scholarly journals Cell Membrane Adaptations Mediate β-Lactam-Induced Resensitization of Daptomycin-Resistant (DAP-R) Staphylococcus aureus In Vitro

2021 ◽  
Vol 9 (5) ◽  
pp. 1028
Author(s):  
Nagendra N. Mishra ◽  
Arnold S. Bayer ◽  
Sarah L. Baines ◽  
Ashleigh S. Hayes ◽  
Benjamin P. Howden ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Cell Membrane ◽  
Surface Charge ◽  
Prolonged Exposure ◽  
Point Mutations ◽  
Carotenoid Content ◽  
Multiple Point ◽  
Genetic Changes ◽  
Allelic Exchange

The reversal of daptomycin resistance in MRSA to a daptomycin-susceptible phenotype following prolonged passage in selected β-lactams occurs coincident with the accumulation of multiple point mutations in the mprF gene. MprF regulates surface charge by modulating the content and translocation of the positively charged cell membrane phospholipid, lysyl-phosphatidylglycerol (LPG). The precise cell membrane adaptations accompanying such β-lactam-induced mprF perturbations are unknown. This study examined key cell membrane metrics relevant to antimicrobial resistance among three daptomycin-resistant MRSA clinical strains, which became daptomycin-susceptible following prolonged exposure to cloxacillin (‘daptomycin-resensitized’). The causal role of such secondary mprF mutations in mediating daptomycin resensitization was confirmed through allelic exchange strategies. The daptomycin-resensitized strains derived either post-cloxacillin passage or via allelic exchange (vs. their respective daptomycin-resistant strains) showed the following cell membrane changes: (i) enhanced BODIPY-DAP binding; (ii) significant reductions in LPG content, accompanied by significant increases in phosphatidylglycerol content (p < 0.05); (iii) no significant changes in positive cell surface charge; (iv) decreased cell membrane fluidity (p < 0.05); (v) enhanced carotenoid content (p < 0.05); and (vi) lower branched chain fatty acid profiles (antiso- vs. iso-), resulting in increases in saturated fatty acid composition (p < 0.05). Overall, the cell membrane characteristics of the daptomycin-resensitized strains resembled those of parental daptomycin-susceptible strains. Daptomycin resensitization with selected β-lactams results in both definable genetic changes (i.e., mprF mutations) and a number of key cell membrane phenotype modifications, which likely facilitate daptomycin activity.

scholarly journals In VitroCross-Resistance to Daptomycin and Host Defense Cationic Antimicrobial Peptides in Clinical Methicillin-Resistant Staphylococcus aureus Isolates

2011 ◽  
Vol 55 (9) ◽  
pp. 4012-4018 ◽  
Author(s):  
Nagendra N. Mishra ◽  
James McKinnell ◽  
Michael R. Yeaman ◽  
Aileen Rubio ◽  
Cynthia C. Nast ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Cell Membrane ◽  
Antimicrobial Peptides ◽  
Surface Charge ◽  
Host Defense ◽  
Cationic Antimicrobial Peptides ◽  
Methicillin Resistant ◽  
Membrane Order

ABSTRACTWe investigated the hypothesis that methicillin-resistantStaphylococcus aureus(MRSA) isolates developing reduced susceptibilities to daptomycin (DAP; a calcium-dependent molecule acting as a cationic antimicrobial peptide [CAP]) may also coevolve reducedin vitrosusceptibilities to host defense cationic antimicrobial peptides (HDPs). Ten isogenic pairs of clinical MRSA DAP-susceptible/DAP-resistant (DAPs/DAPr) strains were tested against two distinct HDPs differing in structure, mechanism of action, and origin (thrombin-induced platelet microbicidal proteins [tPMPs] and human neutrophil peptide-1 [hNP-1]) and one bacterium-derived CAP, polymyxin B (PMB). Seven of 10 DAPrstrains had point mutations in themprFlocus (with or withoutyycoperon mutations), while three DAPrstrains had neither mutation. Several phenotypic parameters previously associated with DAPrwere also examined: cell membrane order (fluidity), surface charge, and cell wall thickness profiles. Compared to the 10 DAPsparental strains, their respective DAPrstrains exhibited (i) significantly reduced susceptibility to killing by all three peptides (P< 0.05), (ii) increased cell membrane fluidity, and (iii) significantly thicker cell walls (P< 0.0001). There was no consistent pattern of surface charge profiles distinguishing DAPsand DAPrstrain pairs. Reducedin vitrosusceptibility to two HDPs and one bacterium-derived CAP tracked closely with DAPrin these 10 recent MRSA clinical isolates. These results suggest that adaptive mechanisms involved in the evolution of DAPralso provide MRSA with enhanced survivability against HDPs. Such adaptations appear to correlate with MRSA variations in cell membrane order and cell wall structure. DAPrstrains with or without mutations in themprFlocus demonstrated significant cross-resistance profiles to these unrelated CAPs.

scholarly journals Fatty Acid Profile, Carotenoid Content, and In Vitro Anticancer Activity of Karimunjawa and Lampung Sea Cucumber

2016 ◽  
Vol 11 (3) ◽  
pp. 117
Author(s):  
Ekowati Chasanah ◽  
Yusro Nuri Fawzya ◽  
Kustiariyah Tarman ◽  
Hedi Indra Januar ◽  
Muhammad Nursid
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Anticancer Activity ◽  
Fatty Acid Profile ◽  
Sea Cucumber ◽  
Raw Material ◽  
Carotenoid Content ◽  
Food Ingredient ◽  
Mcf 7

Fatty acids and carotenoid has been known as an anticancer agent on both preventing and treating cancer disease. This study was conducted to analyze the fatty acid profile, carotenoid and in vitro anticancer activity of 12 sea cucumber harvested from Karimunjawa and Lampung waters. The aim of the study was to determin the potency of sea cucumbers as raw material for nutraceutical products. Fatty acid profile and carotenoid content were characterized by gas chromatography and spectrophotometry techniques, while in vitro anticancer activity was assessed by MTT assay against cervix (HeLa), breast (T47D and MCF-7) and colon (WiDR) cancer cells. Results of the study showed polyunsaturated fatty acid (PUFA) dominated the composition of fatty acids in the samples from both locations. Holothuria sp. was detected to contain the highest amount of carotenoid. Furthermore, the highest in vitro anticancer activity was detected also in the sample of Holothuria sp. The activity of 30 ppm Holothuria sp. extract against HeLa cell was detected to be almost equal to the 5 ppm doxorubicin control. Concentration of 5 ppm Holothuria sp. extract also showed positive result in killing 50% of MCF-7 and T47D, but capable to 100% kill HeLa and WiDR cells. At concentration of 25 ppm, the extract was able to kill all the 4 cells tested. Statistical analysis showed the amount of carotenoid and two particular fatty acid compounds (docosadienoic and eicosapentaenoic acid) significantly (P<0.05) contributed to the cytotoxic activity that was found in the sea cucumber samples. Those compounds were found in highest concentration from Holothuria sp harvested from Lampung waters, thus being the most prospective raw material for nutraceutical or functional food ingredient with anticancer potency.

scholarly journals SilentMutations (SIM): a tool for analyzing long-range RNA-RNA interactions in viral genomes and structured RNAs

2018 ◽  
Author(s):  
Daniel Desirò ◽  
Martin Hölzer ◽  
Bashar Ibrahim ◽  
Manja Marz
Keyword(s):  
Long Range ◽  
Influenza A ◽  
Graphical Representation ◽  
Point Mutations ◽  
Multiple Point ◽  
Coding Region ◽  
Viral Rnas ◽  
Viral Genomes ◽  
Single Nucleotide Change

ABSTRACTBackgroundA single nucleotide change in the coding region can alter the amino acid sequence of a protein. In consequence, natural or artificial sequence changes in viral RNAs may have various effects not only on protein stability, function and structure but also on viral replication.In recent decades, several tools have been developed to predict the effect of mutations in structured RNAs such as viral genomes or non-coding RNAs. Some tools use multiple point mutations and also take coding regions into account. However, none of these tools was designed to specifically simulate the effect of mutations on viral long-range interactions.ResultsHere, we developedSilentMutations (SIM), an easy-to-use tool to analyze the effect of multiple point mutations on the secondary structures of two interacting viral RNAs. The tool can simulate disruptive and compensatory mutants of two interacting single-stranded RNAs. This allows a fast and accurate assessment of key regions potentially involved in functional long-range RNA-RNA interactions and will eventually help virologists and RNA-experts to design appropriate experiments.SIMonly requires two interacting single-stranded RNA regions as input. The output is a plain text file containing the most promising mutants and a graphical representation of all interactions.ConclusionWe applied our tool on two experimentally validated influenza A virus and hepatitis C virus interactions and we were able to predict potential double mutants forin vitrovalidation experiments.AvailabilityThe source code and documentation ofSIMare freely available at github.com/desiro/silentMutations.

scholarly journals Prolonged Exposure to β-Lactam Antibiotics Reestablishes Susceptibility of Daptomycin-Nonsusceptible Staphylococcus aureus to Daptomycin

2020 ◽  
Vol 64 (9) ◽  
Author(s):  
Rachel E. Jenson ◽  
Sarah L. Baines ◽  
Benjamin P. Howden ◽  
Nagendra N. Mishra ◽  
Sabrina Farah ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Surface Charge ◽  
Prolonged Exposure ◽  
Cell Envelope ◽  
Serial Passage ◽  
Single Nucleotide ◽  
Genetic Changes ◽  
Content Type ◽  
Host Defense Peptide ◽  
Time Kill

ABSTRACT Daptomycin-nonsusceptible (DAP-NS) Staphylococcus aureus often exhibits gain-in-function mutations in the mprF gene (involved in positive surface charge maintenance). Standard β-lactams, although relatively inactive against methicillin-resistant S. aureus (MRSA), may prevent the emergence of mprF mutations and DAP-NS. We determined if β-lactams might also impact DAP-NS isolates already possessing an mprF mutation to revert them to DAP-susceptible (DAP-S) phenotypes and, if so, whether this is associated with specific penicillin-binding protein (PBP) targeting. This study included 25 DAP-S/DAP-NS isogenic, clinically derived MRSA bloodstream isolates. MICs were performed for DAP, nafcillin (NAF; PBP-promiscuous), cloxacillin (LOX; PBP-1), ceftriaxone (CRO; PBP-2), and cefoxitin (FOX; PBP-4). Three DAP-NS isolates were selected for a 28-day serial passage in subinhibitory β-lactams. DAP MICs and time-kill assays, host defense peptide (LL-37) susceptibilities, and whole-genome sequencing were performed to associate genetic changes with key phenotypic profiles. Pronounced decreases in baseline MICs were observed for NAF and LOX (but not for CRO or FOX) among DAP-NS versus DAP-S isolates (“seesaw” effect). Prolonged (28-d) β-lactam passage of three DAP-NS isolates significantly reduced DAP MICs. LOX was most impactful (∼16-fold decrease in DAP MIC; 2 to 0.125 mg/liter). In these DAP-NS isolates with preexisting mprF polymorphisms, accumulation of additional mprF mutations occurred with prolonged LOX exposures. This was associated with enhanced LL-37 killing activity and reduced surface charge (both mprF-dependent phenotypes). β-lactams that either promiscuously or specifically target PBP-1 have significant DAP “resensitizing” effects against DAP-NS S. aureus strains. This may relate to the acquisition of multiple mprF single nucleotide polymorphism (SNPs), which, in turn, affect cell envelope function and metabolism.

scholarly journals Analysis of Cell Membrane Characteristics of In Vitro-Selected Daptomycin-Resistant Strains of Methicillin-Resistant Staphylococcus aureus

2009 ◽  
Vol 53 (6) ◽  
pp. 2312-2318 ◽  
Author(s):  
Nagendra N. Mishra ◽  
Soo-Jin Yang ◽  
Ayumi Sawa ◽  
Aileen Rubio ◽  
Cynthia C. Nast ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Membrane ◽  
Surface Charge ◽  
Parental Strain ◽  
Phospholipid Content ◽  
Surface Binding ◽  
Methicillin Resistant ◽  
Positive Surface Charge ◽  
Vancomycin Mics

ABSTRACT Our previous studies of clinical daptomycin-resistant (Dapr) Staphylococcus aureus strains suggested that resistance is linked to the perturbations of several key cell membrane (CM) characteristics, including the CM order (fluidity), phospholipid content and asymmetry, and relative surface charge. In the present study, we examined the CM profiles of a well-known methicillin-resistant Staphylococcus aureus (MRSA) strain (MW2) after in vitro selection for DAP resistance by a 20-day serial passage in sublethal concentrations of DAP. Compared to levels for the parental strain, Dapr strains exhibited (i) decreased CM fluidity, (ii) the increased synthesis of total lysyl-phosphatidylglycerol (LPG), (iii) the increased flipping of LPG to the CM outer bilayer, and (iv) the increased expression of mprF, the gene responsible for the latter two phenotypes. In addition, we found that the expression of the dlt operon, which also increases positive surface charge, was enhanced in the Dapr mutants. These phenotypic and genotypic changes correlated with reduced DAP surface binding, mirroring observations made in clinical Dapr isolates. In this strain, serial exposure to DAP induced an increase in vancomycin MICs into the vancomycin-intermediate S. aureus (VISA) range (4 μg/ml) in parallel with increasing DAP MICs. Also, this Dapr strain exhibited significantly thicker cell walls than the parental strain, potentially correlating with the coevolution of the VISA phenotype and implicating cell wall structure and/or function in the Dapr phenotype. Importantly, despite the overexpression of mprF and dlt, the relative net positive surface charge was decreased in the Dapr mutants, suggesting that other factors contribute to the surface charge alterations and that a simple charge repulsion mechanism could not entirely explain the Dapr phenotype in these strains.

scholarly journals Characterization of a new TEM-derived beta-lactamase produced in a Serratia marcescens strain.

1997 ◽  
Vol 41 (11) ◽  
pp. 2374-2382 ◽  
Author(s):  
M Perilli ◽  
A Felici ◽  
N Franceschini ◽  
A De Santis ◽  
L Pagani ◽  
...  
Keyword(s):  
Serratia Marcescens ◽  
Three Dimensional ◽  
Point Mutations ◽  
Catalytic Efficiency ◽  
Point Of View ◽  
Multiple Point ◽  
Crystallographic Structure ◽  
Beta Lactamase

A natural TEM variant beta-lactamase was isolated from an epidemic strain of Serratia marcescens. Nucleotide gene sequencing revealed multiple point mutations located in the 42-to-44 tripeptide and positions 145 to 146, 178, and 238. In addition, a glutamic acid 212 deletion was also found. The purified enzyme was studied from a kinetic point of view, revealing the highest catalytic efficiency (k[cat]/Km) values for ceftazidime and aztreonam compared with the TEM-1 prototype enzyme. The in vitro resistance correlated with kinetic parameters, and the enzyme also mediated resistance to some penicillins and an ampicillin-clavulanic acid combination. The mutational and kinetic changes are discussed in relation to the three-dimensional crystallographic structure of the wild-type TEM-1 enzyme.

In Vitro Incorporation and Metabolism of Icosapentaenoic and Docosahexaenoic Acids in Human Platelets - Effect on Aggregation

1986 ◽  
Vol 56 (01) ◽  
pp. 057-062 ◽  
Author(s):  
Martine Croset ◽  
M Lagarde
Keyword(s):  
Arachidonic Acid ◽  
Fatty Acid ◽  
Platelet Aggregation ◽  
Fatty Acid Distribution ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Aggregation Response ◽  
Docosahexaenoic Acids ◽  
Membrane Level

SummaryWashed human platelets were pre-loaded with icosapentaenoic acid (EPA), docosahexaenoic acid (DHA) or EPA + DHA and tested for their aggregation response in comparison with control platelets. In fatty acid-rich platelets, an inhibition of the aggregation could be observed when induced by thrombin, collagen or U-46619. The strongest inhibition was observed with DHA-rich platelets and it was reduced when DHA was incorporated in the presence of EPA.Study of fatty acid distribution in cell lipids after loading showed that around 90% of EPA or DHA taken up was acylated into phospholipids and a very small amount (less than 2%) remained in their free and hydroxylated forms. DHA was more efficiently acylated into phosphatidylethanolamine (PE) than into phosphatidylinositol (PI) in contrast to what observed with EPA, and both acids were preferentially incorporated into phosphatidylcholine (PC). EPA inhibited total incorporation of DHA and increased its relative acylation into PE at the expense of PC. In contrast, DHA did not affect the acylation of EPA. Upon stimulation with, thrombin, EPA was liberated from phospholipids and oxygenated (as judged by the formation of its monohydroxy derivative) whereas DHA was much less metabolized, although consistently transferred into PE.It is concluded that EPA and DHA might affect platelet aggregation via different mechanisms when pre-loaded in phospholipids. Whereas EPA is known to alter thromboxane A2 metabolism from endogenous arachidonic acid, by competing with it, DHA might act directly at the membrane level for inhibiting aggregation.

Methods of experimental polyploidization and their use in apple breeding

2020 ◽  
Vol 62 ◽  
pp. 85-90
Author(s):  
L. V. Tashmatova ◽  
O. V. Matsneva ◽  
T. M. Khromova ◽  
V. V. Shakhov
Keyword(s):  
Exposure Time ◽  
Cell Walls ◽  
Prolonged Exposure ◽  
Ornamental Plants ◽  
Colchicine Treatment ◽  
Apple Trees ◽  
Open Ground ◽  
High Concentrations ◽  
Diploid Gametes

The article presents methods of experimental polyploidy of fruit, berry and ornamental plants. The purpose of this review is to highlight the problems and prospects of polyploidization of plants in the open ground and in vitro culture and the possibility of their application for apple trees. For the purpose of obtaining apple tetraploids as donors of diploid gametes, seed seedlings were treated with a solution of colchicine in concentrations of 0.1-0.4 % for 24 and 48 hours. Colchicine concentrations of 0.3 % and 0.4 % at 48 hours of treatment had a detrimental eff ect on their development. As a result, tetraploids and chimeras were obtained from seeds from free pollination of the varieties Orlik, Svezhest, Kandil Orlovsky, as well as from seeds obtained from crossing the varieties Svezhest×Bolotovskoe, Moskovskoe Оzherel’e×Imrus, Girlyanda×Venyaminovskoe. The optimal concentration of colchicine was 0.1 %. Methods of colchicine treatment have been studied: 1) adding to the nutrient medium, colchicine concentration: 0.01%, 0.02%, exposure time 24h-19 days; 2) applying amitotic solution to the growth point, colchicine concentration: 0.1 %, 0.2 %, exposure time 24h-7 days. To increase the penetration of colchicine through the cell walls, a 0.1 % dimexide solution was used. Studies have shown that high concentrations and prolonged exposure to colchicine reduce the viability of explants.

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