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BACKGROUND: Antimicrobial resistance is a major public health threat, and new agents are needed. Computational approaches have been proposed to reduce the cost and time needed for compound screening. AIMS: A machine learning (ML) model was developed for the in silico screening of low molecular weight molecules. METHODS: We used the results of a high-throughput Caenorhabditis elegans methicillin-resistant Staphylococcus aureus (MRSA) liquid infection assay to develop ML models for compound prioritization and quality control. RESULTS: The compound prioritization model achieved an AUC of 0.795 with a sensitivity of 81% and a specificity of 70%. When applied to a validation set of 22,768 compounds, the model identified 81% of the active compounds identified by high-throughput screening (HTS) among only 30.6% of the total 22,768 compounds, resulting in a 2.67-fold increase in hit rate. When we retrained the model on all the compounds of the HTS dataset, it further identified 45 discordant molecules classified as non-hits by the HTS, with 42/45 (93%) having known antimicrobial activity. CONCLUSION: Our ML approach can be used to increase HTS efficiency by reducing the number of compounds that need to be physically screened and identifying potential missed hits, making HTS more accessible and reducing barriers to entry.
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Host-microbe interactions constitute dynamical systems that can be represented by mathematical formulations that determine their dynamic nature and are categorized as deterministic, stochastic, or chaotic. Knowing the type of dynamical interaction is essential for understanding the system under study. Very little experimental work has been done to determine the dynamical characteristics of host-microbe interactions, and its study poses significant challenges. The most straightforward experimental outcome involves an observation of time to death upon infection. However, in measuring this outcome, the internal parameters and the dynamics of each particular host-microbe interaction in a population of interactions are hidden from the experimentalist. To investigate whether a time-to-death (time-to-event) data set provides adequate information for searching for chaotic signatures, we first determined our ability to detect chaos in simulated data sets of time-to-event measurements and successfully distinguished the time-to-event distribution of a chaotic process from a comparable stochastic one. To do so, we introduced an inversion measure to test for a chaotic signature in time-to-event distributions. Next, we searched for chaos in the time-to-death of Caenorhabditis elegans and Drosophila melanogaster infected with Pseudomonas aeruginosa or Pseudomonas entomophila, respectively. We found suggestions of chaotic signatures in both systems but caution that our results are preliminary and highlight the need for more fine-grained and larger data sets in determining dynamical characteristics. If validated, chaos in host-microbe interactions would have important implications for the occurrence and outcome of infectious diseases, the reproducibility of experiments in the field of microbial pathogenesis, and the prediction of microbial threats.IMPORTANCEIs microbial pathogenesis a predictable scientific field? At a time when we are dealing with coronavirus disease 2019, there is intense interest in knowing about the epidemic potential of other microbial threats and new emerging infectious diseases. To know whether microbial pathogenesis will ever be a predictable scientific field requires knowing whether a host-microbe interaction follows deterministic, stochastic, or chaotic dynamics. If randomness and chaos are absent from virulence, there is hope for prediction in the future regarding the outcome of microbe-host interactions. Chaotic systems are inherently unpredictable, although it is possible to generate short-term probabilistic models, as is done in applications of stochastic processes and machine learning to weather forecasting. Information on the dynamics of a system is also essential for understanding the reproducibility of experiments, a topic of great concern in the biological sciences. Our study finds preliminary evidence for chaotic dynamics in infectious diseases.
Assuntos
Doenças Transmissíveis , Interações entre Hospedeiro e Microrganismos , Animais , Drosophila melanogaster , Reprodutibilidade dos Testes , MatemáticaRESUMO
Infections caused by Staphylococcus aureus, notably methicillin-resistant S. aureus (MRSA), pose treatment challenges due to its ability to tolerate antibiotics and develop antibiotic resistance. The former, a mechanism independent of genetic changes, allows bacteria to withstand antibiotics by altering metabolic processes. Here, a potent methylazanediyl bisacetamide derivative, MB6, is described, which selectively targets MRSA membranes over mammalian membranes without observable resistance development. Although MB6 is effective against growing MRSA cells, its antimicrobial activity against MRSA persisters is limited. Nevertheless, MB6 significantly potentiates the bactericidal activity of gentamicin against MRSA persisters by facilitating gentamicin uptake. In addition, MB6 in combination with daptomycin exhibits enhanced anti-persister activity through mutual reinforcement of their membrane-disrupting activities. Crucially, the "triple" combination of MB6, gentamicin, and daptomycin exhibits a marked enhancement in the killing of MRSA persisters compared to individual components or any double combinations. These findings underscore the potential of MB6 to function as a potent and selective membrane-active antimicrobial adjuvant to enhance the efficacy of existing antibiotics against persister cells. The molecular mechanisms of MB6 elucidated in this study provide valuable insights for designing anti-persister adjuvants and for developing new antimicrobial combination strategies to overcome the current limitations of antibiotic treatments.
Assuntos
Daptomicina , Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Animais , Daptomicina/farmacologia , Staphylococcus aureus , Gentamicinas/farmacologia , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , MamíferosRESUMO
Acne vulgaris is a complex skin disease involving infection by Cutibacterium acnes, inflammation, and hyperkeratinization. We evaluated the activity of the retinoid 6-[3-(adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437) and 16 other retinoid analogs as potential anti-C. acnes compounds and found that CD437 displayed the highest antimicrobial activity with an MIC against C. acnes (ATCC 6919 and HM-513) of 1 µg/mL. CD437 demonstrated an MBC of 2 µg/mL compared to up to 64 µg/mL for the retinoid adapalene and up to 16 µg/mL for tetracycline, which are commonly used clinically to treat acne. Membrane permeability assays demonstrated that exposure of C. acnes ATCC 6919 to CD437 damaged the integrity of C. acnes ATCC 6919 bacterial membranes, and this finding was confirmed with scanning electron microscopy. Additionally, CD437 downregulated the expression of C. acnes ATCC 6919 virulence factors, including the genes encoding Christie-Atkins-Munch-Petersen factor 1 (CAMP1), CAMP2, glycerol-ester hydrolase B (GehB), sialidase B, and neuraminidase. In a mouse skin infection model of C. acnes ATCC 6919, topical treatment with CD437 ameliorated skin lesions and reduced the bacterial burden in situ (P < 0.001). In human NHEK primary cells, CD437 reduced the transcriptional levels of the coding genes for inflammatory cytokines (interleukin-1α, ~10-fold; interleukin-6, ~20-fold; interleukin-8, ~30-fold; and tumor necrosis factor-alpha, ~6-fold) and downregulated the transcriptional levels of KRT10 (~10-fold), FLG (~4-fold), and TGM1 (~2-fold), indicating that CD437 can diminish inflammation and hyperkeratinization. In summary, CD437 deserves further attention for its dual function as a potential acne therapeutic that potentially acts on both the pathogen and the host.
Assuntos
Acne Vulgar , Retinoides , Camundongos , Animais , Humanos , Retinoides/metabolismo , Retinoides/uso terapêutico , Acne Vulgar/tratamento farmacológico , Acne Vulgar/microbiologia , Citocinas/metabolismo , Antibacterianos/uso terapêutico , Inflamação , Propionibacterium acnesRESUMO
Host-microbe interactions constitute dynamical systems that can be represented by mathematical formulations that determine their dynamic nature, and are categorized as deterministic, stochastic, or chaotic. Knowing the type of dynamical interaction is essential for understanding the system under study. Very little experimental work has been done to determine the dynamical characteristics of host-microbe interactions and its study poses significant challenges. The most straightforward experimental outcome involves an observation of time to death upon infection. However, in measuring this outcome, the internal parameters, and the dynamics of each particular host-microbe interaction in a population of interactions are hidden from the experimentalist. To investigate whether a time-to-death (time to event) dataset provides adequate information for searching for chaotic signatures, we first determined our ability to detect chaos in simulated data sets of time-to-event measurements and successfully distinguished the time-to-event distribution of a chaotic process from a comparable stochastic one. To do so, we introduced an inversion measure to test for a chaotic signature in time-to-event distributions. Next, we searched for chaos, in time-to-death of Caenorhabditis elegans and Drosophila melanogaster infected with Pseudomonas aeruginosa or Pseudomonas entomophila, respectively. We found suggestions of chaotic signatures in both systems, but caution that our results are preliminary and highlight the need for more fine-grained and larger data sets in determining dynamical characteristics. If validated, chaos in host-microbe interactions would have important implications for the occurrence and outcome of infectious diseases, the reproducibility of experiments in the field of microbial pathogenesis and the prediction of microbial threats.
RESUMO
The opportunistic human pathogen Staphylococcus aureus can evade antibiotics by acquiring antibiotic resistance genes or by entering into a non-growing dormant state. Moreover, the particular circumstances of a specific infection site, such as acidity or anaerobicity, often weaken antibiotic potency. Decreased bacterial susceptibility combined with diminished antibiotic potency is responsible for high failure rates when treating S. aureus infections. Here, we report that the membrane-active antimicrobial agent nTZDpa does not only exhibit enhanced antibiotic activity against multidrug-resistant Gram-positive pathogens in acidic pH, but also retains antimicrobial potency under anaerobic conditions. This agent completely eradicated highly antibiotic-tolerant cells and biofilms formed by methicillin-resistant S. aureus at pH 5.5 at concentrations at which it was not potent at pH 7.4. Furthermore, nTZDpa was more potent at synergistically potentiating gentamicin killing against antibiotic-tolerant MRSA cells at low pH than at high pH. All-atom molecular dynamics simulations combined with membrane-permeabilization assays revealed that the neutral form of nTZDpa, which contains carboxylic acid, is more effective than the deprotonated form at penetrating the bacterial membrane and plays an essential role in membrane activity. An acidic pH increases the proportion of the neutrally charged nTZDpa, which results in antimicrobial enhancement. Our results provide key insights into rational design of pH-sensitive membrane-active antimicrobials and antibiotic adjuvants that are effective in an infection environment. These findings demonstrate that nTZDpa is a promising lead compound for developing new therapeutics against hard-to-cure infections caused by drug-resistant and -tolerant S. aureus.
Assuntos
Staphylococcus aureus Resistente à Meticilina , Antibacterianos/uso terapêutico , Humanos , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana , Staphylococcus aureusRESUMO
Pseudomonas aeruginosa is an opportunistic bacterial pathogen that exhibits pathogenicity in an unusually broad range of plants and animals, and it is of interest to study the roles of particular virulence-related factors in diverse hosts. The production of many P. aeruginosa virulence factors is under the control of a quorum sensing (QS) signaling network, which has three interconnected branches that engage in intricate cross talk: Las, Rhl, and MvfR. Because there has been no systematic comparison of the roles of the three QS systems in mediating P. aeruginosa virulence in various hosts, we compared the virulence of wild-type (WT) P. aeruginosa PA14 and a set of isogenic PA14 QS in-frame deletion mutants in four selected hosts, the reference plant Arabidopsis thaliana (Arabidopsis), the crop plant Brassica napus (canola), the nematode Caenorhabditis elegans, and the fruit fly Drosophila melanogaster. The first letters of the selected host genera, A, B, C, and D, inspired the title of this article and indicate that this work lays the groundwork for future elucidation of the specific roles of each QS branch in mediating virulence in diverse hosts. IMPORTANCE In this study, we performed a systematic comparison of the virulence of WT P. aeruginosa and QS mutants in selected hosts and conditions. This work represents an important contribution to the long-term goal of unraveling the entangled roles of different branches of the P. aeruginosa QS network in different hosts and will serve as a valuable resource for the field of host-pathogen interactions.
Assuntos
Pseudomonas aeruginosa , Percepção de Quorum , Animais , Drosophila melanogaster , Pseudomonas aeruginosa/genética , Virulência , Fatores de Virulência/genéticaRESUMO
Pioneering microbial genomic surveys have revealed numerous untapped biosynthetic gene clusters, unveiling the great potential of new natural products. Here, using a combination of genome mining, mutasynthesis, and activity screening in an infection model comprising Caenorhabditis elegans and Pseudomonas aeruginosa, we identified candidate virulence-blocking amychelin siderophore compounds from actinomycetes. Subsequently, we developed unreported analogs of these virulence-blocking siderophores with improved potency by exploiting an Amycolatopsis methanolica strain 239T chorismate to salicylate a biosynthetic subpathway for mutasynthesis. This allowed us to generate the fluorinated amychelin, fluoroamychelin I, which rescued C. elegans from P. aeruginosa-mediated killing with an EC50 value of 1.4 µM, outperforming traditional antibiotics including ceftazidime and meropenem. In general, this paper describes an efficient platform for the identification and production of classes of anti-microbial compounds with potential unique modes of action.
Assuntos
Mineração de Dados , Genômica , Halogenação , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Sideróforos/química , Sideróforos/farmacologia , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Caenorhabditis elegans/genética , Ceftazidima/farmacologia , Avaliação Pré-Clínica de Medicamentos , Meropeném/farmacologiaRESUMO
Sphingosine-1-phophate (S1P) is a sphingolipid-derived signaling molecule that controls diverse cellular functions including cell growth, homeostasis, and stress responses. In a variety of metazoans, cytosolic S1P is transported into the extracellular space where it activates S1P receptors in a concentration-dependent manner. In the free-living nematode Caenorhabditis elegans, the spin-2 gene, which encodes a S1P transporter, is activated during Gram-positive or Gram-negative bacterial infection of the intestine. However, the role during infection of spin-2 and three additional genes in the C. elegans genome encoding other putative S1P transporters has not been elucidated. Here, we report an evolutionally conserved function for S1P and a non-canonical role for S1P transporters in the C. elegans immune response to bacterial pathogens. We found that mutations in the sphingosine kinase gene (sphk-1) or in the S1P transporter genes spin-2 or spin-3 decreased nematode survival after infection with Pseudomonas aeruginosa or Enterococcus faecalis. In contrast to spin-2 and spin-3, mutating spin-1 leads to an increase in resistance to P. aeruginosa. Consistent with these results, when wild-type C. elegans were supplemented with extracellular S1P, we found an increase in their lifespan when challenged with P. aeruginosa and E. faecalis. In comparison, spin-2 and spin-3 mutations suppressed the ability of S1P to rescue the worms from pathogen-mediated killing, whereas the spin-1 mutation had no effect on the immune-enhancing activity of S1P. S1P demonstrated no antimicrobial activity toward P. aeruginosa and Escherichia coli and only minimal activity against E. faecalis MMH594 (40 µM). These data suggest that spin-2 and spin-3, on the one hand, and spin-1, on the other hand, transport S1P across cellular membranes in opposite directions. Finally, the immune modulatory effect of S1P was diminished in C. eleganssek-1 and pmk-1 mutants, suggesting that the immunomodulatory effects of S1P are mediated by the p38 MAPK signaling pathway.
Assuntos
Caenorhabditis elegans/imunologia , Caenorhabditis elegans/microbiologia , Interações Hospedeiro-Patógeno/fisiologia , Lisofosfolipídeos/metabolismo , Esfingosina/análogos & derivados , Animais , Antibacterianos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Escherichia coli/patogenicidade , Humanos , Longevidade , Lisofosfolipídeos/farmacologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/patogenicidade , Transdução de Sinais , Esfingosina/metabolismo , Esfingosina/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
There is a significant need to combat the growing challenge of antibacterial drug resistance. We have previously developed a whole-animal dual-screening platform that first used the nematode Caenorhabditis elegans, to identify low-toxicity antibacterial hits in a high-throughput format. The hits were then evaluated in the wax moth caterpillar Galleria mellonella infection model to confirm efficacy and low toxicity at a whole animal level. This multi-host approach is a powerful tool for revealing compounds that show antibacterial effects and relatively low toxicity at the whole organism level. This paper reports the use of the multi-host approach to identify and validate five new anti-staphylococcal compounds: (1) 4,4',4â³-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol(PPT), (2) (1S,2S)-2-[2-[[3-(1H-benzimidazol-2-yl)propyl]methylamino]ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-(1-methylethyl)-2-naphthalenyl cyclopropanecarboxylate dihydrochloride(NNC), (3) 4,5,6,7-tetrabromobenzotriazole (TBB), (4) 3-[2-[2-chloro-4-[3-(2,6-dichlorophenyl)-5-(1-methylethyl)-4-isoxazolyl]methoxy]phenyl]ethenyl] benzoic acid(GW4064), and (5) N-(cyclopropylmethoxy)-3,4,5-trifluoro-2-[(4-iodo-2-methylphenyl)amino] benzamide(PD198306). The compounds reduced the severity of methicillin-resistant Staphylococcus aureus (MRSA, strain MW2) infections in both C. elegans and G. mellonella and showed minimal inhibitory concentrations (MICs) in the range of 2-8 µg/mL. Compounds NNC, PPT, and TBB permeabilized MRSA-MW2 cells to SYTOX green, suggesting that they target bacterial membranes. Compound TBB showed synergistic activity with doxycycline and oxacillin against MRSA-MW2, and compounds PPT, NNC, GW4064, and PD198306 synergized with doxycycline, polymyxin-B, gentamicin, and erythromycin, respectively. The study demonstrates the utility of the multi-host approach with follow-up hit characterization for prioritizing anti-MRSA compounds for further evaluation.
RESUMO
Resistance or tolerance to traditional antibiotics is a challenging issue in antimicrobial chemotherapy. Moreover, traditional bactericidal antibiotics kill only actively growing bacterial cells, whereas nongrowing metabolically inactive cells are tolerant to and therefore "persist" in the presence of legacy antibiotics. Here, we report that the diarylurea derivative PQ401, previously characterized as an inhibitor of the insulin-like growth factor I receptor, kills both antibiotic-resistant and nongrowing antibiotic-tolerant methicillin-resistant Staphylococcus aureus (MRSA) by lipid bilayer disruption. PQ401 showed several beneficial properties as an antimicrobial lead compound, including rapid killing kinetics, low probability for resistance development, high selectivity to bacterial membranes compared to mammalian membranes, and synergism with gentamicin. In contrast to well-studied membrane-disrupting cationic antimicrobial low-molecular-weight compounds and peptides, molecular dynamic simulations supported by efficacy data demonstrate that the neutral form of PQ401 penetrates and subsequently embeds into bacterial lipid bilayers more effectively than the cationic form. Lastly, PQ401 showed efficacy in both the Caenorhabditis elegans and Galleria mellonella models of MRSA infection. These data suggest that PQ401 may be a lead candidate for repurposing as a membrane-active antimicrobial and has potential for further development as a human antibacterial therapeutic for difficult-to-treat infections caused by both drug-resistant and -tolerant S. aureusIMPORTANCE Membrane-damaging antimicrobial agents have great potential to treat multidrug-resistant or multidrug-tolerant bacteria against which conventional antibiotics are not effective. However, their therapeutic applications are often hampered due to their low selectivity to bacterial over mammalian membranes or their potential for cross-resistance to a broad spectrum of cationic membrane-active antimicrobial agents. We discovered that the diarylurea derivative compound PQ401 has antimicrobial potency against multidrug-resistant and multidrug-tolerant Staphylococcus aureus PQ401 selectively disrupts bacterial membrane lipid bilayers in comparison to mammalian membranes. Unlike cationic membrane-active antimicrobials, the neutral form of PQ401 rather than its cationic form exhibits maximum membrane activity. Overall, our results demonstrate that PQ401 could be a promising lead compound that overcomes the current limitations of membrane selectivity and cross-resistance. Also, this work provides deeper insight into the design and development of new noncharged membrane-targeting therapeutics to combat hard-to-cure bacterial infections.
Assuntos
Aminoquinolinas/farmacologia , Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Compostos de Fenilureia/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Animais , Caenorhabditis elegans/microbiologia , Sinergismo Farmacológico , Gentamicinas/farmacologia , Hemólise/efeitos dos fármacos , Humanos , Larva/microbiologia , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Mariposas/microbiologia , Infecções Estafilocócicas/microbiologiaRESUMO
BACKGROUND: Multicellular animals and bacteria frequently engage in predator-prey and host-pathogen interactions, such as the well-studied relationship between Pseudomonas aeruginosa and the nematode Caenorhabditis elegans. This study investigates the genomic and genetic basis of bacterial-driven variability in P. aeruginosa virulence towards C. elegans to provide evolutionary insights into host-pathogen relationships. RESULTS: Natural isolates of P. aeruginosa that exhibit diverse genomes display a broad range of virulence towards C. elegans. Using gene association and genetic analysis, we identify accessory genome elements that correlate with virulence, including both known and novel virulence determinants. Among the novel genes, we find a viral-like mobile element, the teg block, that impairs virulence and whose acquisition is restricted by CRISPR-Cas systems. Further genetic and genomic evidence suggests that spacer-targeted elements preferentially associate with lower virulence while the presence of CRISPR-Cas associates with higher virulence. CONCLUSIONS: Our analysis demonstrates substantial strain variation in P. aeruginosa virulence, mediated by specific accessory genome elements that promote increased or decreased virulence. We exemplify that viral-like accessory genome elements that decrease virulence can be restricted by bacterial CRISPR-Cas immune defense systems, and suggest a positive, albeit indirect, role for host CRISPR-Cas systems in virulence maintenance.
Assuntos
Caenorhabditis elegans/microbiologia , Interações Hospedeiro-Patógeno/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Animais , Sistemas CRISPR-Cas , Genoma Bacteriano , Sequências Repetitivas DispersasRESUMO
Treatment of Staphylococcus aureus infections is complicated by the development of antibiotic tolerance, a consequence of the ability of S. aureus to enter into a nongrowing, dormant state in which the organisms are referred to as persisters. We report that the clinically approved anthelmintic agent bithionol kills methicillin-resistant S. aureus (MRSA) persister cells, which correlates with its ability to disrupt the integrity of Gram-positive bacterial membranes. Critically, bithionol exhibits significant selectivity for bacterial compared with mammalian cell membranes. All-atom molecular dynamics (MD) simulations demonstrate that the selectivity of bithionol for bacterial membranes correlates with its ability to penetrate and embed in bacterial-mimic lipid bilayers, but not in cholesterol-rich mammalian-mimic lipid bilayers. In addition to causing rapid membrane permeabilization, the insertion of bithionol increases membrane fluidity. By using bithionol and nTZDpa (another membrane-active antimicrobial agent), as well as analogs of these compounds, we show that the activity of membrane-active compounds against MRSA persisters positively correlates with their ability to increase membrane fluidity, thereby establishing an accurate biophysical indicator for estimating antipersister potency. Finally, we demonstrate that, in combination with gentamicin, bithionol effectively reduces bacterial burdens in a mouse model of chronic deep-seated MRSA infection. This work highlights the potential repurposing of bithionol as an antipersister therapeutic agent.
Assuntos
Antibacterianos/farmacologia , Membrana Celular/efeitos dos fármacos , Reposicionamento de Medicamentos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Animais , Bitionol/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Colesterol/química , Modelos Animais de Doenças , Sinergismo Farmacológico , Gentamicinas/farmacologia , Bicamadas Lipídicas/química , Fluidez de Membrana/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/ultraestrutura , Simulação de Dinâmica Molecular , Fosfatidilcolinas/química , Relação Estrutura-Atividade , Lipossomas UnilamelaresRESUMO
My trajectory to becoming a plant biologist was shaped by a complex mix of scientific, political, sociological, and personal factors. I was trained as a microbiologist and molecular biologist in the late 1960s and early 1970s, a time of political upheaval surrounding the Vietnam War. My political activism taught me to be wary of the potential misuses of scientific knowledge and to promote the positive applications of science for the benefit of society. I chose agricultural science for my postdoctoral work. Because I was not trained as a plant biologist, I devised a postdoctoral project that took advantage of my microbiological training, and I explored using genetic technologies to transfer the ability to fix nitrogen from prokaryotic nitrogen-fixing species to the model plant Arabidopsis thaliana with the ultimate goal of engineering crop plants. The invention of recombinant DNA technology greatly facilitated the cloning and manipulation of bacterial nitrogen-fixation ( nif) genes, but it also forced me to consider how much genetic engineering of organisms, including human beings, is acceptable. My laboratory has additionally studied host-pathogen interactions using Arabidopsis and the nematode Caenorhabditis elegans as model hosts.
Assuntos
Arabidopsis/genética , Caenorhabditis elegans/genética , Interações Hospedeiro-Patógeno/genética , Simbiose/genética , Animais , Arabidopsis/microbiologia , Biologia/história , Caenorhabditis elegans/microbiologia , História do Século XX , História do Século XXI , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/patogenicidade , Fixação de Nitrogênio/genética , Sinorhizobium meliloti/genética , Sinorhizobium meliloti/patogenicidadeRESUMO
Conventional antibiotics are not effective in treating infections caused by drug-resistant or persistent nongrowing bacteria, creating a dire need for the development of new antibiotics. We report that the small molecule nTZDpa, previously characterized as a nonthiazolidinedione peroxisome proliferator-activated receptor gamma partial agonist, kills both growing and persistent Staphylococcus aureus cells by lipid bilayer disruption. S. aureus exhibited no detectable development of resistance to nTZDpa, and the compound acted synergistically with aminoglycosides. We improved both the potency and selectivity of nTZDpa against MRSA membranes compared to mammalian membranes by leveraging synthetic chemistry guided by molecular dynamics simulations. These studies provide key insights into the design of selective and potent membrane-active antibiotics effective against bacterial persisters.
Assuntos
Antibacterianos/farmacologia , Descoberta de Drogas , Indóis/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Sulfetos/farmacologia , Eritrócitos/efeitos dos fármacos , Humanos , Bicamadas Lipídicas/metabolismo , Testes de Sensibilidade Microbiana , Staphylococcus aureus/efeitos dos fármacosRESUMO
High throughput DNA sequencing methodology (next generation sequencing; NGS) has rapidly evolved over the past 15 years and new methods are continually being commercialized. As the technology develops, so do increases in the number of corresponding applications for basic and applied science. The purpose of this review is to provide a compendium of NGS methodologies and associated applications. Each brief discussion is followed by web links to the manufacturer and/or web-based visualizations. Keyword searches, such as with Google, may also provide helpful internet links and information. © 2018 by John Wiley & Sons, Inc.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Genoma , HumanosRESUMO
Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.
Assuntos
Elementos de DNA Transponíveis/genética , Mutagênese/genética , Pseudomonas aeruginosa/química , Animais , Humanos , Mutagênese InsercionalRESUMO
A challenge in the treatment of Staphylococcus aureus infections is the high prevalence of methicillin-resistant S. aureus (MRSA) strains and the formation of non-growing, dormant 'persister' subpopulations that exhibit high levels of tolerance to antibiotics and have a role in chronic or recurrent infections. As conventional antibiotics are not effective in the treatment of infections caused by such bacteria, novel antibacterial therapeutics are urgently required. Here we used a Caenorhabditis elegans-MRSA infection screen to identify two synthetic retinoids, CD437 and CD1530, which kill both growing and persister MRSA cells by disrupting lipid bilayers. CD437 and CD1530 exhibit high killing rates, synergism with gentamicin, and a low probability of resistance selection. All-atom molecular dynamics simulations demonstrated that the ability of retinoids to penetrate and embed in lipid bilayers correlates with their bactericidal ability. An analogue of CD437 was found to retain anti-persister activity and show an improved cytotoxicity profile. Both CD437 and this analogue, alone or in combination with gentamicin, exhibit considerable efficacy in a mouse model of chronic MRSA infection. With further development and optimization, synthetic retinoids have the potential to become a new class of antimicrobials for the treatment of Gram-positive bacterial infections that are currently difficult to cure.
Assuntos
Antibacterianos/classificação , Antibacterianos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Retinoides/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Animais , Antibacterianos/efeitos adversos , Antibacterianos/uso terapêutico , Benzoatos/química , Benzoatos/farmacologia , Benzoatos/uso terapêutico , Benzoatos/toxicidade , Caenorhabditis elegans/efeitos dos fármacos , Caenorhabditis elegans/microbiologia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Gentamicinas/farmacologia , Gentamicinas/uso terapêutico , Humanos , Bicamadas Lipídicas/química , Staphylococcus aureus Resistente à Meticilina/citologia , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Camundongos , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Mutação , Naftóis/química , Naftóis/farmacologia , Naftóis/uso terapêutico , Naftóis/toxicidade , Retinoides/química , Retinoides/uso terapêutico , Retinoides/toxicidadeRESUMO
When Drosophila melanogaster feeds on Pseudomonas aeruginosa, some bacteria cross the intestinal barrier and eventually proliferate in the hemocoel. This process is limited by hemocytes through phagocytosis. P. aeruginosa requires the quorum-sensing regulator RhlR to elude the cellular immune response of the fly. RhlI synthesizes the autoinducer signal that activates RhlR. Here, we show that rhlI mutants are unexpectedly more virulent than rhlR mutants, both in fly and in nematode intestinal infection models, suggesting that RhlR has RhlI-independent functions. We also report that RhlR protects P. aeruginosa from opsonization mediated by the Drosophila thioester-containing protein 4 (Tep4). RhlR mutant bacteria show higher levels of Tep4-mediated opsonization, as compared to rhlI mutants, which prevents lethal bacteremia in the Drosophila hemocoel. In contrast, in a septic model of infection, in which bacteria are introduced directly into the hemocoel, Tep4 mutant flies are more resistant to wild-type P. aeruginosa, but not to the rhlR mutant. Thus, depending on the infection route, the Tep4 opsonin can either be protective or detrimental to host defense.
Assuntos
Proteínas de Bactérias/genética , RNA Helicases DEAD-box/genética , Ligases/genética , Fagocitose , Pseudomonas aeruginosa/genética , Percepção de Quorum/genética , Fatores de Transcrição/genética , Animais , Caenorhabditis elegans/microbiologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/imunologia , Drosophila melanogaster/imunologia , Drosophila melanogaster/microbiologia , Regulação Bacteriana da Expressão Gênica , Intestinos/imunologia , Intestinos/microbiologia , Pseudomonas aeruginosa/patogenicidade , Receptores de Reconhecimento de Padrão/imunologia , VirulênciaRESUMO
The innate immune response of the nematode Caenorhabditis elegans has been extensively studied and a variety of Toll-independent immune response pathways have been identified. Surprisingly little, however, is known about how pathogens activate the C. elegans immune response. Enterococcus faecalis and Enterococcus faecium are closely related enterococcal species that exhibit significantly different levels of virulence in C. elegans infection models. Previous work has shown that activation of the C. elegans immune response by Pseudomonas aeruginosa involves P. aeruginosa-mediated host damage. Through ultrastructural imaging, we report that infection with either E. faecalis or E. faecium causes the worm intestine to become distended with proliferating bacteria in the absence of extensive morphological changes and apparent physical damage. Genetic analysis, whole-genome transcriptional profiling, and multiplexed gene expression analysis demonstrate that both enterococcal species, whether live or dead, induce a rapid and similar transcriptional defense response dependent upon previously described immune signaling pathways. The host response to E. faecium shows a stricter dependence upon stress response signaling pathways than the response to E. faecalis. Unexpectedly, we find that E. faecium is a C. elegans pathogen and that an active wild-type host defense response is required to keep an E. faecium infection at bay. These results provide new insights into the mechanisms underlying the C. elegans immune response to pathogen infection.