pfmdr1 mutations associated with chloroquine resistance incur a fitness cost in Plasmodium falciparum

Efforts to control malaria worldwide have been hindered by the development and expansion of parasite populations resistant to many first-line antimalarial compounds. Two of the best-characterized determinants of drug resistance in the human malaria parasite Plasmodium falciparum are pfmdr1 and pfcrt, although the mechanisms by which resistance is mediated by these genes is still not clear. In order to determine whether mutations in pfmdr1 associated with chloroquine resistance affect the capacity of the parasite to persist when drug pressure is removed, we conducted competition experiments between P. falciparum strains in which the endogenous pfmdr1 locus was modified by allelic exchange. In the absence of selective pressure, the component of chloroquine resistance attributable to mutations at codons 1034, 1042 and 1246 in the pfmdr1 gene also gave rise to a substantial fitness cost in the intraerythrocytic asexual stage of the parasite. The loss of fitness incurred by these mutations was calculated to be 25% with respect to an otherwise genetically identical strain in which wild-type polymorphisms had been substituted at these three codons. At least part of the fitness loss may be attributed to a diminished merozoite viability. These in vitro results support recent in vivo observations that in several countries where chloroquine use has been suspended because of widespread resistance, sensitive strains are re-emerging.     

Cloning and characterization of the parC and parE genes of Streptococcus pneumoniae encoding DNA topoisomerase IV: role in fluoroquinolone resistance.

DNA topoisomerase IV mediates chromosome segregation and is a potential target for antibacterial agents including new antipneumococcal fluoroquinolones. We have used hybridization to a Staphylococcus aureus gyrB probe in concert with chromosome walking to isolate the Streptococcus pneumoniae parE-parC locus, lying downstream of a putative new insertion sequence and encoding 647-residue ParE and 823-residue ParC subunits of DNA topoisomerase IV. These proteins exhibited greatest homology respectively to the GrlB (ParE) and GrlA (ParC) subunits of S. aureus DNA topoisomerase IV. When combined, whole-cell extracts of Escherichia coli strains expressing S. pneumoniae ParC or ParE proteins reconstituted a salt-insensitive ATP-dependent decatenase activity characteristic of DNA topoisomerase IV. A second gyrB homolog isolated from S. pneumoniae encoded a 648-residue protein which we identified as GyrB through its close homology both to counterparts in S. aureus and Bacillus subtilis and to the product of the S. pneumoniae nov-1 gene that confers novobiocin resistance. gyrB was not closely linked to gyrA. To examine the role of DNA topoisomerase IV in fluoroquinolone action and resistance in S. pneumoniae, we isolated mutant strains stepwise selected for resistance to increasing concentrations of ciprofloxacin. We analysed four low-level resistant mutants and showed that Ser-79 of ParC, equivalent to resistance hotspots Ser-80 of GrlA and Ser-84 of GyrA in S. aureus, was in each case substituted with Tyr. These results suggest that DNA topoisomerase IV is an important target for fluoroquinolones in S. pneumoniae and establish this organism as a useful gram-positive system for resistance studies.     

From resistance to stimulation: the evolution of a virus in the presence of a dominant lethal inhibitory scaffolding protein

By acquiring resistance to an inhibitor, viruses can become dependent on that inhibitor for optimal fitness. However, inhibitors rarely, if ever, stimulate resistant strain fitness to values that equal or exceed the uninhibited wild-type level. This would require an adaptive mechanism that converts the inhibitor into a beneficial replication factor. Using a plasmid-encoded inhibitory external scaffolding protein that blocks φX174 assembly, we previously demonstrated that such mechanisms are possible. The resistant strain, referred to as the evolved strain, contains four mutations contributing to the resistance phenotype. Three mutations confer substitutions in the coat protein, whereas the fourth mutation alters the virus-encoded external scaffolding protein. To determine whether stimulation by the inhibitory protein coevolved with resistance or whether it was acquired after resistance was firmly established, the strain temporally preceding the previously characterized mutant, referred to as the intermediary strain, was isolated and characterized. The results of the analysis indicated that the mutation in the virus-encoded external scaffolding protein was primarily responsible for stimulating strain fitness. When the mutation was placed in a wild-type background, it did not confer resistance. The mutation was also placed in cis with the plasmid-encoded dominant lethal mutation. In this configuration, the stimulating mutation exhibited no activity, regardless of the genotype (wild type, evolved, or intermediary) of the infecting virus. Thus, along with the coat protein mutations, stimulation required two external scaffolding protein genes: the once inhibitory gene and the mutant gene acquired during evolution.     

Role of the kdr and super-kdr sodium channel mutations in pyrethroid resistance: correlation of allelic frequency to resistance level in wild and laboratory populations of horn flies (Haematobia irritans)

The kdr and super-kdr point mutations found in the insect sodium channel gene are postulated to confer knockdown resistance (kdr) to pyrethroids. Using an allele-specific PCR assay to detect these mutations in individual horn flies, Haematobia irritans (L.), we determined the allelic frequency of the kdr and super-kdr mutations in several wild and laboratory populations. Wild populations with very similar allelic frequencies had resistance levels that ranged widely from 3- to 18-fold relative to a susceptible population. Conversely, the kdr allele frequency in a lab population with 17-fold resistance was nearly double that found in a heavily pressured wild population with 18-fold resistance. We conclude that, although the kdr mutation confers significant levels of pyrethroid resistance, a substantial component of resistance in insecticidally pressured populations is conferred by mechanisms that are PBO-suppressible. High super-kdr allele frequencies were detected in two resistant lab populations, but in wild populations with equivalent resistance the super-kdr allele frequency was very low. Interestingly, in over 1200 individuals assayed, the super-kdr mutation was never detected in the absence of the kdr mutation.     

Adaptation to the cost of resistance: a model of compensation, recombination, and selection in a haploid organism

Populations of pathogenic organisms often evolve resistance in response to the use of pesticides or antibiotics. This rise of resistance may be followed by a fall when chemical control is suspended and resistance alleles carry a fitness cost. Another possibility is that mutations at secondary loci compensate for the cost, usually without loss of resistance. This enables resistant types to withstand invasion by the susceptible wild-type; resistance then persists in the population, which reduces the efficacy of future pesticide or antibiotic use. We examined a two-locus model of a haploid organism that adapts to the cost of resistance by a single compensatory mutation. We addressed the question how different combinations of cost and compensation and different levels of recombination affect the consequences of a single pesticide application. Resistance will become fixed in the population when the fraction of the population exposed to pesticide exceeds the cost of resistance. Compensatory mutations reduce the cost of resistance and therefore this threshold level of pesticide use. In the absence of pesticide, recombination promotes stability of equilibria. In the presence of pesticide, recombination accelerates the fixation of resistance and compensating alleles; recombination may also enable the persistence of compensated resistant types after pesticide use.     

Role of penicillin-binding protein 2 (PBP2) in the antibiotic susceptibility and cell wall cross-linking of Staphylococcus aureus: evidence for the cooperative functioning of PBP2, PBP4, and PBP2A

Ceftizoxime, a beta-lactam antibiotic with high selective affinity for penicillin-binding protein 2 (PBP2) of Staphylococcus aureus, was used to select a spontaneous resistant mutant of S. aureus strain 27s. The stable resistant mutant ZOX3 had an increased ceftizoxime MIC and a decreased affinity of its PBP2 for ceftizoxime and produced peptidoglycan in which the proportion of highly cross-linked muropeptides was reduced. The pbpB gene of ZOX3 carried a single C-to-T nucleotide substitution at nucleotide 1373, causing replacement of a proline with a leucine at amino acid residue 458 of the transpeptidase domain of the protein, close to the SFN conserved motif. Experimental proof that this point mutation was responsible for the drug-resistant phenotype, and also for the decreased PBP2 affinity and reduced cell wall cross-linking, was provided by allelic replacement experiments and site-directed mutagenesis. Disruption of pbpD, the structural gene of PBP4, in either the parental strain or the mutant caused a large decrease in the highly cross-linked muropeptide components of the cell wall and in the mutant caused a massive accumulation of muropeptide monomers as well. Disruption of pbpD also caused increased sensitivity to ceftizoxime in both the parental cells and the ZOX3 mutant, while introduction of the plasmid-borne mecA gene, the genetic determinant of the beta-lactam resistance protein PBP2A, had the opposite effects. The findings provide evidence for the cooperative functioning of two native S. aureus transpeptidases (PBP2 and PBP4) and an acquired transpeptidase (PBP2A) in staphylococcal cell wall biosynthesis and susceptibility to antimicrobial agents.     

The correlation between phenotypes and genotypes of macrolides, lincosamides and streptogramins B resistance in Staphylococcus aureus

For 31 clinical strains of S. aureus the correlation between phenotype and genotype of resistance to macrolides, lincosamides and streptogramins B (MLSB) was established.. Phenotypes were determined on the basis of: susceptibility to erythromycin and clindamycin and the ability to an induction of the resistance (phenotypes S, susceptible; R , constitutive resistant, D, resistant after induction with erythromycin, D+, resistant after induction with erythromycin and with a presence of the small colonies inside inhibition zone between erythromycin and clindamycin discs), and on the basis of the resistance to spectinomycin (spR, resistant, spS, susceptible). Among examined S. aureus strains eight phenotypes of resistance to MLSB were recognized (the corresponding genotypes are given in brackets). Six phenotypes were typical: SspS (lack of MLS-B resistance genes), NEGspS (msrA/B, 1 strain), D+spS (ermCi, 4 strains),. DspR (ermAi, 11 strains and ermAi + msrA/B, 2 strains), RspR (ermAc, 4 strains and ermA + msrA/B,1 strain and ermA + ermC, 1 strain) and RspS (ermCc, 6 strains and ermB, 1 strain). Two rare phenotypes in two single strains were observed: SspR (ermAi, the strain with altered inducibility, inductor other than erythromycin) and DspS (ermAi, presumably mutation or lack of spc in Tn554).     

水稻细菌性条斑病和白叶枯病抗性遗传研究

分析了Ｈａｓｈｉｋａｌｍｉ,Ｄｕｌａｒ和９０ＩＲＢＢＮ４４三个抗源品种对水稻细菌性条斑病Ｓ－１０３菌株和白叶枯病Ｐ１菌系的抗性遗传。结果表明,Ｈａｓｈｉｋａｌｍｉ和Ｄｕｌａｒ对Ｓ－１０３的抗性均由２对隐性基因所控制,９０ＩＲＢＢＮ４４则带有１对隐性抗性基因。经等位性测定表明,Ｈａｓｈｉｋａｌｍｉ和Ｄｕｌａｒ的２对基因中至少有１对是等位的,但它们与９０ＩＲＢＢＮ４４的１对基因均不等位。３个抗源品种对Ｐ１的抗性都受１对隐性基因控制,该基因与ｘａ－５等位。连锁遗传分析表明,Ｈａｓｈｉｋａｌｍｉ和Ｄｕｌａｒ对Ｓ－１０３的２对抗细条病基因中的１对与ｘａ－５相连锁,而９０ＩＲＢＢＮ４４的１对抗性基因与ｘａ－５呈独立遗传。本文还就开展水稻抗细菌性条斑病育种进行了讨论。     

Cell wall components in Staphylococcus aureus with double resistance to gramicidin S and actinomycin D]

Cell walls of Staphylococcus aureus R9/80 resistant to gramicidin S and actinomycin D were investigated. The strain was isolated after passages of a previously isolated strain of S. aureus with resistance to gramicidin and definite changes in the cell walls, a medium with increasing concentrations of actinomycin being used for the passages. The data on the study of the cell walls of the strain with the double resistance were compared with the results of the investigation of the cell walls of the strain susceptible to gramicidin, the gramicidin resistant strain (initial for strain R9/80) and the actinomycin adapted strain that also showed changes in the cell walls. The cell walls of the resistant strains had no significant changes in the peptidoglycane and glucosamine levels, as well as in the peptidoglycane amino acid composition. Teichoic acids of all the strains had different levels of substitution of ribite by D-alanine (a factor influencing the negative charge of teichoic acids and the wall at large). It was noted that all the strains resistant to the tested antibiotics had lower levels of teichoic acids in the cell walls. The resistant cells showed some increase of the lipid component in the walls: from 1.6% in the susceptible strain to 2.1-2.9% in the resistant cells. The main trend of the changes in the resistance development was revealed to be the thickening of the cell wall and its consolidation. The development of resistance to gramicidin, actinomycin and to both the antibiotics provoked respectively a 2.4-, 4- and 5.4-fold increase of the content of the main cell component. i.e. peptidoglycane in the cell biomass. The barrier role of the cell walls in the resistant strains and their ability to bind the antibiotic is discussed.     

Understanding the Mechanism of Atovaquone Drug Resistance in Plasmodium falciparum Cytochrome b Mutation Y268S Using Computational Methods

The rapid appearance of resistant malarial parasites after introduction of atovaquone (ATQ) drug has prompted the search for new drugs as even single point mutations in the active site of Cytochrome b protein can rapidly render ATQ ineffective. The presence of Y268 mutations in the Cytochrome b (Cyt b) protein is previously suggested to be responsible for the ATQ resistance in Plasmodium falciparum (P. falciparum). In this study, we examined the resistance mechanism against ATQ in P. falciparum through computational methods. Here, we reported a reliable protein model of Cyt bc1 complex containing Cyt b and the Iron-Sulphur Protein (ISP) of P. falciparum using composite modeling method by combining threading, ab initio modeling and atomic-level structure refinement approaches. The molecular dynamics simulations suggest that Y268S mutation causes ATQ resistance by reducing hydrophobic interactions between Cyt bc1 protein complex and ATQ. Moreover, the important histidine contact of ATQ with the ISP chain is also lost due to Y268S mutation. We noticed the induced mutation alters the arrangement of active site residues in a fashion that enforces ATQ to find its new stable binding site far away from the wild-type binding pocket. The MM-PBSA calculations also shows that the binding affinity of ATQ with Cyt bc1 complex is enough to hold it at this new site that ultimately leads to the ATQ resistance.     

Construction of a yrA plasmid for genetic characterization of fluoroquinolone-resistant Staphylococcus aureus

Abstract We have constructed a gyrA trans -complementation plasmid, pAT512, by cloning the wild-type gyrA gene of Staphylococcus aurues into expression vector pAT392. Introduction by electrotransformation of pAT512 into a high-level fluoroquinolone resistant mutant of S. aureus (ciprofloxacin MIC= 16 μ g ml⁻¹) having a gyrA mutation which results in a Ser-84 to Leu substitution, reduced the MICs of norfloxacin and ciprofloxacin for the host of four- and eight-fold, respectively.     

Repression of Invasion Genes and Decreased Invasion in a High-Level Fluoroquinolone-Resistant Salmonella Typhimurium Mutant

Background

Nalidixic acid resistance among Salmonella Typhimurium clinical isolates has steadily increased, whereas the level of ciprofloxacin resistance remains low. The main objective of this study was to characterize the fluoroquinolone resistance mechanisms acquired in a S. Typhimurium mutant selected with ciprofloxacin from a susceptible isolate and to investigate its invasion ability.

Methodology/Principal Findings

Three different amino acid substitutions were detected in the quinolone target proteins of the resistant mutant (MIC of ciprofloxacin, 64 µg/ml): D87G and G81C in GyrA, and a novel mutation, E470K, in ParE. A protein analysis revealed an increased expression of AcrAB/TolC and decreased expression of OmpC. Sequencing of the marRAB, soxRS, ramR and acrR operons did not show any mutation and neither did their expression levels in a microarray analysis. A decreased percentage of invasion ability was detected when compared with the susceptible clinical isolate in a gentamicin protection assay. The microarray results revealed a decreased expression of genes which play a role during the invasion process, such as hilA, invF and the flhDC operon. Of note was the impaired growth detected in the resistant strain. A strain with a reverted phenotype (mainly concerning the resistance phenotype) was obtained from the resistant mutant.

Conclusions/Significance

In conclusion, a possible link between fluoroquinolone resistance and decreased cell invasion ability may exist explaining the low prevalence of fluoroquinolone-resistant S. Typhimurium clinical isolates. The impaired growth may appear as a consequence of fluoroquinolone resistance acquisition and down-regulate the expression of the invasion genes.     

Bidirectional selection for body mass and correlated response of pyrethroid resistance and fitness in Sitophilus zeamais

Responses to artificial selection on body mass in the maize weevil Sitophilus zeamais (Coleoptera: Curculionidae) were investigated to determine whether changes in body mass are associated with insecticide susceptibility, rate of population growth, and metabolic rate. Two strains of the maize weevil differing in susceptibility to pyrethroid insecticides were subjected to bidirectional selection on body mass. The susceptible strain responded to selection resulting in individuals with lower or higher body mass, but the resistant strain responded significantly only to selection for lower body mass. The resistant strain selected for low body mass increased its level of deltamethrin resistance in 44 × . In contrast, selection for low body mass in the susceptible parental strain led to increased deltamethrin susceptibility (50 × ) and selection for high body mass increased deltamethrin resistance (4 × ). Thus, the correlated response of insecticide resistance to selection for body mass differed between strains, a likely consequence of their distinct genetic background. Regardless, body mass was positively correlated with fitness (reproductive output) (r = 0.79; P < 0.001), while such correlation with respiration rate was significant only at P = 0.07 (r = 0.44). Therefore, the association between body mass and deltamethrin resistance is population‐dependent in the maize weevil, and the confluence of deltamethrin resistance and high body mass in a given strain will likely favour its energy metabolism and lead to the mitigation of fitness costs usually associated with insecticide resistance. The genetic background and selection history of insecticide resistant populations should not be neglected since they may favour the confluence of insecticide resistance with mitigation mechanisms of its associated fitness costs limiting the tactics available to their management.