Role of MurT C-Terminal Domain in the Amidation of Staphylococcus aureus Peptidoglycan

Fundacao para a Ciencia e a Tecnologia (FCT) through grants PTDC/FIS-NAN/ 0117/2014 and PTDC/BIA-MIC/31645/2017. project UID/Multi/04378/2019 (Unidade de Ciencias Biomoleculares Aplicadas-UCIBIO), funded by FCT/MCTES; project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular), funded by FEDER through COMPETE2020-Programa Operacional Competitividade e Internacionalizacao (POCI); national funds through FCT; by project ONEIDA (LISBOA-01-0145-FEDER-016417), cofunded by FEEI (Fundos Europeus Estruturais e de Investimento) from the Programa Operacional Regional Lisboa 2020; and by national funds from FCT. Funding was also provided by European Society of Clinical Microbiology and Infectious Diseases research grant 2015, awarded to R.G.S. B.V.G., T.A.F., and I.R.G. were supported by fellowships SFRH/BD/131623/2017 respectively. J.S.D. acknowledges the National NMR Network (PTNMR) and Infrastructure Project ROTEIRO/0031/2013-PINFRA/22161/2016 (cofinanced by FEDER through COMPETE 2020, POCI, PORL, and FCT through PIDDAC).Glutamate amidation, a secondary modification of the peptidoglycan, was first identified in Staphylococcus aureus. It is catalyzed by the protein products of the murT and gatD genes, which are conserved and colocalized in the genomes of most sequenced Gram-positive bacterial species. The MurT-GatD complex is required for cell viability, full resistance to β-lactam antibiotics, and resistance to human lysozyme and is recognized as an attractive target for new antimicrobials. Great effort has been invested in the study of this step, culminating recently in three independent reports addressing the structural elucidation of the MurT-GatD complex. In this work, we demonstrate through the use of nonstructural approaches the critical and multiple roles of the C-terminal domain of MurT, annotated as DUF1727, in the MurT-GatD enzymatic complex. This domain provides the physical link between the two enzymatic activities and is essential for the amidation reaction. Copurification of recombinant MurT and GatD proteins and bacterial two-hybrid assays support the observation that the MurT-GatD interaction occurs through this domain. Most importantly, we provide in vivo evidence of the effect of substitutions at specific residues in DUF1727 on cell wall peptidoglycan amidation and on the phenotypes of oxacillin resistance and bacterial growth.publishersversionpublishe

Genomic identification of cryptic susceptibility to penicillins and β-lactamase inhibitors in methicillin-resistant Staphylococcus aureus.

Antibiotic resistance in bacterial pathogens threatens the future of modern medicine. One such resistant pathogen is methicillin-resistant Staphylococcus aureus (MRSA), which is resistant to nearly all β-lactam antibiotics, limiting treatment options. Here, we show that a significant proportion of MRSA isolates from different lineages, including the epidemic USA300 lineage, are susceptible to penicillins when used in combination with β-lactamase inhibitors such as clavulanic acid. Susceptibility is mediated by a combination of two different mutations in the mecA promoter region that lowers mecA-encoded penicillin-binding protein 2a (PBP2a) expression, and in the majority of isolates by either one of two substitutions in PBP2a (E246G or M122I) that increase the affinity of PBP2a for penicillin in the presence of clavulanic acid. Treatment of S. aureus infections in wax moth and mouse models shows that penicillin/β-lactamase inhibitor susceptibility can be exploited as an effective therapeutic choice for 'susceptible' MRSA infection. Finally, we show that isolates with the PBP2a E246G substitution have a growth advantage in the presence of penicillin but the absence of clavulanic acid, which suggests that penicillin/β-lactamase susceptibility is an example of collateral sensitivity (resistance to one antibiotic increases sensitivity to another). Our findings suggest that widely available and currently disregarded antibiotics could be effective in a significant proportion of MRSA infections.MRC - G1001787/1 MRC - MR/N002660/1 WT098600 HICF-T5-342 MR/S00291X/1 201344/Z/16/Z MR/P007201/

Tackling bacterial resistance using antibiotics as ionic liquids and organic salts

Bacterial resistance to current antibiotics has a major impact on worldwide human health, leading to 700K deaths every year. The development of novel antibiotics did not present significant progress, namely regarding clinical trials, over the last years due to low returns. Thus, innovative alternatives must be devised to tackle the continuous rise of antimicrobial resistance. Ionic Liquids and Organic Salts from Active Pharmaceutical Ingredients (API-OSILs) have risen in academia for over 10 years as an efficient formulation for drugs with low bioavailability and permeability, as well as reduction or elimination of polymorphism, thereby potentially enhancing their pharmaceutical efficiency. To the best of our knowledge, our group is the first to perform research on the development of API-OSILs from antibiotics as a way to improve their efficiency. More specifically, we have successfully combined ampicillin, penicillin and amoxicillin as anions with biocompatible organic cations such as choline, alkylpyridiniums and alkylimidazoliums. In this communication, we present our latest developments in the synthesis and physicochemical (DSC) characterization of OSILs from these antibiotics, in addition to in vitro antimicrobial activity data, in particular towards MRSA and multi-resistant E. coli, as well as sensitive strains of gram-positive and gram-negative bacteria.info:eu-repo/semantics/publishedVersio

Antifouling napyradiomycins from marine-derived actinomycetes streptomyces aculeolatus

UID/Multi/04378/2019 UID/Multi/04423/2019 FP7/2007–2013 DFRH/WIIA/102/2011 SFRH/BI/52130/ 2013 IF/00700/2014 SFRH/BPD/110020/2015 PTDC/BIA-MIC/31645/2017 PTDC/BTA-GES/ 32359/2017 PTDC/BTA-BTA/31422/2017 POCI-01-0145-FEDER-031422The undesired attachment of micro and macroorganisms on water-immersed surfaces, known as marine biofouling, results in severe prevention and maintenance costs (billions €/year) for aquaculture, shipping and other industries that rely on coastal and off-shore infrastructures. To date, there are no sustainable, cost-effective and environmentally safe solutions to address this challenging phenomenon. Therefore, we investigated the antifouling activity of napyradiomycin derivatives that were isolated from actinomycetes from ocean sediments collected off the Madeira Archipelago. Our results revealed that napyradiomycins inhibited ≥80% of the marine biofilm-forming bacteria assayed, as well as the settlement of Mytilus galloprovincialis larvae (EC50 15), without viability impairment. In silico prediction of toxicity end points are of the same order of magnitude of standard approved drugs and biocides. Altogether, napyradiomycins disclosed bioactivity against marine micro and macrofouling organisms, and non-toxic effects towards the studied species, displaying potential to be used in the development of antifouling products.publishersversionpublishe

Seawater carbonate chemistry and leaf coloration, photophysiology and photosynthetic pigments of seagrass Zostera noltii

Seagrasses play an essential ecological role within coastal habitats and their worldwide population decline has been linked to different types of anthropogenic forces. We investigated, for the first time, the combined effects of future ocean warming and acidification on fundamental biological processes of Zostera noltii, including shoot density, leaf coloration, photophysiology (electron transport rate, ETR; maximum PSII quantum yield, Fv/Fm) and photosynthetic pigments. Shoot density was severely affected under warming conditions, with a concomitant increase in the frequency of brownish colored leaves (seagrass die-off). Warming was responsible for a significant decrease in ETR and Fv/Fm (particularly under control pH conditions), while promoting the highest ETR variability (among experimental treatments). Warming also elicited a significant increase in pheophytin and carotenoid levels, alongside an increase in carotenoid/chlorophyll ratio and De-Epoxidation State (DES). Acidification significantly affected photosynthetic pigments content (antheraxanthin, beta-carotene, violaxanthin and zeaxanthin), with a significant decrease being recorded under the warming scenario. No significant interaction between ocean acidification and warming was observed. Our findings suggest that future ocean warming will be a foremost determinant stressor influencing Z. noltii survival and physiological performance. Additionally, acidification conditions to occur in the future will be unable to counteract deleterious effects posed by ocean warming

The Diversity, Metabolomics Profiling, and the Pharmacological Potential of Actinomycetes Isolated from the Estremadura Spur Pockmarks (Portugal)

The Estremadura Spur pockmarks are a unique and unexplored ecosystem located in the North Atlantic, off the coast of Portugal. A total of 85 marine-derived actinomycetes were isolated and cultured from sediments collected from this ecosystem at a depth of 200 to 350 m. Nine genera, Streptomyces, Micromonospora, Saccharopolyspora, Actinomadura, Actinopolymorpha, Nocardiopsis, Saccharomonospora, Stackebrandtia, and Verrucosispora were identified by 16S rRNA gene sequencing analyses, from which the first two were the most predominant. Non-targeted LC-MS/MS, in combination with molecular networking, revealed high metabolite diversity, including several known metabolites, such as surugamide, antimycin, etamycin, physostigmine, desferrioxamine, ikarugamycin, piericidine, and rakicidin derivatives, as well as numerous unidentified metabolites. Taxonomy was the strongest parameter influencing the metabolite production, highlighting the different biosynthetic potentials of phylogenetically related actinomycetes; the majority of the chemical classes can be used as chemotaxonomic markers, as the metabolite distribution was mostly genera-specific. The EtOAc extracts of the actinomycete isolates demonstrated antimicrobial and antioxidant activity. Altogether, this study demonstrates that the Estremadura Spur is a source of actinomycetes with potential applications for biotechnology. It highlights the importance of investigating actinomycetes from unique ecosystems, such as pockmarks, as the metabolite production reflects their adaptation to this habitat

Genomic identification of cryptic susceptibility to penicillins and β-lactamase inhibitors in methicillin-resistant Staphylococcus aureus.

Antibiotic resistance in bacterial pathogens threatens the future of modern medicine. One such resistant pathogen is methicillin-resistant Staphylococcus aureus (MRSA), which is resistant to nearly all β-lactam antibiotics, limiting treatment options. Here, we show that a significant proportion of MRSA isolates from different lineages, including the epidemic USA300 lineage, are susceptible to penicillins when used in combination with β-lactamase inhibitors such as clavulanic acid. Susceptibility is mediated by a combination of two different mutations in the mecA promoter region that lowers mecA-encoded penicillin binding protein 2a (PBP2a) expression, and in the majority of isolates by either one of two substitutions in PBP2a (E246G or M122I) that increase the affinity of PBP2a for penicillin in the presence of clavulanic acid. Treatment of S. aureus infections in wax moth and mouse models demonstrate that penicillin/β-lactamase inhibitor susceptibility can be exploited as an effective therapeutic choice for ‘susceptible’ MRSA infection. Finally, we show that isolates with the PBP2a E246G substitution have a growth advantage in the presence of penicillin, but the absence of clavulanic acid, which suggests that penicillin/β-lactamase susceptibility is an example of collateral sensitivity (resistance to one antibiotic increases sensitivity to another). Our findings suggest that widely available and currently disregarded antibiotics could be effective in a significant proportion of MRSA infections