Trimethoprim Resistance of Dihydrofolate Reductase Variants from Clinical Isolates of Pneumocystis jirovecii (original) (raw)
Related papers
Biochemistry, 2009
To understand the role of specific active site residues in conferring selective dihydrofolate reductase (DHFR) inhibition from pathogenic organisms such as Pneumocystis carinii (pc) or Pneumocystis jirovecii (pj), the causative agent in AIDS pneumonia, it is necessary to evaluate the role of these residues in the human enzyme. We report the first kinetic parameters for DHFR from pjDHFR and pcDHFR with methotrexate (MTX), trimethoprim (TMP), and its potent derivative, PY957. We also report the mutagenesis and kinetic analysis of active site mutant proteins at positions 35 and 64 of human (h) DHFR and the crystal structure determinations of four hDHFR ternary complexes of NADPH and PY957 with the wild type DHFR enzyme, the single mutant protein, Gln35Lys1, and two double mutant proteins, Gln35Ser/Asn64Ser and Gln35Ser/Asn64Phe. These substitutions place into human DHFR amino acids found at those sites in the opportunistic pathogens pcDHFR (Q35K/ N64F) and pjDHFR (Q35S/N64S). The K i inhibition constant for PY957 showed greatest potency of the compound for the N64F single mutant protein (5.2 nM), followed by wild type pcDHFR (K i 22 nM), and then wild type hDHFR enzyme (K i 230 nM). Structural data reveal significant conformational changes in the binding interactions of PY957 in the hDHFR Q35S/N64F mutant protein complex compared to the other hDHFR mutant protein complexes and the pcDHFR ternary complex. The conformation of PY957 in the wild type DHFR is similar to that observed for the single mutant protein. These data support the hypothesis that the enhanced selectivity of PY957 for pcDHFR is in part due to the in the design more selective inhibitors that target these opportunistic pathogens. 1 The numbering used throughout this paper for the human DHFR sequence is based on the first position being Val-1 rather than Met-1 as observed in the gene sequence listing. This numbering has been used in previous publications of the kinetic and structural data for hDHFR and is being used here for continuity. unique contributions at positions 37 and 69 (pcDHFR numbering). This insight will help
Biochemistry, 2009
To understand the role of specific active site residues in conferring selective dihydrofolate reductase (DHFR) inhibition from pathogenic organisms such as Pneumocystis carinii (pc) or Pneumocystis jirovecii (pj), the causative agent in AIDS pneumonia, it is necessary to evaluate the role of these residues in the human enzyme. We report the first kinetic parameters for DHFR from pjDHFR and pcDHFR with methotrexate (MTX), trimethoprim (TMP), and its potent derivative, PY957. We also report the mutagenesis and kinetic analysis of active site mutant proteins at positions 35 and 64 of human (h) DHFR and the crystal structure determinations of four hDHFR ternary complexes of NADPH and PY957 with the wild type DHFR enzyme, the single mutant protein, Gln35Lys1, and two double mutant proteins, Gln35Ser/Asn64Ser and Gln35Ser/Asn64Phe. These substitutions place into human DHFR amino acids found at those sites in the opportunistic pathogens pcDHFR (Q35K/ N64F) and pjDHFR (Q35S/N64S). The K i inhibition constant for PY957 showed greatest potency of the compound for the N64F single mutant protein (5.2 nM), followed by wild type pcDHFR (K i 22 nM), and then wild type hDHFR enzyme (K i 230 nM). Structural data reveal significant conformational changes in the binding interactions of PY957 in the hDHFR Q35S/N64F mutant protein complex compared to the other hDHFR mutant protein complexes and the pcDHFR ternary complex. The conformation of PY957 in the wild type DHFR is similar to that observed for the single mutant protein. These data support the hypothesis that the enhanced selectivity of PY957 for pcDHFR is in part due to the in the design more selective inhibitors that target these opportunistic pathogens.
The Journal of Infectious Diseases, 1998
Multidrug-resistant Streptococcus pneumoniae strains have emerged over the past decade at an alarming rate. The molecular mechanism of trimethoprim resistance was investigated in 5 pneumococcal strains isolated in the Washington, DC, area from patients with invasive infections. Cloning and sequencing of the trimethoprim resistance determinant from these pneumococci indicated that an altered chromosome-encoded dihydrofolate reductase (DHFR) was responsible for the observed resistance. Comparison of DHFR sequences from pneumococcal strains with various susceptibilities to trimethoprim, together with site-directed mutagenesis, revealed that substitution of isoleucine-100 with a leucine residue resulted in trimethoprim resistance. Hydrogen bonding between the carbonyl oxygen of isoleucine-100 and the 4-amino group of trimethoprim is proposed to play a critical role in the inhibition of DHFR by trimethoprim. This enzyme-substrate model should facilitate the design of new antibacterial agents with improved activity against S. pneumoniae.
bioRxiv (Cold Spring Harbor Laboratory), 2023
Pneumocystis jirovecii is a fungal pathogen that causes pneumocystis pneumonia, a disease that mainly affects immunocompromised individuals. This fungus has historically been hard to study because of our inability to grow it in vitro. One of the main drug targets in P. jirovecii is its dihydrofolate reductase (PjDHFR). Here, by using functional complementation of the baker's yeast ortholog, we show that PjDHFR can be inhibited by the antifolate methotrexate in a dose-dependent manner. Using deep mutational scanning of PjDHFR, we identify mutations conferring resistance to methotrexate. Thirty-one sites spanning the protein have at least one mutation that leads to resistance, for a total of 355 high-confidence resistance mutations. Most resistanceinducing mutations are found inside the active site, and many are structurally equivalent to mutations known to lead to resistance to different antifolates in other organisms. Some sites show specific resistance mutations, where only a single substitution confers resistance, whereas others are more permissive, as several substitutions at these sites confer resistance. Surprisingly, one of the permissive sites (F199) is without direct contact to either ligand or cofactor, suggesting that it acts through an allosteric mechanism. Modeling changes in binding energy between F199 mutants and drug shows that most mutations destabilize interactions between the protein and the drug. This evidence points towards a more important role of this position in resistance than previously estimated and highlights potential unknown allosteric mechanisms of resistance to antifolate in DHFRs. Our results offer unprecedented resources for the interpretation of mutation effects in the main drug target of an uncultivable fungal pathogen. .
Journal of Infectious Diseases, 1999
Recent studies of the human Pneumocystis carinii dihydropteroate synthase (DHPS) gene suggest that P. carinii is developing resistance to sulfamethoxazole (SMX) and dapsone. To explore whether P. carinii is also developing resistance to trimethoprim (TMP), the human P. carinii dihydrofolate reductase (DHFR) gene was cloned, DHFR and DHPS genes in 37 P. carinii isolates from 35 patients were sequenced, and the relationship between TMP-SMX or dapsone use and gene mutations was analyzed. The DHFR gene sequences were identical in all isolates except 1 with a synonymous substitution. In contrast, the DHPS gene sequences showed mutations in 16 of the 37 isolates; prior sulfa/sulfone prophylaxis was associated with the presence of these mutations ( ). In addition to suggesting that there is less selective P ! .001 pressure on DHFR than on DHPS, this study reinforces the hypothesis that mutations in the DHPS gene are likely involved in the development of sulfa resistance in P. carinii.
Trimethoprim resistance in Haemophilus influenzae is due to altered dihydrofolate reductase(s)
The Biochemical journal, 1991
We characterized a highly purified preparation of the chromosomally encoded dihydrofolate reductase (DHFR) from a trimethoprim-susceptible (Tmp8; strain MAP) and two trimethoprim-resistant (TmpR) strains (MAP/47 and MAP/42) of Haemophilus influenzae. The enzymes were purified between 650- and 3000-fold by gel-filtration and dye-ligand chromatography. The apparent molecular mass of the three proteins was 18400 Da by PAGE under denaturing and nondenaturing conditions. Total enzyme activity was greater in all fractions from the TmpR strains compared with the Tmp8 isolate. The three enzymes had a similar Km for dihydrofolate (7, 9 and 5 microM) and NADPH (2, 5 and 6 microM). However, the Tmp IC50 (the concentration necessary for 50% inhibition of DHFR activity) for the Tmp8 strain MAP was 0.001 microM, whereas DHFR from the TmpR strains MAP/47 and MAP/42 had values of 0.1 microM and 0.3 microM respectively. The methotrexate IC50 of the MAP/42 DHFR was 0.06 microM in comparison with the ...
Journal of Structural Biology, 2011
In order to produce a more potent replacement for trimethoprim (TMP) used as a therapy for Pneumocystis pneumonia and targets dihydrofolate reductase from Pneumocystis jirovecii (pjDHFR), it is necessary to understand the determinants of potency and selectivity against DHFR from the mammalian host and fungal pathogen cells. To this end, active site residues in human (h)DHFR were replaced with those from pjDHFR. Structural data are reported for two complexes of TMP with the double mutants Gln35Ser/Asn64Phe (Q35S/N64F) and Gln35Lys/Asn64Phe (Q35K/N64F) of hDHFR that unexpectedly show evidence for the binding of two molecules of TMP: one molecule that binds in the normal folate binding site and the second molecule that binds in a novel subpocket site such that the mutated residue Phe64 is involved in van der Waals contacts to the trimethoxyphenyl ring of the second TMP molecule. Kinetic data for the binding of TMP to hDHFR and pjDHFR reveal an 84-fold selectivity of TMP against pjDHFR (K i 49 nM) compared to hDHFR (K i 4093 nM). Two mutants that contain one substitution from pj-and one from the closely related Pneumocystis carinii DHFR (pcDHFR) (Q35K/N64F and Q35S/N64F) show K i values of 593 and 617 nM, respectively; these K i values are well above both the K i for pjDHFR and are similar to pcDHFR (Q35K/N64F) and Q35S/N64F) (305 nM). These results suggest that active site residues 35 and 64 play key roles in determining selectivity for pneumocystis DHFR, but that other residues contribute to the unique binding of inhibitors to these enzymes. † This work was supported in part by grants from the National Institutes of Health GM051670 (VC). ‡ Coordinates and crystallographic structure factors for mutant human DHFR TMP complexes have been deposited in the Protein Data Bank under the accession codes 3S3V and 3N0H.
1997
Streptococcus pneumoniae isolates resistant to several antimicrobial agent classes including trimethoprim- sulfamethoxazole have been reported with increasing frequency throughout the world. The MICs of tri- methoprim, sulfamethoxazole, and trimethoprim-sulfamethoxazole (1:19) for 259 clinical isolates from South Africa were determined, and 166 of these 259 (64%) isolates were resistant to trimethoprim-sulfamethoxazole (MICs >20 mg/liter). Trimethoprim resistance was found to be more
Antimicrobial Agents and Chemotherapy, 2013
ABSTRACTA major concern of immunocompromised patients, in particular those with AIDS, is susceptibility to infection caused by opportunistic pathogens such asPneumocystis jirovecii, which is a leading cause of pneumonia in immunocompromised patients. We report the first kinetic and structural data for 2,4-diamino-6-[(2′,5′-dichloro anilino)methyl]pyrido[2,3-d]pyrimidine (OAAG324), a potent inhibitor of dihydrofolate reductase (DHFR) fromP. jirovecii(pjDHFR), and also for trimethoprim (TMP) and methotrexate (MTX) with pjDHFR,Pneumocystis cariniiDHFR (pcDHFR), and human DHFR (hDHFR). OAAG324 shows a 9.0-fold selectivity for pjDHFR (Ki, 2.7 nM) compared to its selectivity for hDHFR (Ki, 24.4 nM), whereas there is only a 2.3-fold selectivity for pcDHFR (Ki, 6.3 nM). In order to understand the determinants of inhibitory potency, active-site mutations of pj-, pc-, and hDHFR were explored to make these enzymes more like each other. The most unexpected observations were that the variant pcD...
Journal of infection in developing countries, 2015
Pneumocystis pneumonia (PCP) is an opportunistic life-threatening infection, especially for immunocompromised individuals. A trimethoprim-sulfamethoxazole (TMP-SMX) combination is commonly used for the treatment of PCP, targeting both dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) enzymes. Several studies have already shown that polymorphisms in the DHPS gene are associated with drug resistance. The present study analyzed DHFR gene polymorphisms in Pneumocystis jirovecii recovered from clinical samples from patients admitted to a tertiary care health center in New Delhi, India. Detection of P. jirovecii was performed using Gomori methenamine silver staining (GMS) and nested polymerase chain reaction (PCR) assay targeting the mitochondrial large subunit ribosomal RNA (mt LSU rRNA) gene. The DHFR gene was amplified using nested PCR protocol and was sequenced for detection of polymorphisms. Of 180 clinical samples, only 4% (7/180) were positive by GMS staining, and ...