Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 22
Filtrar
1.
Mol Cell Proteomics ; : 100851, 2024 Oct 02.
Artículo en Inglés | MEDLINE | ID: mdl-39366656

RESUMEN

The bacillus Calmette-Guérin BCG vaccine (Mycobacterium bovis) is primarily used to prevent tuberculosis (TB) infections but has wide-ranging immunogenic effects. One of its most notable properties is its ability to induce trained immunity, a memory-like response in innate immune cells such as macrophages. Through targeted analyses of well-established histone marks, prior research has shown that these changes are generated through epigenetic modification. Mass spectrometry-based proteomic approaches provide a way to globally profile various aspects of the proteome, providing data to further identify unexplored mechanisms of BCG-mediated immunomodulation. Here we use multi-level proteomics (total, histone, and phospho to identify networks and potential mechanisms that mediate BCG induced immunomodulation in macrophages. Histone-focused proteomics and total proteomics were performed at the University of Cape Town (data available via ProteomeXchange with identifier PXD051187), while phosphoproteomics data was retrieved from the ProteomeXchange Repository (identifier PXD013171). We identify several epigenetic mechanisms that may drive BCG-induced training phenotypes. Evidence across the proteomics and histone-focused proteomics data sets pair 6 epigenetic effectors (NuA4, NuRD, NSL, Sin3A, SIRT2, SIRT6) and their substrates.

2.
Chemistry ; 30(8): e202303047, 2024 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-37966101

RESUMEN

Glycosylphosphatidylinositols (GPIs) need to interact with other components in the cell membrane to transduce transmembrane signals. A bifunctional GPI probe was employed for photoaffinity-based proximity labelling and identification of GPI-interacting proteins in the cell membrane. This probe contained the entire core structure of GPIs and was functionalized with photoreactive diazirine and clickable alkyne to facilitate its crosslinking with proteins and attachment of an affinity tag. It was disclosed that this probe was more selective than our previously reported probe containing only a part structure of the GPI core for cell membrane incorporation and an improved probe for studying GPI-cell membrane interaction. Eighty-eight unique membrane proteins, many of which are related to GPIs/GPI-anchored proteins, were identified utilizing this probe. The proteomics dataset is a valuable resource for further analyses and data mining to find new GPI-related proteins and signalling pathways. A comparison of these results with those of our previous probe provided direct evidence for the profound impact of GPI glycan structure on its interaction with the cell membrane.


Asunto(s)
Glicosilfosfatidilinositoles , Polisacáridos , Glicosilfosfatidilinositoles/química , Membrana Celular/metabolismo , Polisacáridos/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal
3.
Anal Chem ; 94(51): 17810-17818, 2022 12 27.
Artículo en Inglés | MEDLINE | ID: mdl-36520113

RESUMEN

Liquid chromatography (LC)-mass spectrometry (MS)/MS lipidomic normalization is generally performed by equalizing pre-extraction sample materials or via DNA or protein pre-quantitation methods, which have known measurement inaccuracies. We propose the use of the sulfo-phospho-vanillin assay (SPVA), a total lipid colorimetric analysis, as a pre-quantitation method to normalize lipids in lipidomic LC-MS/MS applications. The assay has been applied to a 300 µL well volume in a 96-well plate and tested using Avanti total lipid standards of porcine brain and E. coli. Assay parameters for lipid sample volume, sulfuric acid, vanillin/phosphoric acid, post-reaction incubation time, and wavelength are optimized for robust application to biologically sourced lipid samples. Standard test samples were prepared using three concentrations covering approximately 100 µg/mL range. The optimized assay yielded test sample errors less than 10%, indicating a precise and accurate assay performance. The test samples were then analyzed by LC-MS/MS and normalized using SPVA pre-quantitation and pseudo-mass normalization. The detected lipids showed smaller standard deviations and greater relative concentration differences compared to the pseudo-mass normalized lipids, showing promise as a normalization method.


Asunto(s)
Lipidómica , Espectrometría de Masas en Tándem , Animales , Porcinos , Cromatografía Liquida/métodos , Espectrometría de Masas en Tándem/métodos , Escherichia coli , Lípidos/análisis
4.
Anal Chem ; 93(6): 3154-3162, 2021 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-33534538

RESUMEN

Glycosphingolipids (GSLs) play a key role in various biological and pathological events. Thus, determination of the complete GSL compositions in human tissues is essential for comparative and functional studies of GSLs. In this work, a new strategy was developed for GSL characterization and glycolipidomics analysis based on two-stage matching of experimental and reference MS/MS spectra. In the first stage, carbohydrate fragments, which contain only glycans and thus are conserved within a GSL species, are directly matched to yield a species identification. In the second stage, glycolipid fragments from the matched GSL species, which contain both the lipid and glycans and thus shift due to lipid structural changes, are treated according to lipid rule-based matching to characterize the lipid compositions. This new strategy uses the whole spectrum for GSL characterization. Furthermore, simple databases containing only a single lipid form per GSL species can be utilized to identify multiple GSL lipid forms. It is expected that this method will help accelerate glycolipidomics analysis and disclose new and diverse lipid forms of GSLs.


Asunto(s)
Glicoesfingolípidos , Espectrometría de Masas en Tándem , Cromatografía Liquida , Humanos , Lípidos , Polisacáridos
5.
J Am Chem Soc ; 142(32): 13645-13650, 2020 08 12.
Artículo en Inglés | MEDLINE | ID: mdl-32702987

RESUMEN

The nematode Caenorhabditis elegans produces a broad family of pheromones, known as the ascarosides, that are modified with a variety of groups derived from primary metabolism. These modifications are essential for the diverse activities of the ascarosides in development and various behaviors, including attraction, aggregation, avoidance, and foraging. The mechanism by which these different groups are added to the ascarosides is poorly understood. Here, we identify a family of over 30 enzymes, which are homologous to mammalian carboxylesterase (CES) enzymes, and show that a number of these enzymes are responsible for the selective addition of specific modifications to the ascarosides. Through stable isotope feeding experiments, we demonstrate the in vivo activity of the CES-like enzymes and provide direct evidence that the acyl-CoA synthetase ACS-7, which was previously implicated in the attachment of certain modifications to the ascarosides in C. elegans, instead activates the side chains of certain ascarosides for shortening through ß-oxidation. Our data provide a key to the combinatorial logic that gives rise to different modified ascarosides, which should greatly facilitate the exploration of the specific biological functions of these pheromones in the worm.


Asunto(s)
Caenorhabditis elegans/enzimología , Carboxilesterasa/metabolismo , Coenzima A Ligasas/metabolismo , Animales , Glucolípidos/biosíntesis , Glucolípidos/química , Estructura Molecular
6.
Artículo en Inglés | MEDLINE | ID: mdl-33106266

RESUMEN

Mycobacterium abscessus causes serious infections that often require over 18 months of antibiotic combination therapy. There is no standard regimen for the treatment of M. abscessus infections, and the multitude of combinations that have been used clinically have had low success rates and high rates of toxicities. With ß-lactam antibiotics being safe, double ß-lactam and ß-lactam/ß-lactamase inhibitor combinations are of interest for improving the treatment of M. abscessus infections and minimizing toxicity. However, a mechanistic approach for building these combinations is lacking since little is known about which penicillin-binding protein (PBP) target receptors are inactivated by different ß-lactams in M. abscessus We determined the preferred PBP targets of 13 ß-lactams and 2 ß-lactamase inhibitors in two M. abscessus strains and identified PBP sequences by proteomics. The Bocillin FL binding assay was used to determine the ß-lactam concentrations that half-maximally inhibited Bocillin binding (50% inhibitory concentrations [IC50s]). Principal component analysis identified four clusters of PBP occupancy patterns. Carbapenems inactivated all PBPs at low concentrations (0.016 to 0.5 mg/liter) (cluster 1). Cephalosporins (cluster 2) inactivated PonA2, PonA1, and PbpA at low (0.031 to 1 mg/liter) (ceftriaxone and cefotaxime) or intermediate (0.35 to 16 mg/liter) (ceftazidime and cefoxitin) concentrations. Sulbactam, aztreonam, carumonam, mecillinam, and avibactam (cluster 3) inactivated the same PBPs as cephalosporins but required higher concentrations. Other penicillins (cluster 4) specifically targeted PbpA at 2 to 16 mg/liter. Carbapenems, ceftriaxone, and cefotaxime were the most promising ß-lactams since they inactivated most or all PBPs at clinically relevant concentrations. These first PBP occupancy patterns in M. abscessus provide a mechanistic foundation for selecting and optimizing safe and effective combination therapies with ß-lactams.


Asunto(s)
Mycobacterium abscessus , Inhibidores de beta-Lactamasas , Antibacterianos/farmacología , Pruebas de Sensibilidad Microbiana , Proteínas de Unión a las Penicilinas/genética , Penicilinas , Inhibidores de beta-Lactamasas/farmacología , beta-Lactamas/farmacología
7.
Proteomics ; 19(13): e1800394, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31070281

RESUMEN

Exosomes are membrane-bound vesicles that traffic small molecular cargos. These cargos participate in cell-cell communication and contribute to the pathogenesis of many disease including cancer. How these mechanisms contribute to communication within the pancreatic adenocarcinoma (PDAC) microenvironment and how they contribute to PDAC biology are poorly understood. Performed in this study are comprehensive, quantitative comparisons of the proteomes of three PDAC cell lines to those of the exosomes they produce. Approximately 35% of whole cell proteins sort into exosomes. Analysis of composition of microbiomes (ANCOM) determined a cluster of 98 enriched pancreatic cancer exosome core proteins (ePC-ECPs). Further, these proteins are predicted by ingenuity pathway analysis (IPA) as actively involved in signaling pathways regulating cell death and survival, cellular movement, and cell-to-cell signaling and interaction in particular (top three p-value significant pathways). Significant enrichment of canonical pathways of acute phase response signaling (inflammatory response signaling pathways) and FXR and RXR activation in biosynthetic pathways are also predicted; 97 ePC-ECPs are associated with cancer and among them, 34 are specifically associated with PDAC. In conclusion, exosomes from PDAC are enriched with cancer-associated signaling proteins. Further assessment of these proteins as PDAC biomarkers or therapeutic targets is warranted.


Asunto(s)
Adenocarcinoma/patología , Exosomas/metabolismo , Neoplasias Pancreáticas/patología , Proteoma/metabolismo , Biomarcadores de Tumor/metabolismo , Comunicación Celular/fisiología , Línea Celular Tumoral , Movimiento Celular/fisiología , Supervivencia Celular/fisiología , Humanos , Transducción de Señal , Microambiente Tumoral , Neoplasias Pancreáticas
8.
Cell Chem Biol ; 31(5): 1011-1022.e6, 2024 May 16.
Artículo en Inglés | MEDLINE | ID: mdl-38183989

RESUMEN

Methyl ketone (MK)-ascarosides represent essential components of several pheromones in Caenorhabditis elegans, including the dauer pheromone, which triggers the stress-resistant dauer larval stage, and the male-attracting sex pheromone. Here, we identify an acyl-CoA thioesterase, ACOT-15, that is required for the biosynthesis of MK-ascarosides. We propose a model in which ACOT-15 hydrolyzes the ß-keto acyl-CoA side chain of an ascaroside intermediate during ß-oxidation, leading to decarboxylation and formation of the MK. Using comparative metabolomics, we identify additional ACOT-15-dependent metabolites, including an unusual piperidyl-modified ascaroside, reminiscent of the alkaloid pelletierine. The ß-keto acid generated by ACOT-15 likely couples to 1-piperideine to produce the piperidyl ascaroside, which is much less dauer-inducing than the dauer pheromone, asc-C6-MK (ascr#2, 1). The bacterial food provided influences production of the piperidyl ascaroside by the worm. Our work shows how the biosynthesis of MK- and piperidyl ascarosides intersect and how bacterial food may impact chemical signaling in the worm.


Asunto(s)
Caenorhabditis elegans , Feromonas , Animales , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Feromonas/metabolismo , Feromonas/biosíntesis , Feromonas/química , Proteínas de Caenorhabditis elegans/metabolismo , Tioléster Hidrolasas/metabolismo
9.
bioRxiv ; 2024 Mar 13.
Artículo en Inglés | MEDLINE | ID: mdl-37398242

RESUMEN

Despite an increasingly detailed picture of the molecular mechanisms of phage-bacterial interactions, we lack an understanding of how these interactions evolve and impact disease within patients. Here we report a year-long, nation-wide study of diarrheal disease patients in Bangladesh. Among cholera patients, we quantified Vibrio cholerae (prey) and its virulent phages (predators) using metagenomics and quantitative PCR, while accounting for antibiotic exposure using quantitative mass spectrometry. Virulent phage (ICP1) and antibiotics suppressed V. cholerae to varying degrees and were inversely associated with severe dehydration depending on resistance mechanisms. In the absence of anti-phage defenses, predation was 'effective,' with a high predator:prey ratio that correlated with increased genetic diversity among the prey. In the presence of anti-phage defenses, predation was 'ineffective,' with a lower predator:prey ratio that correlated with increased genetic diversity among the predators. Phage-bacteria coevolution within patients should therefore be considered in the deployment of phage-based therapies and diagnostics.

10.
Science ; 384(6693): eadj3166, 2024 Apr 19.
Artículo en Inglés | MEDLINE | ID: mdl-38669570

RESUMEN

Despite an increasingly detailed picture of the molecular mechanisms of bacteriophage (phage)-bacterial interactions, we lack an understanding of how these interactions evolve and impact disease within patients. In this work, we report a year-long, nationwide study of diarrheal disease patients in Bangladesh. Among cholera patients, we quantified Vibrio cholerae (prey) and its virulent phages (predators) using metagenomics and quantitative polymerase chain reaction while accounting for antibiotic exposure using quantitative mass spectrometry. Virulent phage (ICP1) and antibiotics suppressed V. cholerae to varying degrees and were inversely associated with severe dehydration depending on resistance mechanisms. In the absence of antiphage defenses, predation was "effective," with a high predator:prey ratio that correlated with increased genetic diversity among the prey. In the presence of antiphage defenses, predation was "ineffective," with a lower predator:prey ratio that correlated with increased genetic diversity among the predators. Phage-bacteria coevolution within patients should therefore be considered in the deployment of phage-based therapies and diagnostics.


Asunto(s)
Bacteriófagos , Cólera , Variación Genética , Vibrio cholerae , Cólera/microbiología , Vibrio cholerae/genética , Vibrio cholerae/virología , Bacteriófagos/genética , Bacteriófagos/fisiología , Humanos , Bangladesh , Antibacterianos/uso terapéutico , Índice de Severidad de la Enfermedad , Adulto , Metagenómica
11.
Invest Ophthalmol Vis Sci ; 64(1): 7, 2023 01 03.
Artículo en Inglés | MEDLINE | ID: mdl-36630141

RESUMEN

Purpose: To determine correlations between lipids in the fluid reservoir (FR) and the severity of midday fogging (MDF) in scleral lens (SL) wear. Methods: SL neophytes were recruited to wear custom SL for 4 days, examined after 8 hours on days 1 and 4. Lens vault and MDF were quantified from anterior segment optical coherence tomography (AS-OCT), and the FR was collected and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Relative abundance of lipids was compared to MDF scores using nonparametric correlation testing (Spearman rank). Ocular surface and SL fitting characteristics (lens vault, fitting curves) were likewise compared to MDF. Results: Thirteen participants (26 eyes, 69% female, 28 ± 9 years old) were included in this study. MDF severity after 8 hours of SL wear was 33 ± 29 units on day 1 and 28 ± 24 units on day 4 (r = .94; P < 0.01). Twelve samples were analyzed using LC-MS/MS, and a total of 170 distinct lipid species were detected. The lipid classes with greatest correlation to MDF were the wax esters (r = .73, P = 0.01), cholesteryl esters (r = .59; P = 0.049), and triacylglycerols (r = .64, P = 0.03). Polar lipids were observed abundantly in all samples. None of the measured ocular surface or fitting outcomes were correlated to MDF. Conclusions: Nonpolar lipids were the greatest contributors to MDF among these normal participants. Polar lipids may be due to cellular debris, although they do not appear contributory to MDF.


Asunto(s)
Lentes de Contacto Hidrofílicos , Espectrometría de Masas en Tándem , Humanos , Femenino , Adulto Joven , Adulto , Masculino , Cromatografía Liquida , Esclerótica , Lípidos
12.
PLoS One ; 18(7): e0280169, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37418364

RESUMEN

The spiny mouse (Acomys) is gaining popularity as a research organism due to its phenomenal regenerative capabilities. Acomys recovers from injuries to several organs without fibrosis. For example, Acomys heals full thickness skin injuries with rapid re-epithelialization of the wound and regeneration of hair follicles, sebaceous glands, erector pili muscles, adipocytes, and dermis without scarring. Understanding mechanisms of Acomys regeneration may uncover potential therapeutics for wound healing in humans. However, access to Acomys colonies is limited and primary fibroblasts can only be maintained in culture for a limited time. To address these obstacles, we generated immortalized Acomys dermal fibroblast cell lines using two methods: transfection with the SV40 large T antigen and spontaneous immortalization. The two cell lines (AcoSV40 and AcoSI-1) maintained the morphological and functional characteristics of primary Acomys fibroblasts, including maintenance of key fibroblast markers and ECM deposition. The availability of these cells will lower the barrier to working with Acomys as a model research organism, increasing the pace at which new discoveries to promote regeneration in humans can be made.


Asunto(s)
Murinae , Regeneración , Humanos , Animales , Regeneración/fisiología , Murinae/fisiología , Piel/metabolismo , Cicatrización de Heridas/fisiología , Fibroblastos/fisiología
13.
Protein Sci ; 32(1): e4537, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36482787

RESUMEN

Oxalate decarboxylase from Bacillus subtilis is a binuclear Mn-dependent acid stress response enzyme that converts the mono-anion of oxalic acid into formate and carbon dioxide in a redox neutral unimolecular disproportionation reaction. A π-stacked tryptophan dimer, W96 and W274, at the interface between two monomer subunits facilitates long-range electron transfer between the two Mn ions and plays an important role in the catalytic mechanism. Substitution of W96 with the unnatural amino acid 5-hydroxytryptophan leads to a persistent EPR signal which can be traced back to the neutral radical of 5-hydroxytryptophan with its hydroxyl proton removed. 5-Hydroxytryptophan acts as a hole sink preventing the formation of Mn(III) at the N-terminal active site and strongly suppresses enzymatic activity. The lower boundary of the standard reduction potential for the active site Mn(II)/Mn(III) couple can therefore be estimated as 740 mV against the normal hydrogen electrode at pH 4, the pH of maximum catalytic efficiency. Our results support the catalytic importance of long-range electron transfer in oxalate decarboxylase while at the same time highlighting the utility of unnatural amino acid incorporation and specifically the use of 5-hydroxytryptophan as an energetic sink for hole hopping to probe electron transfer in redox proteins.


Asunto(s)
5-Hidroxitriptófano , Electrones , 5-Hidroxitriptófano/metabolismo , Manganeso/química , Oxidación-Reducción , Ácido Oxálico , Espectroscopía de Resonancia por Spin del Electrón
14.
Cell Chem Biol ; 30(2): 203-213.e17, 2023 02 16.
Artículo en Inglés | MEDLINE | ID: mdl-36750097

RESUMEN

Proteolysis targeting chimeras (PROTACs) are bifunctional molecules that degrade target proteins through recruiting E3 ligases. However, their application is limited in part because few E3 ligases can be recruited by known E3 ligase ligands. In this study, we identified piperlongumine (PL), a natural product, as a covalent E3 ligase recruiter, which induces CDK9 degradation when it is conjugated with SNS-032, a CDK9 inhibitor. The lead conjugate 955 can potently degrade CDK9 in a ubiquitin-proteasome-dependent manner and is much more potent than SNS-032 against various tumor cells in vitro. Mechanistically, we identified KEAP1 as the E3 ligase recruited by 955 to degrade CDK9 through a TurboID-based proteomics study, which was further confirmed by KEAP1 knockout and the nanoBRET ternary complex formation assay. In addition, PL-ceritinib conjugate can degrade EML4-ALK fusion oncoprotein, suggesting that PL may have a broader application as a covalent E3 ligase ligand in targeted protein degradation.


Asunto(s)
Factor 2 Relacionado con NF-E2 , Ubiquitina-Proteína Ligasas , Proteolisis , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ligandos
15.
Curr Eye Res ; 47(6): 832-842, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35317695

RESUMEN

PURPOSE: Contact lens complications occur more often in older teenagers and young adults compared to children. This study explored differences in tear proteomics between children and young adults wearing soft contact lens (SCL), orthokeratology or spectacles for >3 years. METHODS: Twelve children and 12 sex- and correction-matched young adults were enrolled. Tears were collected via Schirmer strips for tear proteomic analysis using mass spectrometry. Proteome Discoverer was used for protein identification. Label-Free Quantitation was generated using Scaffold software; Fisher's Exact tests were used to compare proteins by age and correction groups. Generalized linear models were used to assess differences in overall protein levels by age and correction groups. A secondary analysis of proteins presented in >50% of samples of each group was conducted using the R/Bioconductor limma package. RESULTS: There were 385 proteins present only in young adults while 183 were unique in children. There were 528 unique proteins to SCL, 96 to orthokeratology and 149 to spectacle wearers. Based on Fisher's Exact analyses, 126 proteins were higher in young adults than children (all P < 0.048). Forty-seven protein levels were higher in SCL compared to orthokeratology (all P < 0.01), 33 protein levels were higher in SCL compared to spectacles (all P < 0.01), 15 protein levels were higher in orthokeratology compared to spectacle wearers (all P < 0.01). Based on generalized linear models, young adults had higher overall protein levels than children (P = 0.001), SCL had higher protein levels than spectacle wearers (P < 0.001) but no differences were found between orthokeratology and spectacle wearers (P = 0.79). Based on the secondary analysis, only Antileukoproteinase was higher in the young adult orthokeratology group compared to other groups (P < 0.01). CONCLUSIONS: Tear protein type and abundance differ by age and correction. Further research is needed to understand the effects of contact lens correction in children and young adults on the tear proteome.


Asunto(s)
Lentes de Contacto Hidrofílicos , Oftalmopatías , Adolescente , Niño , Oftalmopatías/metabolismo , Anteojos , Humanos , Proyectos Piloto , Proteoma/metabolismo , Proteómica , Lágrimas/metabolismo , Adulto Joven
16.
ACS Omega ; 7(7): 6403-6411, 2022 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-35224401

RESUMEN

Glycosphingolipids (GSLs), including gangliosides, are essential components of the cell membrane. Because of their vital biological functions, a facile method for the analysis and comparison of GSLs in biological issues is desired. To this end, a new method for GSL analysis was developed based on two-stage matching of the carbohydrate and glycolipid product ions of experimental and reference MS/MS spectra of GSLs. The applicability of this method to the analysis of gangliosides in biological tissues was verified using human plasma and mouse brains spiked with standards. The method was then used to characterize endogenous gangliosides in mouse and human brains. It was shown that each endogenous ganglioside species had varied lipid forms and that mouse and human brains had different compositions of ganglioside species and lipid forms. Moreover, a 36-carbon ceramide is found to represent the major lipid form for mouse brain gangliosides, while the major lipid form for most human brain gangliosides is a 38-carbon ceramide. This study has verified that the two-stage MS/MS spectral matching method could be used to study gangliosides or GSLs and their lipid forms in complex biological samples, thereby having a broad application.

17.
Microb Genom ; 8(12)2022 12.
Artículo en Inglés | MEDLINE | ID: mdl-36748512

RESUMEN

The antibiotic formulary is threatened by high rates of antimicrobial resistance (AMR) among enteropathogens. Enteric bacteria are exposed to anaerobic conditions within the gastrointestinal tract, yet little is known about how oxygen exposure influences AMR. The facultative anaerobe Vibrio cholerae was chosen as a model to address this knowledge gap. We obtained V. cholerae isolates from 66 cholera patients, sequenced their genomes, and grew them under anaerobic and aerobic conditions with and without three clinically relevant antibiotics (ciprofloxacin, azithromycin, doxycycline). For ciprofloxacin and azithromycin, the minimum inhibitory concentration (MIC) increased under anaerobic conditions compared to aerobic conditions. Using standard resistance breakpoints, the odds of classifying isolates as resistant increased over 10 times for ciprofloxacin and 100 times for azithromycin under anaerobic conditions compared to aerobic conditions. For doxycycline, nearly all isolates were sensitive under both conditions. Using genome-wide association studies, we found associations between genetic elements and AMR phenotypes that varied by oxygen exposure and antibiotic concentrations. These AMR phenotypes were more heritable, and the AMR-associated genetic elements were more often discovered, under anaerobic conditions. These AMR-associated genetic elements are promising targets for future mechanistic research. Our findings provide a rationale to determine whether increased MICs under anaerobic conditions are associated with therapeutic failures and/or microbial escape in cholera patients. If so, there may be a need to determine new AMR breakpoints for anaerobic conditions.


Asunto(s)
Cólera , Vibrio cholerae , Humanos , Vibrio cholerae/genética , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Cólera/microbiología , Azitromicina/farmacología , Doxiciclina/uso terapéutico , Estudio de Asociación del Genoma Completo , Anaerobiosis , Farmacorresistencia Bacteriana/genética , Ciprofloxacina/farmacología , Oxígeno
18.
Nat Commun ; 12(1): 4912, 2021 08 13.
Artículo en Inglés | MEDLINE | ID: mdl-34389721

RESUMEN

Polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) hybrid systems typically use complex protein-protein interactions to facilitate direct transfer of intermediates between these multimodular megaenzymes. In the canal-associated neurons (CANs) of Caenorhabditis elegans, PKS-1 and NRPS-1 produce the nemamides, the only known hybrid polyketide-nonribosomal peptides biosynthesized by animals, through a poorly understood mechanism. Here, we use genome editing and mass spectrometry to map the roles of individual PKS-1 and NRPS-1 enzymatic domains in nemamide biosynthesis. Furthermore, we show that nemamide biosynthesis requires at least five additional enzymes expressed in the CANs that are encoded by genes distributed across the worm genome. We identify the roles of these enzymes and discover a mechanism for trafficking intermediates between a PKS and an NRPS. Specifically, the enzyme PKAL-1 activates an advanced polyketide intermediate as an adenylate and directly loads it onto a carrier protein in NRPS-1. This trafficking mechanism provides a means by which a PKS-NRPS system can expand its biosynthetic potential and is likely important for the regulation of nemamide biosynthesis.


Asunto(s)
Vías Biosintéticas/genética , Proteínas de Caenorhabditis elegans/genética , Péptido Sintasas/genética , Péptidos/metabolismo , Sintasas Poliquetidas/genética , Policétidos/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Cromatografía Liquida/métodos , Enzimas/genética , Enzimas/metabolismo , Expresión Génica , Espectrometría de Masas/métodos , Estructura Molecular , Mutación , Neuronas/metabolismo , Péptido Sintasas/metabolismo , Péptidos/química , Sintasas Poliquetidas/metabolismo , Policétidos/química
19.
Appl Biol Chem ; 64(1): 72, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34722955

RESUMEN

The biochemical composition of organic fertilizers largely determines their nutrient supply characteristics following soil application as well as their potential impact on soil microbial communities. Yet, limited information is available regarding the biochemical composition of organic fertilizers derived from different nutrient sources. Here, we qualitatively analyzed the presence and abundance of proteins, lipids, and metabolites in a liquid fish fertilizer (LFF) product and a type of granular organic fertilizer (GOF) commonly used in organic vegetable production, using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our results suggest that the presence and abundance of proteins, lipids, and metabolites differ greatly between GOF and LFF. The qualitative analysis shows LFF as a rich source of metabolites, while complex proteins and long-chain saturated fatty acids are dominant in GOF. The degree of biochemical composition complexity may help explain the varying impacts of different types of organic fertilizers on nutrient availability, soil health, and environmental quality. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13765-021-00625-2.

20.
Clin Pharmacol Ther ; 109(4): 1000-1020, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33576025

RESUMEN

Multidrug-resistant bacteria are causing a serious global health crisis. A dramatic decline in antibiotic discovery and development investment by pharmaceutical industry over the last decades has slowed the adoption of new technologies. It is imperative that we create new mechanistic insights based on latest technologies, and use translational strategies to optimize patient therapy. Although drug development has relied on minimal inhibitory concentration testing and established in vitro and mouse infection models, the limited understanding of outer membrane permeability in Gram-negative bacteria presents major challenges. Our team has developed a platform using the latest technologies to characterize target site penetration and receptor binding in intact bacteria that inform translational modeling and guide new discovery. Enhanced assays can quantify the outer membrane permeability of ß-lactam antibiotics and ß-lactamase inhibitors using multiplex liquid chromatography tandem mass spectrometry. While ß-lactam antibiotics are known to bind to multiple different penicillin-binding proteins (PBPs), their binding profiles are almost always studied in lysed bacteria. Novel assays for PBP binding in the periplasm of intact bacteria were developed and proteins identified via proteomics. To characterize bacterial morphology changes in response to PBP binding, high-throughput flow cytometry and time-lapse confocal microscopy with fluorescent probes provide unprecedented mechanistic insights. Moreover, novel assays to quantify cytosolic receptor binding and intracellular drug concentrations inform target site occupancy. These mechanistic data are integrated by quantitative and systems pharmacology modeling to maximize bacterial killing and minimize resistance in in vitro and mouse infection models. This translational approach holds promise to identify antibiotic combination dosing strategies for patients with serious infections.


Asunto(s)
Técnicas Bacteriológicas/métodos , Descubrimiento de Drogas/métodos , Farmacorresistencia Bacteriana Múltiple/fisiología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Gramnegativas/fisiología , Animales , Membrana Celular/fisiología , Modelos Animales de Enfermedad , Humanos , Modelos Teóricos , Proteínas de Unión a las Penicilinas/fisiología , beta-Lactamas/farmacología
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA