folP Gene Research Articles

Resistance to trimethoprim/sulfamethoxazole and Tropheryma whipplei

Whipple's disease (WD) is a chronic infection caused by Tropheryma whipplei. A 1-year treatment of oral trimethoprim/sulfamethoxazole (SXT) is commonly used. Advances in the culture of T. whipplei have allowed for full genome sequencing and antibiotic susceptibility testing, which has demonstrated resistance of T. whipplei to trimethoprim. Several mutations in the folP gene that encodes dihydropteroate synthase, the target of sulphonamides, has been reported for one patient with clinically acquired resistance to SXT. Here we report three new patients who experienced clinically acquired resistance to SXT during treatment and one patient with biological failure. Sixty-two folP sequences from DNA samples of 59 WD patients were also obtained. Among the detected amino acid changes, two positions (N4S and S234F) significantly predicted secondary sulfamethoxazole failure (four of five). We suggest that these mutations should be detected at the time of WD diagnosis by sequencing folP in order to avoid sulfamethoxazole monotherapy.

A novel method for simple detection of mutations conferring drug resistance in Mycobacterium leprae, based on a DNA microarray, and its applicability in developing countries

A simple method to detect mutations in the genome of Mycobacterium leprae that confer resistance to key drugs for leprosy was exploited on the basis of a reverse hybridization system. A series of oligonucleotide probes corresponding to each mutation in the folP1, rpoB and gyrA genes for dapsone, rifampicin and ofloxacin resistance, respectively, were selected and fixed on a glass slide as capture probes, to develop a DNA microarray termed the leprosy drug susceptibility-DNA microarray (LDS-DA). Mutations in clinical isolates of M. leprae were successfully identified by the LDS-DA. Feasibility studies were conducted to evaluate the performance of the LDS-DA in two developing countries, Myanmar and the Philippines. The high concordance of the results obtained by this method with the results of nucleotide sequencing strongly supports the applicability of the LDS-DA as a drug susceptibility test in place of sequencing, a time-consuming and costly procedure. This is a rapid and simple method for the simultaneous susceptibility testing of three front-line drugs for leprosy, and solves the problems of previously reported methods.

A bioinformatic approach to understanding antibiotic resistance in intracellular bacteria through whole genome analysis

Intracellular bacteria survive within eukaryotic host cells and are difficult to kill with certain antibiotics. As a result, antibiotic resistance in intracellular bacteria is becoming commonplace in healthcare institutions. Owing to the lack of methods available for transforming these bacteria, we evaluated the mechanisms of resistance using molecular methods and in silico genome analysis. The objective of this review was to understand the molecular mechanisms of antibiotic resistance through in silico comparisons of the genomes of obligate and facultative intracellular bacteria. The available data on in vitro mutants reported for intracellular bacteria were also reviewed. These genomic data were analysed to find natural mutations in known target genes involved in antibiotic resistance and to look for the presence or absence of different resistance determinants. Our analysis revealed the presence of tetracycline resistance protein (Tet) in Bartonella quintana, Francisella tularensis and Brucella ovis; moreover, most of the Francisella strains possessed the blaA gene, AmpG protein and metallo-β-lactamase family protein. The presence or absence of folP (dihydropteroate synthase) and folA (dihydrofolate reductase) genes in the genome could explain natural resistance to co-trimoxazole. Finally, multiple genes encoding different efflux pumps were studied. This in silico approach was an effective method for understanding the mechanisms of antibiotic resistance in intracellular bacteria. The whole genome sequence analysis will help to predict several important phenotypic characteristics, in particular resistance to different antibiotics. In the future, stable mutants should be obtained through transformation methods in order to demonstrate experimentally the determinants of resistance in intracellular bacteria.

Estudio de resistencia a la rifampicina y la dapsona en tres pacientes con recurrencia de lepra

To detect the presence of rifampin- and dapsone-resistant strains of Mycobacterium leprae in three patients with recurring leprosy and clinically-suspected antimicrobial resistance through molecular techniques. A retrospective, descriptive study was conducted of three multibacillary patients at the "Agua de Dios" Sanitarium in Cundinamarca, Colombia, that presented leprosy relapses that were documented by medical history, bacilloscopy, and biopsy. Biopsies were taken of the skin lesions and the bacteria were subject to DNA extraction and purification. Regions of the rpoB and folP1 genes associated with antimicrobial resistance were amplified and subjected to touch-down polymerase chain reaction and the amplified products were sequenced using the Sanger method. A punctual mutation was identified in nucleotide 1367 of the rpoB gene in two of the samples studied. This mutation was not found in the folP1 gene of any of the three patients. The mutation identified showed strains of rifampin-resistant M. leprae in two of the three patients with recurring leprosy. Mutations that indicate dapsone-resistance were not detected in any of the three patients.

The frequency of drug resistance mutations in Mycobacterium leprae isolates in untreated and relapsed leprosy patients from Myanmar, Indonesia and the Philippines

The magnitude of drug resistance in Mycobacterium leprae to dapsone, rifampicin, and ofloxacin was studied in three Southeast Asian countries with a high prevalence of leprosy. M. leprae from the skin of leprosy patients was collected in North Maluku and North Sulawesi in Indonesia, Yangon in Myanmar, and Cebu in the Philippines. Mutations in the drug resistance determining regions in the folP1, rpoB, and gyrA genes, which have been proven to confer resistance, were analysed. In addition, samples from 51 newly diagnosed cases and 13 patients with leprosy relapse in Cebu were submitted for susceptibility testing in the mouse footpad. Of 252 isolates obtained from new cases, 3% were dapsone resistant and 2% were rifampicin resistant. In samples taken from patients with relapsed leprosy (n = 53), significantly more resistance mutations were detected: 15% had dapsone resistance mutations, and 8% had rifampicin resistance mutations. Two patients with relapsed leprosy had mutations for both dapsone and rifampicin resistance. No mutations conferring quinolone resistance were detected. No mutations were detected in the folP1 gene of M. leprae isolates with a low degree of resistance to dapsone. Detection of drug-resistant cases by mutation detection in the drug resistance determining region of the genome is a practical method for monitoring resistance. A comparison of the results obtained in this study with previous data obtained prior to the use of multidrug therapy (MDT), does not indicate clearly whether the magnitude of drug resistance has changed. Larger studies of resistance mutations in M. leprae isolated from patients with relapsed leprosy are needed to confirm our results. We recommend monitoring the magnitude of drug resistance globally, by testing M. leprae DNA from relapse cases and a representative sample of new cases.

Internal promoter characterization and expression of theDeinococcus radiodurans pprI-folPgene cluster

PprI is a general gene switch responsible for the extraordinary radioresistance of Deinococcus radiodurans. From NCBI DNA sequence analysis, it was predicted that the translation start codon of the downstream folP (DR0168) gene overlaps the pprI (DR0167) stop codon, suggesting that these genes may form an operon. In this study, we show that a mutant containing an inserted sequence in folP does not grow unless folate is added to the medium, but is not affected in extreme radioresistance, whereas a pprI disruptant strain could grow in the absence of folate. It was found that expression of a pprI-lacZ fusion is constitutive and unaltered following ionizing radiation as is the production of the PprI protein. PprI protein is not expressed if its promoter is deleted and the transcription from the entire pprI promoter is essential for radioresistance of D. radiodurans. However, the deletion of pprI promoter has no effect on the expression of the folP-lacZ fusion. Primer extension analysis of the folP promoter region shows that folP is transcribed from its own promoter located within the pprI structural gene. All these results do support neither the existence of a pprI-folP operon nor a regulatory role of FolP in pprI expression.

Variations in gene organization and DNA uptake signal sequence in the folP region between commensal and pathogenic Neisseria species

BackgroundHorizontal gene transfer is an important source of genetic variation among Neisseria species and has contributed to the spread of resistance to penicillin and sulfonamide drugs in the pathogen Neisseria meningitidis. Sulfonamide resistance in Neisseria meningitidis is mediated by altered chromosomal folP genes. At least some folP alleles conferring resistance have been horizontally acquired from other species, presumably from commensal Neisseriae. In this work, the DNA sequence surrounding folP in commensal Neisseria species was determined and compared to corresponding regions in pathogenic Neisseriae, in order to elucidate the potential for inter-species DNA transfer within this region.ResultsThe upstream region of folP displayed differences in gene order between species, including an insertion of a complete Correia element in Neisseria lactamica and an inversion of a larger genomic segment in Neisseria sicca, Neisseria subflava and Neisseria mucosa. The latter species also had DNA uptake signal sequences (DUS) in this region that were one base different from the DUS in pathogenic Neisseriae. Another interesting finding was evidence of a horizontal transfer event from Neisseria lactamica or Neisseria cinerea that introduced a novel folP allele to the meningococcal population.ConclusionGenetic recombination events immediately upstream of folP and horizontal transfer have resulted in sequence differences in the folP region between the Neisseria species. This variability could be a consequence of the selective pressure on this region exerted by the use of sulfonamide drugs.

Dihydropteroate Synthase Mutations in the folP1 Gene Predict Dapsone Resistance in Relapsed Cases of Leprosy

Molecular detection was compared with the mouse footpad inoculation test for detection of dapsone resistance in 38 strains of Mycobacterium leprae. Mutations of the folP1 gene (at codons 53 or 55) were found in 6 of 6 strains with high-level resistance, in 3 of 4 strains with intermediate-level resistance, and in 1 of 6 strains with low-level resistance, but not in 22 dapsone-susceptible strains. In cases of infection with strains of M. leprae carrying the folP1 mutation, therapy with dapsone may be replaced by therapy with a fluoroquinolone.

Genome Comparison Analysis of Molecular Mechanisms of Resistance to Antibiotics in the Rickettsia Genus

In this study we describe molecular mechanisms of resistance to several classes of antibiotics within drug targets by in silico genome comparisons for bacteria of the genus Rickettsia. Apart from the mutations in the rpoB gene in naturally rifampin-resistant Rickettsia species previously reported by our team, we found that typhus group (TG) rickettsiae had a triple amino acid difference in the highly conserved region of the L22 ribosomal protein as compared to the spotted fever group rickettsiae (SFG), which could explain the natural resistance of SFG rickettsia to erythromycin. We found also that the genome of R. conorii contains an aminoglycoside 3'-phosphotransferase. Finally, either folA gene (encoding dihydrofolate reductase) and/or folP gene (encoding dihydropteroate synthase) was missing in the genome of rickettsial strains explaining the natural resistance to cotrimoxazole. Finally, multiple genes encoding for pump efflux were found especially in the genome of R. conorii that could be involved in resistance to antibiotics. Five specific ORFs related to antibiotic resistance have been identified in the genome of R. felis including a streptomycin resistance protein homologue, a class C beta-lactamase, a class D beta-lactamase, a penicillin acylase homologue, and an ABC-type multidrug transporter system. For the first time, using this approach, an experimental beta-lactamase activity has been shown for this bacterium. We believe that whole genome sequence analysis may help to predict several phenotypic characters, in particular resistance to antibiotics for obligate intracellular bacteria.

Mutations in genes related to drug resistance in Mycobacterium leprae isolates from leprosy patients in Korea

Objective. Identification of the presence and drug resistance of Mycobacterium leprae is key to the diagnosis and treatment of leprosy in non-endemic country like Korea. The aim of this study was to screen the drug target DNA such as folP, rpoB, gyr, and 23S rRNA of drug resistance strain of M. leprae. Patients and methods. Sequences of those genes were analyzed for the 104 bacterial index positive cases out of 171 leprosy patients in Korea using touchdown PCR, single stranded conformational polymorphism. Results. Twenty (19.2%) cases have shown the mutations in folP gene of dapsone-resistant M. leprae in which three (2.89%) cases were mutations in two genes, folP and rpoB, of multidrugs resistant strains to dapsone and rifampin, and two (1.92%) cases in folP and gyr genes of resistance to dapsone and oflaxacin, respectively. Besides double mutation for folP gene was one case (0.96%) and for rpoB gene one case, respectively. There was no mutant isolates in 23S rRNA gene against clarithromycin. Conclusions. This result should leads to a better understanding of the status of multidrug resistant leprosy in Korea and may assist in the rapid diagnosis of drug resistant M. leprae and the choice of the appropriate treatment regimens.

薬剤耐性らい菌の出現と迅速検出方法の開発

DNA sequences of Mycobacterium leprae in particular regions of the gyrA, rpoB, and folP genes responsible for resistance to new quinolones, rifampicin and dapsone, respectively, were analyzed. Among 88 isolates of M. leprae from leprosy patients in Japan, Haiti, Indonesia, Pakistan, and the Philippines, eleven isolates had mutational changes in 2 genes (resistance to 2 drugs), and 2 isolates (Shinsei-1 and Zensho-4) showed mutations in 3 genes (resistance to 3 drugs). These findings are suggesting emergence of multi-drug resistant M. leprae.

Folate Biosynthesis Pseudogenes, ΨfolPand ΨfolK, and an O-Sialoglycoprotein Endopeptidase Gene Homolog in the Phytoplasma Genome

Phytoplasmas are wall-less phytopathogenic prokaryotes of small genome sizes that are obligate parasites of insect vectors and plant hosts. We have cloned a clover phyllody (CPh) phytoplasma DNA locus containing five potential coding sequences. Two were identified as pseudogenes (PsifolP and PsifolK) homologous to folP and folK genes, which encode dihydropteroate synthase (DHPS) and 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK), respectively, in other bacteria. Evolution of the phytoplasma presumably involved loss of functions through the formation of these and other pseudogenes during adaptation to obligate parasitism. The findings suggest that the phytoplasma lacks capacity for de novo folate biosynthesis and possesses a transport system for absorption of preformed folate from host cells. The PsifolP-PsifolK region was flanked by three open reading frames (ORFs) encoding a DegV family protein, a hypothetical protein with a P60-like lipoprotein domain homologous with the P60-like Mycoplasma hominis protein, and a glycoprotease (Gcp) protein that possibly functions as a host adaptation or virulence factor.

PCR and restriction endonuclease assay for detection of a novel mutation associated with sulfonamide resistance in Neisseria meningitidis.

We identified a previously undocumented mutation in the dihydropteroate synthase (folP) gene associated with Neisseria meningitidis sulfonamide resistance. A PCR-based assay to detect this mutation, which is 100% predictive of sulfonamide resistance, was developed.

Detection of gene mutations related with drug resistance in Mycobacterium leprae from leprosy patients using Touch-Down (TD) PCR

The lack of methods to identify Mycobacterium leprae with the resistance against multi-drugs quickly and specifically has hindered effective chemotherapy against M. leprae infection. To screen M. leprae with resistance against multi-drugs, the Touch-Down (TD)-PCR has been used in this study. Sequences of the folP, rpoA, B, and gyrA, B genes were analyzed for isolates of M. leprae from leprosy patients in Korea. We amplified designated region of several genes in M. leprae involved in drug resistance and could obtain the PCR products of each gene. The mutations in the particular region of folP, rpoB, and gyrB gene were certified by TD-PCR single-stranded conformational polymorphism and DNA sequencing, respectively.

Multidrug resistant Mycobacterium leprae from patients with leprosy.

Sequences of the folP1, rpoB, and gyrA genes were analyzed for 88 isolates of Mycobacterium leprae from leprosy patients in Japan, Haiti, Indonesia, Pakistan, and the Philippines. Thirteen isolates (14.8%) showed representative mutations in more than two genes, suggesting the emergence of multidrug-resistant M. leprae.

Amino acid repetitions in the dihydropteroate synthase of Streptococcus pneumoniae lead to sulfonamide resistance with limited effects on substrate K(m).

Sulfonamide resistance in Streptococcus pneumoniae is due to changes in the chromosomal folP (sulA) gene coding for dihydropteroate synthase (DHPS). The first reported laboratory-selected sulfonamide-resistant S. pneumoniae isolate had a 6-bp repetition, the sul-d mutation, leading to a repetition of the amino acids Ile(66) and Glu(67) in the gene product DHPS. More recently, clinical isolates showing this and other repetitions have been reported. WA-5, a clinical isolate from Washington State, contains a 6-bp repetition in the folP gene, identical to the sul-d mutation. The repetition was deleted by site-directed mutagenesis. Enzyme kinetic measurements showed that the deletion was associated with a 35-fold difference in K(i) for sulfathiazole but changed the K(m) for p-aminobenzoic acid only 2.5-fold and did not significantly change the K(m) for 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine pyrophosphate. The enzyme characteristics of the deletion variant were identical to those of DHPS from a sulfonamide-susceptible strain. DHPS from clinical isolates with repetitions of Ser(61) had very similar enzyme characteristics to the DHPS from WA-5. The results confirm that the repetitions are sufficient for development of a resistant enzyme and suggest that the fitness cost to the organism of developing resistance may be very low.

Sulfonamide resistance: mechanisms and trends.

Sulfonamides were the first drugs acting selectively on bacteria which could be used systemically. Today they are infrequently used, in part due to widespread resistance. The target of sulfonamides, and the basis for their selectivity, is the enzyme dihydropteroate synthase (DHPS) in the folic acid pathway. Mammalian cells are not dependent on endogenous synthesis of folic acid and generally lack DHPS. Instead, they have a folate uptake system which most prokaryotes lack. Laboratory mutants in the dhps (folP) gene can be easily isolated and show a trade off between sulfonamide resistance and DHPS enzyme performance. Clinical resistant mutants, however, have additional compensatory mutations in DHPS that allow it to function normally. In many pathogenic bacteria sulfonamide resistance is mediated by the horizontal transfer of foreign folP or parts of it. Clinical resistance in gram-negative enteric bacteria is plasmid-borne and is effected by genes encoding alternative drug-resistance variants of the DHPS enzymes. Two such genes, sul1 and sul2, have been sequenced and are found at roughly the same frequency among clinical isolates. Remarkably, the corresponding DHPS enzymes show pronounced insensitivity to sulfonamides but normal binding to the p -aminobenzoic acid substrate, despite the close structural similarity between substrate and inhibitor. Copyright 2000 Harcourt Publishers Ltd.

Dapsone resistance inMycobacterium leprae

The folP1 gene of Mycobacterium leprae, which encodes dihydropteroate synthase (DHPS), was studied for the presence of mutations associated with resistance to dapsone (DDS). When the folP1 of several DDS-resistant clinical isolates of M. leprae were sequenced, two missense mutations were identified. One mutation occurred at codon 53, substituting isoleucine for threonine in DHPS-1, and a second mutation occurred in codon 55, substituting arginine for proline. DNA sequencing of strains of M. leprae resistant to 0.01 g% DDS in the mouse diet revealed that 13 of 14 strains contained either the 53 or 55 folP1 mutation. None of the susceptible strains and only one of five strains resistant to 0.001 g% DDS revealed a mutation in folP1, suggesting that only high-level DDS resistance is associated with the mutations identified in folP1. Development and application of simple molecular tests to assess drug-related mutations in M. leprae could establish current levels of drug resistance in leprosy as a reference point for future monitoring of drug resistance at the global level.