scholarly journals Differential Adaptations of Methicillin-ResistantStaphylococcus aureusto SerialIn VitroPassage in Daptomycin: Evolution of Daptomycin Resistance and Role of Membrane Carotenoid Content and Fluidity

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Nagendra N. Mishra ◽  
Aileen Rubio ◽  
Cynthia C. Nast ◽  
Arnold S. Bayer
Keyword(s):  
Production Cross ◽  
Serial Passage ◽  
Carotenoid Content ◽  
Cross Resistance ◽  
Nucleotide Polymorphisms ◽  
Host Defense Peptides ◽  
Identical Manner ◽  
Mrsa Strains

Previous studies showed serial 20 din vitropassage of MRSA strain MW2 in sublethal daptomycin (DAP) resulted in diverse perturbations in both cell membrane (CM) and cell wall (CW) characteristics, including increased CM rigidity; increased CW thickness; “gain-in-function” single nucleotide polymorphisms (SNPs) in themprFlocus (i.e., increased synthesis and translocation of lysyl-phosphatidylglycerol (L-PG)); progressive accumulation of SNPs inyycandrpolocus genes; reduced carotenoid production; cross-resistance to innate host defense peptides. The current study was designed to characterize the reproducibility of these phenotypic and genotypic modifications followingin vitroserial passages of the same parental strain. After a second 20d serialin vitropassage of parental MW2, emergence of DAP-R was associated with evolution of several phenotypes closely mirroring previous passage outcomes. However, in contrast to the initial serial passage strain set, we observed (i) only modest increase in L-PG synthesis and no increase in L-PG outer CM translocation; (ii) significantly increased carotenoid synthesis (P<0.05); (iii) a different order of SNP accumulations (mprF≫rpoB≫yycG); (iv) a different cadre and locations of such SNPs. Thus, MRSA strains are not “pre-programmed” to phenotypically and/or genotypically adapt in an identical manner during induction of DAP resistance.

scholarly journals Mechanistic Fingerprinting Reveals Kinetic Signatures of Resistance to Daptomycin and Host Defense Peptides in Streptococcus mitis-oralis

Antibiotics ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 404
Author(s):  
Michael R. Yeaman ◽  
Liana C. Chan ◽  
Nagendra N. Mishra ◽  
Arnold S. Bayer
Keyword(s):  
Flow Cytometry ◽  
Host Defense ◽  
Durable Resistance ◽  
Cross Resistance ◽  
Streptococcus Mitis ◽  
Host Defense Peptides ◽  
Strain Pair ◽  
Defense Peptides

Streptococcus mitis-oralis (S. mitis-oralis) infections are increasingly prevalent in specific populations, including neutropenic cancer and endocarditis patients. S. mitis-oralis strains have a propensity to evolve rapid, high-level and durable resistance to daptomycin (DAP-R) in vitro and in vivo, although the mechanism(s) involved remain incompletely defined. We examined mechanisms of DAP-R versus cross-resistance to cationic host defense peptides (HDPs), using an isogenic S. mitis-oralis strain-pair: (i) DAP-susceptible (DAP-S) parental 351-WT (DAP MIC = 0.5 µg/mL), and its (ii) DAP-R variant 351-D10 (DAP MIC > 256 µg/mL). DAP binding was quantified by flow cytometry, in-parallel with temporal (1–4 h) killing by either DAP or comparative prototypic cationic HDPs (hNP-1; LL-37). Multicolor flow cytometry was used to determine kinetic cell responses associated with resistance or susceptibility to these molecules. While overall DAP binding was similar between strains, a significant subpopulation of 351-D10 cells hyper-accumulated DAP (>2–4-fold vs. 351-WT). Further, both DAP and hNP-1 induced cell membrane (CM) hyper-polarization in 351-WT, corresponding to significantly greater temporal DAP-killing (vs. 351-D10). No strain-specific differences in CM permeabilization, lipid turnover or regulated cell death were observed post-exposure to DAP, hNP-1 or LL-37. Thus, the adaptive energetics of the CM appear coupled to the outcomes of interactions of S. mitis-oralis with DAP and selected HDPs. In contrast, altered CM permeabilization, proposed as a major mechanism of action of both DAP and HDPs, did not differentiate DAP-S vs. DAP-R phenotypes in this S. mitis-oralis strain-pair.

scholarly journals Allelic variation contributes to bacterial host specificity

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Min Yue ◽  
Xiangan Han ◽  
Leon De Masi ◽  
Chunhong Zhu ◽  
Xun Ma ◽  
...  
Keyword(s):  
Allelic Variation ◽  
Multinomial Logistic Regression ◽  
Gene Gain ◽  
Nucleotide Polymorphisms ◽  
Genome Wide ◽  
Serovar Typhimurium ◽  
Functional Analyses ◽  
High Degree

Abstract Understanding the molecular parameters that regulate cross-species transmission and host adaptation of potential pathogens is crucial to control emerging infectious disease. Although microbial pathotype diversity is conventionally associated with gene gain or loss, the role of pathoadaptive nonsynonymous single-nucleotide polymorphisms (nsSNPs) has not been systematically evaluated. Here, our genome-wide analysis of core genes within Salmonella enterica serovar Typhimurium genomes reveals a high degree of allelic variation in surface-exposed molecules, including adhesins that promote host colonization. Subsequent multinomial logistic regression, MultiPhen and Random Forest analyses of known/suspected adhesins from 580 independent Typhimurium isolates identifies distinct host-specific nsSNP signatures. Moreover, population and functional analyses of host-associated nsSNPs for FimH, the type 1 fimbrial adhesin, highlights the role of key allelic residues in host-specific adherence in vitro. Together, our data provide the first concrete evidence that functional differences between allelic variants of bacterial proteins likely contribute to pathoadaption to diverse hosts.

scholarly journals Identification of a novel tedizolid resistance mutation in rpoB of MRSA after in vitro serial passage

2020 ◽  
Author(s):  
Tianwei Shen ◽  
Kelsi Penewit ◽  
Adam Waalkes ◽  
Libin Xu ◽  
Stephen J Salipante ◽  
...  
Keyword(s):  
Protein Function ◽  
Homology Modelling ◽  
Resistance Mutation ◽  
Laboratory Strain ◽  
Serial Passage ◽  
Cross Resistance ◽  
Σ Factor ◽  
Rpob Mutation ◽  
Stable Mutant

Abstract Objectives Tedizolid is an oxazolidinone antimicrobial with activity against Gram-positive bacteria, including MRSA. Tedizolid resistance is uncommon and tedizolid’s capacity to select for cross-resistance to other antimicrobials is incompletely understood. The objective of this study was to further explore the phenotypic and genetic basis of tedizolid resistance in MRSA. Methods We selected for tedizolid resistance in an MRSA laboratory strain, N315, by serial passage until an isolate with an MIC ≥1 log2 dilution above the breakpoint for resistance (≥2 mg/L) was recovered. This isolate was subjected to WGS and susceptibility to a panel of related and unrelated antimicrobials was tested in order to determine cross-resistance. Homology modelling was performed to evaluate the potential impact of the mutation on target protein function. Results After 10 days of serial passage we recovered a phenotypically stable mutant with a tedizolid MIC of 4 mg/L. WGS revealed only one single nucleotide variant (A1345G) in rpoB, corresponding to amino acid substitution D449N. MICs of linezolid, chloramphenicol, retapamulin and quinupristin/dalfopristin increased by ≥2 log2 dilutions, suggesting the emergence of the so-called ‘PhLOPSa’ resistance phenotype. Susceptibility to other drugs, including rifampicin, was largely unchanged. Homology models revealed that the mutated residue of RNA polymerase would be unlikely to directly affect oxazolidinone action. Conclusions To the best of our knowledge, this is the first time that an rpoB mutation has been implicated in resistance to PhLOPSa antimicrobials. The mechanism of resistance remains unclear, but is likely indirect, involving σ-factor binding or other alterations in transcriptional regulation.

scholarly journals Plasmid-Mediated Resistance to Thrombin-Induced Platelet Microbicidal Protein in Staphylococci: Role of theqacA Locus

1999 ◽  
Vol 43 (10) ◽  
pp. 2395-2399 ◽  
Author(s):  
Leon Iri Kupferwasser ◽  
Ronald A. Skurray ◽  
Melissa H. Brown ◽  
Neville Firth ◽  
Michael R. Yeaman ◽  
...  
Keyword(s):  
Multidrug Resistance ◽  
Efflux Pump ◽  
Endothelial Damage ◽  
Cross Resistance ◽  
Cationic Peptide ◽  
Multidrug Resistance Gene ◽  
Phenotypic Resistance ◽  
Platelet Microbicidal Protein

ABSTRACT Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a small, cationic peptide released from rabbit platelets following thrombin stimulation. In vitro resistance to this peptide among strains of Staphylococcus aureus correlates with the survival advantage of such strains at sites of endothelial damage in humans as well as in experimental endovascular infections. The mechanisms involved in the phenotypic resistance of S. aureus to tPMP-1 are not fully delineated. The plasmid-encoded staphylococcal gene qacA mediates multidrug resistance to multiple organic cations via a proton motive force-dependent efflux pump. We studied whether the qacA gene might also confer resistance to cationic tPMP-1. Staphylococcal plasmids encoding qacA were found to confer resistance to tPMP-1 in an otherwise susceptible parental strain. Deletions which removed the region containing theqacA gene in the S. aureus multiresistance plasmid pSK1 abolished tPMP-1 resistance. Resistance to tPMP-1 in theqacA-bearing strains was inoculum independent but peptide concentration dependent, with the level of resistance decreasing at higher peptide concentrations for a given inoculum. There was no apparent cross-resistance in qacA-bearing strains to other endogenous cationic antimicrobial peptides which are structurally distinct from tPMP-1, including human neutrophil defensin 1, protamine, or the staphylococcal lantibiotics pep5 and nisin. These data demonstrate that the staphylococcal multidrug resistance geneqacA also mediates in vitro resistance to cationic tPMP-1.

scholarly journals The Role of Long Non-Coding RNAs in Endometriosis

2021 ◽  
Vol 22 (21) ◽  
pp. 11425
Author(s):  
Quanah J. Hudson ◽  
Katharina Proestling ◽  
Alexandra Perricos ◽  
Lorenz Kuessel ◽  
Heinrich Husslein ◽  
...  
Keyword(s):  
Treatment Options ◽  
Regulatory Elements ◽  
Nucleotide Polymorphisms ◽  
Delay In Diagnosis ◽  
Model Studies ◽  
Non Coding Rnas ◽  
Gynecological Disorder ◽  
Transcriptomic Studies

Endometriosis is a chronic gynecological disorder affecting the quality of life and fertility of many women around the world. Heterogeneous and non-specific symptoms may lead to a delay in diagnosis, with treatment options limited to surgery and hormonal therapy. Hence, there is a need to better understand the pathogenesis of the disease to improve diagnosis and treatment. Long non-coding RNAs (lncRNAs) have been increasingly shown to be involved in gene regulation but remain relatively under investigated in endometriosis. Mutational and transcriptomic studies have implicated lncRNAs in the pathogenesis of endometriosis. Single-nucleotide polymorphisms (SNPs) in lncRNAs or their regulatory regions have been associated with endometriosis. Genome-wide transcriptomic studies have identified lncRNAs that show deregulated expression in endometriosis, some of which have been subjected to further experiments, which support a role in endometriosis. Mechanistic studies indicate that lncRNAs may regulate genes involved in endometriosis by acting as a molecular sponge for miRNAs, by directly targeting regulatory elements via interactions with chromatin or transcription factors or by affecting signaling pathways. Future studies should concentrate on determining the role of uncharacterized lncRNAs revealed by endometriosis transcriptome studies and the relevance of lncRNAs implicated in the disease by in vitro and animal model studies.

scholarly journals In Vitro Selection and Analysis of Human Immunodeficiency Virus Type 1 Resistant to Derivatives of β-2′,3′-Didehydro-2′,3′-Dideoxy-5-Fluorocytidine

2005 ◽  
Vol 49 (9) ◽  
pp. 3930-3932 ◽  
Author(s):  
Jennifer L. Hammond ◽  
Urvi M. Parikh ◽  
Dianna L. Koontz ◽  
Susan Schlueter-Wirtz ◽  
Chung K. Chu ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
In Vitro Selection ◽  
Serial Passage ◽  
Phenotypic Analysis ◽  
Immunodeficiency Virus

ABSTRACT Serial passage of human immunodeficiency virus type 1 in MT-2 cells in increasing concentrations of the d- and l-enantiomers of β-2′,3′-didehydro-2′,3′-dideoxy-5-fluorocytidine (d4FC) resulted in the selection of viral variants with reverse transcriptase substitutions M184I or M184V for l-d4FC and I63L, K65R, K70N, K70E, or R172K for d-d4FC. Phenotypic analysis of site-directed mutants defined the role of these mutations in reducing susceptibility to l- or d-d4FC.

scholarly journals Heterogeneity ofmprFSequences in Methicillin-Resistant Staphylococcus aureus Clinical Isolates: Role in Cross-Resistance between Daptomycin and Host Defense Antimicrobial Peptides

2014 ◽  
Vol 58 (12) ◽  
pp. 7462-7467 ◽  
Author(s):  
Arnold S. Bayer ◽  
Nagendra N. Mishra ◽  
George Sakoulas ◽  
Poochit Nonejuie ◽  
Cynthia C. Nast ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Host Defense ◽  
Hot Spot ◽  
Cross Resistance ◽  
Phospholipid Content ◽  
Nucleotide Polymorphisms ◽  
Gain Of Function ◽  
Host Defense Peptides ◽  
Methicillin Resistant ◽  
Content Type

ABSTRACTOver the past several years, single-nucleotide polymorphisms (SNPs) within themprFopen reading frame (ORF) have been proposed to be associated with a gain-of-function phenotype in terms of daptomycin (DAP) nonsusceptibility (referred to as daptomycin resistance [DAP-R] herein for ease of presentation) inStaphylococcus aureus. We investigated the frequencies of SNPs within themprFORF and the relationships of such SNPs to cross-resistance between DAP and cationic host defense peptides (HDPs). Thirty-five well-characterized, unique DAP-susceptible (DAP-S) and DAP-R methicillin-resistantS. aureus(MRSA) isolates of the clonal complex 5 genotype were used. In addition tomprFSNPs and DAP-HDP cross-resistance, several other key genotypic and phenotypic metrics often associated with DAP-R were delineated, as follows: (i)mprFexpression, (ii) membrane phospholipid content, (iii) positive surface charge, (iv) DAP binding, and (v) cell wall thickness profiles. A number of DAP-S strains (MICs of ≤1 μg/ml) exhibitedmprFSNPs, occasionally with high-levelmprFsequence variation from the genotype reference strain. However, none of these SNPs were localized to well-chronicledmprFhot spot locations associated with DAP-R inS. aureus. In contrast, all 8 DAP-R isolates demonstrated SNPs within such knownmprFhot spots. Moreover, only the DAP-R strains showed MprF gain-of-function phenotypes, enhancedmprFexpression, higher survival against two prototypical HDPs, and reduced DAP binding. Although a heterogenous array ofmprFSNPs were often found in DAP-S strains, only selected hot spot SNPs, combined with concurrentmprFdysregulation, were associated with the DAP-R phenotype.

scholarly journals Copy number variations of E2F1: a new genetic risk factor for testicular cancer

2017 ◽  
Vol 24 (3) ◽  
pp. 119-125 ◽  
Author(s):  
Maria Santa Rocca ◽  
Andrea Di Nisio ◽  
Arianna Marchiori ◽  
Marco Ghezzi ◽  
Giuseppe Opocher ◽  
...  
Keyword(s):  
Copy Number ◽  
Testicular Germ Cell Tumor ◽  
Testicular Tissue ◽  
Copy Number Variations ◽  
Nucleotide Polymorphisms ◽  
Testicular Germ Cell ◽  
Mtor Phosphorylation ◽  
First Time

Testicular germ cell tumor (TGCT) is one of the most heritable forms of cancer. In last years, many evidence suggested that constitutional genetic factors, mainly single nucleotide polymorphisms, can increase its risk. However, the possible contribution of copy number variations (CNVs) in TGCT susceptibility has not been substantially addressed. Indeed, an increasing number of studies have focused on the effect of CNVs on gene expression and on the role of these structural genetic variations as risk factors for different forms of cancer. E2F1 is a transcription factor that plays an important role in regulating cell growth, differentiation, apoptosis and response to DNA damage. Therefore, deficiency or overexpression of this protein might significantly influence fundamental biological processes involved in cancer development and progression, including TGCT. We analyzed E2F1 CNVs in 261 cases with TGCT and 165 controls. We found no CNVs in controls, but 17/261 (6.5%) cases showed duplications in E2F1. Blot analysis demonstrated higher E2F1 expression in testicular samples of TGCT cases with three copies of the gene. Furthermore, we observed higher phosphorylation of Akt and mTOR in samples with E2F1 duplication. Interestingly, normal, non-tumoral testicular tissue in patient with E2F1 duplication showed lower expression of E2F1 and lower AKT/mTOR phosphorylation with respect to adjacent tumor tissue. Furthermore, increased expression of E2F1 obtained in vitro in NTERA-2 testicular cell line induced increased AKT/mTOR phosphorylation. This study suggests for the first time an involvement of E2F1 CNVs in TGCT susceptibility and supports previous preliminary data on the importance of AKT/mTOR signaling pathway in this cancer.

Factor VII activating protease

2011 ◽  
Vol 31 (03) ◽  
pp. 174-178 ◽  
Author(s):  
M. Etscheid ◽  
S. M. Kanse
Keyword(s):  
The Elderly ◽  
Factor Vii ◽  
Nucleotide Polymorphisms ◽  
Protease Activated Receptors ◽  
Single Nucleotide ◽  
Fibrinolytic Enzymes ◽  
Inflammatory Processes ◽  
Acid Exchange

SummaryFactor VII activating protease (FSAP) is a circulating serine protease with high homology to fibrinolytic enzymes. A role in the regulation of coagulation and fibrinolysis is suspected based on in vitro studies demonstrating activation of FVII or pro-urokinase plasminogen activator (uPA). However, considering the paucity of any studies in animal models or any correlative studies in humans the role of FSAP in haemostasis remains unclear. In relation to vascular remodeling processes or inflammation it has been convincingly shown that FSAP interacts with growth factors as well as protease activated receptors (PAR). Against this sparse background there are a plethora of studies which have investigated the linkage of single nucleotide polymorphisms (SNP) in the FSAP gene (HABP2) to various diseases. The G534E SNP of FSAP is associated with a low proteolytic activity due to an amino acid exchange in the protease domain. This and other SNPs have been linked to carotid stenosis, stroke as well as thrombosis in the elderly and plaque calcification. These SNP analyses indicate an important role for FSAP in the regulation of the haemostasis system as well as fibroproliferative inflammatory processes.

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