Trade-offs with stability modulate innate and mutationally acquired drug resistance in bacterial dihydrofolate reductase enzymes

2018 ◽  
Vol 475 (12) ◽  
pp. 2107-2125 ◽  
Author(s):  
Nishad Matange ◽  
Swapnil Bodkhe ◽  
Maitri Patel ◽  
Pooja Shah
Keyword(s):  
Drug Resistance ◽  
Structural Stability ◽  
Dihydrofolate Reductase ◽  
Mutational Analysis ◽  
Hydrophobic Interactions ◽  
Wild Type ◽  
Resistance Mutations ◽  
Acquired Drug Resistance ◽  
Trade Offs ◽  
And Function

Structural stability is a major constraint on the evolution of protein sequences. However, under strong directional selection, mutations that confer novel phenotypes but compromise structural stability of proteins may be permissible. During the evolution of antibiotic resistance, mutations that confer drug resistance often have pleiotropic effects on the structure and function of antibiotic-target proteins, usually essential metabolic enzymes. In the present study, we show that trimethoprim (TMP)-resistant alleles of dihydrofolate reductase from Escherichia coli (EcDHFR) harboring the Trp30Gly, Trp30Arg or Trp30Cys mutations are significantly less stable than the wild-type, making them prone to aggregation and proteolysis. This destabilization is associated with a lower expression level, resulting in a fitness cost and negative epistasis with other TMP-resistant mutations in EcDHFR. Using structure-based mutational analysis, we show that perturbation of critical stabilizing hydrophobic interactions in wild-type EcDHFR enzyme explains the phenotypes of Trp30 mutants. Surprisingly, though crucial for the stability of EcDHFR, significant sequence variation is found at this site among bacterial dihydrofolate reductases (DHFRs). Mutational and computational analyses in EcDHFR and in DHFR enzymes from Staphylococcus aureus and Mycobacterium tuberculosis demonstrate that natural variation at this site and its interacting hydrophobic residues modulates TMP resistance in other bacterial DHFRs as well, and may explain the different susceptibilities of bacterial pathogens to TMP. Our study demonstrates that trade-offs between structural stability and function can influence innate drug resistance as well as the potential for mutationally acquired drug resistance of an enzyme.

scholarly journals Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Mutational Analysis ◽  
Wild Type ◽  
Content Type ◽  
Growth Defects ◽  
C Terminus ◽  
K 12 ◽  
And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

scholarly journals Midkine Mediates Intercellular Crosstalk between Drug-Resistant and Drug-Sensitive Neuroblastoma Cells In Vitro and In Vivo

ISRN Oncology ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-12
Author(s):  
Fei Chu ◽  
Jessica A. Naiditch ◽  
Sandra Clark ◽  
Yi-Yong Qiu ◽  
Xin Zheng ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Chemotherapy Resistance ◽  
Neuroblastoma Cells ◽  
Cytotoxic Agents ◽  
Drug Resistant ◽  
Wild Type ◽  
Cytoprotective Effect ◽  
Acquired Drug Resistance

Resistance to cytotoxic agents has long been known to be a major limitation in the treatment of human cancers. Although many mechanisms of drug resistance have been identified, chemotherapies targeting known mechanisms have failed to lead to effective reversal of drug resistance, suggesting that alternative mechanisms remain undiscovered. Previous work identified midkine (MK) as a novel putative survival molecule responsible for cytoprotective signaling between drug-resistant and drug-sensitive neuroblastoma, osteosarcoma and breast carcinoma cells in vitro. In the present study, we provide further in vitro and in vivo studies supporting the role of MK in neuroblastoma cytoprotection. MK overexpressing wild type neuroblastoma cells exhibit a cytoprotective effect on wild type cells when grown in a co-culture system, similar to that seen with doxorubicin resistant cells. siRNA knockdown of MK expression in doxorubicin resistant neuroblastoma and osteosarcoma cells ameliorates this protective effect. Overexpression of MK in wild type neuroblastoma cells leads to acquired drug resistance to doxorubicin and to the related drug etoposide. Mouse studies injecting various ratios of doxorubicin resistant or MK transfected cells with GFP transfected wild type cells confirm this cytoprotective effect in vivo. These findings provide additional evidence for the existence of intercellular cytoprotective signals mediated by MK which contribute to chemotherapy resistance in neuroblastoma.

scholarly journals Virologic failure and HIV drug resistance on simplified, dolutegravir-based maintenance therapy: Systematic review and meta-analysis

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 1359 ◽  
Author(s):  
Gilles Wandeler ◽  
Marta Buzzi ◽  
Nanina Anderegg ◽  
Delphine Sculier ◽  
Charles Béguelin ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Viral Load ◽  
Maintenance Therapy ◽  
Virologic Failure ◽  
Meta Analysis ◽  
Dual Therapy ◽  
Undetectable Viral Load ◽  
Resistance Mutations ◽  
Hiv Viral Load ◽  
Acquired Drug Resistance

Background: Dolutegravir-containing maintenance therapy is a promising simplification strategy for virologically suppressed HIV-infected individuals. However, most of the available data to inform this strategy come from small, uncontrolled studies. We estimated the proportion of HIV-infected patients experiencing virological failure (VF) and developing drug resistance on dolutegravir (DTG)-based maintenance therapy. Methods: We searched Medline, Embase, Cochrane Central, Web of Science, and conference abstracts for studies assessing VF on DTG-based maintenance therapy. Studies including ≥5 adults with an undetectable viral load on antiretroviral therapy (ART) who switched to a DTG-based mono- or dual therapy were included. Pooled proportions of VF were estimated using random-intercept logistic meta-regression and acquired drug resistance mutations described for each strategy. Results: Of 1719 studies considered, 21 met our selection criteria, including seven interventional and 14 observational studies. Eight studies including 251 patients assessed VF on DTG monotherapy and fourteen studies including 1670 participants VF on dual therapy. The participant’s median age ranged from 43 to 63 years, their median nadir CD4 count from 90 to 399 cells/µl, and 27.6% were female. The proportion of participants experiencing VF on DTG-monotherapy was 3.6% (95% confidence interval [CI] 1.9-6.7) at 24 weeks and 8.9% (95% CI 4.7-16.2) at 48 weeks. Resistance mutations developed in seven (3.6%) participants on DTG-monotherapy. Among patients on dual therapy, ten (0.7%, 95% CI 0.4-1.3) experienced VF by 48 weeks and none developed resistance to DTG. In adjusted analyses, VF at 24 weeks was less likely on dual therapy than on monotherapy (adjusted odds ratio: 0.10, 95% CI 0.03-0.30). Conclusions: Whereas VF is relatively common on DTG maintenance monotherapy, DTG-based dual therapy appears to be a promising simplification strategy for individuals with a suppressed HIV viral load on triple-ART.

scholarly journals Virological outcome among HIV infected patients transferred from pediatric care to adult units in Madrid, Spain (1997–2017)

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Carolina Beltrán-Pavez ◽  
◽  
Miguel Gutiérrez-López ◽  
Marina Rubio-Garrido ◽  
Ana Valadés-Alcaraz ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Public Hospitals ◽  
Pediatric Care ◽  
Study Cohort ◽  
Resistance Mutations ◽  
Adult Care ◽  
Genotypic Resistance ◽  
Acquired Drug Resistance ◽  
Virological Outcome ◽  
Transferred Patients

Abstract The aim of this transversal study was to describe the virological and immunological features of HIV-infected youths transferred from pediatric to adult care units since 1997 vs. the non-transferred patients from the Madrid Cohort of HIV-infected children and adolescents in Spain. We included 106 non-transferred and 184 transferred patients under clinical follow-up in 17 public hospitals in Madrid by the end of December 2017. Virological and immunological outcomes were compared in transferred vs. non-transferred patients. ART drug resistance mutations and HIV-variants were analyzed in all subjects with available resistance pol genotypes and/or genotypic resistance profiles. Among the study cohort, 133 (72.3%) of 184 transferred and 75 (70.7%) of 106 non-transferred patients had available resistance genotypes. Most (88.9%) of transferred had ART experience at sampling. A third (33.3%) had had a triple-class experience. Acquired drug resistance (ADR) prevalence was significantly higher in pretreated transferred than non-transferred patients (71.8% vs. 44%; p = 0.0009), mainly to NRTI (72.8% vs. 31.1%; p < 0.0001) and PI (29.1% vs. 12%; p = 0.0262). HIV-1 non-B variants were less frequent in transferred vs. non-transferred (6.9% vs. 32%; p < 0.0001). In conclusion, the frequent resistant genotypes found in transferred youths justifies the reinforcement of HIV resistance monitoring after the transition to avoid future therapeutic failures.

scholarly journals Combined Drug Resistance Mutations Substantially Enhance Enzyme Production inPaenibacillus agaridevorans

2018 ◽  
Vol 200 (17) ◽  
Author(s):  
Kazumi Funane ◽  
Yukinori Tanaka ◽  
Takeshi Hosaka ◽  
Kiriko Murakami ◽  
Takatsugu Miyazaki ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Enzyme Production ◽  
Scale Production ◽  
Wild Type ◽  
Resistance Mutations ◽  
Triple Mutant ◽  
Content Type ◽  
Wide Applicability ◽  
Drug Resistance Mutations ◽  
Industrial Enzyme

ABSTRACTThis study shows that sequential introduction of drug resistance mutations substantially increased enzyme production inPaenibacillus agaridevorans. The triple mutant YT478 (rsmGGln225→stop codon,rpsLK56R, andrpoBR485H), generated by screening for resistance to streptomycin and rifampin, expressed a 1,100-fold-larger amount of the extracellular enzyme cycloisomaltooligosaccharide glucanotransferase (CITase) than the wild-type strain. These mutants were characterized by higher intracellularS-adenosylmethionine concentrations during exponential phase and enhanced protein synthesis activity during stationary phase. Surprisingly, the maximal expression of CITase mRNA was similar in the wild-type and triple mutant strains, but the mutant showed greater CITase mRNA expression throughout the growth curve, resulting in enzyme overproduction. A metabolome analysis showed that the triple mutant YT478 had higher levels of nucleic acids and glycolysis metabolites than the wild type, indicating that YT478 mutant cells were activated. The production of CITase by the triple mutant was further enhanced by introducing a mutation conferring resistance to the rare earth element, scandium. This combined drug resistance mutation method also effectively enhanced the production of amylases, proteases, and agarases byP. agaridevoransandStreptomyces coelicolor. This method also activated the silent or weak expression of theP. agaridevoransCITase gene, as shown by comparisons of the CITase gene loci ofP. agaridevoransT-3040 and another cycloisomaltooligosaccharide-producing bacterium,Paenibacillussp. strain 598K. The simplicity and wide applicability of this method should facilitate not only industrial enzyme production but also the identification of dormant enzymes by activating the expression of silent or weakly expressed genes.IMPORTANCEEnzyme use has become more widespread in industry. This study evaluated the molecular basis and effectiveness of ribosome engineering in markedly enhancing enzyme production (>1,000-fold). This method, due to its simplicity, wide applicability, and scalability for large-scale production, should facilitate not only industrial enzyme production but also the identification of novel enzymes, because microorganisms contain many silent or weakly expressed genes which encode novel antibiotics or enzymes. Furthermore, this study provides a new mechanism for strain improvement, with a consistent rather than transient high expression of the key gene(s) involved in enzyme production.

scholarly journals Trade-offs between enzyme fitness and solubility illuminated by deep mutational scanning

2017 ◽  
Vol 114 (9) ◽  
pp. 2265-2270 ◽  
Author(s):  
Justin R. Klesmith ◽  
John-Paul Bacik ◽  
Emily E. Wrenbeck ◽  
Ryszard Michalczyk ◽  
Timothy A. Whitehead
Keyword(s):  
Surface Display ◽  
Protein Solubility ◽  
Fitness Landscapes ◽  
Yeast Surface Display ◽  
Crystallographic Structure ◽  
Missense Mutations ◽  
Classification Models ◽  
Wild Type ◽  
Trade Offs ◽  
And Function

Proteins are marginally stable, and an understanding of the sequence determinants for improved protein solubility is highly desired. For enzymes, it is well known that many mutations that increase protein solubility decrease catalytic activity. These competing effects frustrate efforts to design and engineer stable, active enzymes without laborious high-throughput activity screens. To address the trade-off between enzyme solubility and activity, we performed deep mutational scanning using two different screens/selections that purport to gauge protein solubility for two full-length enzymes. We assayed a TEM-1 beta-lactamase variant and levoglucosan kinase (LGK) using yeast surface display (YSD) screening and a twin-arginine translocation pathway selection. We then compared these scans with published experimental fitness landscapes. Results from the YSD screen could explain 37% of the variance in the fitness landscapes for one enzyme. Five percent to 10% of all single missense mutations improve solubility, matching theoretical predictions of global protein stability. For a given solubility-enhancing mutation, the probability that it would retain wild-type fitness was correlated with evolutionary conservation and distance to active site, and anticorrelated with contact number. Hybrid classification models were developed that could predict solubility-enhancing mutations that maintain wild-type fitness with an accuracy of 90%. The downside of using such classification models is the removal of rare mutations that improve both fitness and solubility. To reveal the biophysical basis of enhanced protein solubility and function, we determined the crystallographic structure of one such LGK mutant. Beyond fundamental insights into trade-offs between stability and activity, these results have potential biotechnological applications.

High rate of virological failure and HIV drug resistance in semi-rural Gabon and implications for dolutegravir-based regimen efficacy

2020 ◽  
Author(s):  
Jéordy D Engone-Ondo ◽  
Augustin Mouinga-Ondémé ◽  
Sonia E Lékana-Douki ◽  
Abdoulaye Diané ◽  
Antony I Mamimandjiami ◽  
...  
Keyword(s):  
Drug Resistance ◽  
High Rate ◽  
Resistance Mutations ◽  
First Line ◽  
Rural Regions ◽  
Hiv Drug Resistance ◽  
Acquired Drug Resistance ◽  
Resource Limited ◽  
Study Population ◽  
Study Participants

Abstract Background The projected UNAIDS goal of ending AIDS by 2030 requires significant global efforts to improve current and future ART strategies. In this study, we assessed viral load (VL) suppression and acquired drug resistance, as well as future efficacy of dolutegravir-based combinations for patients living in semi-rural regions of Gabon. Methods Eligible study participants were adults receiving ART and recruited between 2018 and 2019 in Franceville, Gabon. VL testing was conducted to assess VL suppression and HIV drug resistance (HIVDR) testing was performed to identify resistance mutations and assess their impact on ongoing and future ART regimens. Results We recruited 219 participants overall. The median time on ART was 27 months and 216/219 participants were on first-line ART. VL suppression (VL &lt; 1000 copies/mL) was 57.1% (95% CI 50.5–63.8) overall; 59.4% (51.4–67.5) and 52.2% (40.3–64.2) for women and men, respectively. The overall prevalence of HIVDR was 21.9% among the study population and 67.2% among those who failed ART. Presence of both NRTI and NNRTI mutations was found in 84.6% of sequences with drug resistance mutations, and full activity of a dolutegravir-based first-line regimen including tenofovir disoproxil fumarate/lamivudine/dolutegravir was expected only for 5/39 patients with a resistant virus. Conclusions This study shows a very low rate of VL suppression in a semi-rural context in Africa. Moreover, the high burden of HIVDR has affected both current and newly recommended ART strategies. Better management of ART in resource-limited settings is still a challenging ambition.

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