteristic manner. The most active quaternary ammonium fluoroquinolones, in contrast to ciprofloxacin, reduce the total biomass of P. aeruginosa ATCC 15442 biofilm in post-exposure experiments. The latter effect may be due to the dual mechanism of action of the quaternary fluoroquinolones, which also involves disruption of bacterial cell membranes. IAM-HPLC chromatographic experiments with immobilized artificial membranes (phospholipids) showed that the most active compounds were those with moderate lipophilicity and containing a cyclopropyl group at the N1 nitrogen atom in the fluoroquinolone core.
Assuntos
Compostos de Amônio , Humanos , Animais , Camundongos , Fluoroquinolonas/farmacologia , Fluoroquinolonas/química , Antibacterianos/farmacologia , Antibacterianos/química , Ciprofloxacina , Bactérias , Testes de Sensibilidade MicrobianaRESUMO
The antibiotic-tolerant biofilms present in tuberculous granulomas add an additional layer of complexity when treating mycobacterial infections, including tuberculosis (TB). For a more efficient treatment of TB, the biofilm forms of mycobacteria warrant specific attention. Here, we used Mycobacterium marinum (Mmr) as a biofilm-forming model to identify the abundant proteins covering the biofilm surface. We used biotinylation/streptavidin-based proteomics on the proteins exposed at the Mmr biofilm matrices in vitro to identify 448 proteins and ex vivo proteomics to detect 91 Mmr proteins from the mycobacterial granulomas isolated from adult zebrafish. In vitro and ex vivo proteomics data are available via ProteomeXchange with identifiers PXD033425 and PXD039416, respectively. Data comparisons pinpointed the molecular chaperone GroEL2 as the most abundant Mmr protein within the in vitro and ex vivo proteomes, while its paralog, GroEL1, with a known role in biofilm formation, was detected with slightly lower intensity values. To validate the surface exposure of these targets, we created in-house synthetic nanobodies (sybodies) against the two chaperones and identified sybodies that bind the mycobacterial biofilms in vitro and those present in ex vivo granulomas. Taken together, the present study reports a proof-of-concept showing that surface proteomics in vitro and ex vivo proteomics combined is a valuable strategy to identify surface-exposed proteins on the mycobacterial biofilm. Biofilm surface-binding nanobodies could be eventually used as homing agents to deliver biofilm-targeting treatments to the sites of persistent biofilm infection. IMPORTANCE With the currently available antibiotics, the treatment of TB takes months. The slow response to treatment is caused by antibiotic tolerance, which is especially common among bacteria that form biofilms. Such biofilms are composed of bacterial cells surrounded by the extracellular matrix. Both the matrix and the dormant lifestyle of the bacterial cells are thought to hinder the efficacy of antibiotics. To be able to develop faster-acting treatments against TB, the biofilm forms of mycobacteria deserve specific attention. In this work, we characterize the protein composition of Mmr biofilms in bacterial cultures and in mycobacteria extracted from infected adult zebrafish. We identify abundant surface-exposed targets and develop the first sybodies that bind to mycobacterial biofilms. As nanobodies can be linked to other therapeutic compounds, in the future, they can provide means to target therapies to biofilms.
Assuntos
Mycobacterium marinum , Anticorpos de Domínio Único , Tuberculose , Animais , Proteômica , Peixe-Zebra , Antibacterianos , Tuberculose/microbiologia , BiofilmesRESUMO
In Staphylococcus aureus, ClpP proteases were previously shown to be essential for virulence and stress tolerance in strains derived from NCTC8325. Because these strains exhibit a severely reduced activity of the alternative sigma factor, SigB, we here reassessed the role of ClpP in SigB-proficient clinical strains. To this end, clpP was deleted in strains COL, Newman, and SA564, and the strains were characterized phenotypically. The proteomic changes accomplished by the clpP deletion in the different strains were analyzed using the 2-D DIGE technique. The proteomic analyses revealed mostly conserved changes in the protein profiles of the ClpP-deficient strains. Among the strain-specific changes were the up-regulation of prophage proteins that coincided with an increased spontaneous release of prophages and the relatively poorer growth of the clpP mutants in some strain backgrounds. Interestingly, the effect of ClpP on the expression of selected virulence genes was strain-dependent despite the fact that the expression of the global virulence regulators RNAIII, mgrA, sarZ, sarR, and arlRS was similarly changed in all clpP mutants. ClpP affected the expression of sarS in a strain-dependent manner, and we propose that the differential expression of sarS is central to the strain-dependent effect of ClpP on the expression of virulence genes.
Assuntos
Endopeptidase Clp/genética , Staphylococcus aureus/fisiologia , Estresse Fisiológico , Fatores de Virulência/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Fenótipo , Prófagos , Proteoma/genética , Proteoma/isolamento & purificação , Proteoma/metabolismo , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Transativadores/genética , Transativadores/metabolismo , Eletroforese em Gel Diferencial Bidimensional , Urease/metabolismo , VirulênciaRESUMO
BACKGROUND: Trichinellosis is a zoonotic disease in humans caused by Trichinella spp. The present study was undertaken to discover excretory-secretory (E-S) proteins from T. spiralis and T. britovi muscle larvae (ML) that hold promise for species-specific diagnostics. To that end, the purified E-S proteins were analyzed by fluorescent two-dimensional difference gel electrophoresis (2-D DIGE) coupled with protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). To search for immunoreactive proteins that are specifically recognized by host antibodies the E-S proteins were subjected to two-dimensional (2-DE) immunoblotting with antisera derived from pigs experimentally infected with T. spiralis or T. britovi. RESULTS: According to 2-D DIGE analysis, a total of twenty-two proteins including potentially immunogenic proteins and proteins produced only by one of the two Trichinella species were subjected to LC-MS/MS for protein identification. From these proteins seventeen could be identified, of which many were identified in multiple spots, suggesting that they have undergone post-translational modification, possibly involving glycosylation and/or proteolysis. These proteins included 5'-nucleotidase, serine-type protease/proteinase, and p43 glycoprotein (gp43) as well as 49 kDa E-S protein (p49). Our findings also suggest that some of the commonly identified proteins were post-translationally modified to different extents, which in certain cases seemed to result in species-specific modification. Both commonly and specifically recognized immunoreactive proteins were identified by 2-DE immunoblotting; shared antigens were identified as gp43 and different protease variants, whereas those specific to T. britovi included multiple isoforms of the 5'-nucleotidase. CONCLUSIONS: Both 2-D DIGE and 2-DE immunoblotting approaches indicate that T. spiralis and T. britovi produce somewhat distinctive antigen profiles, which contain E-S antigens with potential as species-specific diagnostic markers for Trichinella. Our results also demonstrate the value of 2-D DIGE as a versatile tool to compare secretomes of different Trichinella species for pinpointing factors contributing to the interaction with the host.