title: Trimethoprim resistance of dihydrofolate reductase variants from clinical isolates of Pneumocystis jirovecii.
abstract: Pneumocystis jirovecii is an opportunistic pathogen that causes serious pneumonia in immunosuppressed patients. Standard therapy and prophylaxis include trimethoprim (TMP)-sulfamethoxazole; trimethoprim in this combination targets dihydrofolate reductase (DHFR). Fourteen clinically observed variants of P. jirovecii DHFR were produced recombinantly to allow exploration of the causes of clinically observed failure of therapy and prophylaxis that includes trimethoprim. Six DHFR variants (S31F, F36C, L65P, A67V, V79I, and I158V) showed resistance to inhibition by trimethoprim, with Ki values for trimethoprim 4-fold to 100-fold higher than those for the wild-type P. jirovecii DHFR. An experimental antifolate with more conformational flexibility than trimethoprim showed strong activity against one trimethoprim-resistant variant. The two variants that were most resistant to trimethoprim (F36C and L65P) also had increased Km values for dihydrofolic acid (DHFA). The catalytic rate constant (kcat) was unchanged for most variant forms of P. jirovecii DHFR but was significantly lowered in F36C protein; one naturally occurring variant with two amino acid substitutions (S106P and E127G) showed a doubling of kcat, as well as a Km for NADPH half that of the wild type. The strongest resistance to trimethoprim occurred with amino acid changes in the binding pocket for DHFA or trimethoprim, and the strongest effect on binding of NADPH was linked to a mutation involved in binding the phosphate group of the cofactor. This study marks the first confirmation that naturally occurring mutations in the gene for DHFR from P. jirovecii produce variant forms of DHFR that are resistant to trimethoprim and may contribute to clinically observed failures of standard therapy or prophylaxis.
authors.affiliation: Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana, USA.
authors.forename: S F
authors.initials: SF
authors.lastname: Queener
authors.forename: V
authors.initials: V
authors.lastname: Cody
authors.forename: J
authors.initials: J
authors.lastname: Pace
authors.forename: P
authors.initials: P
authors.lastname: Torkelson
authors.forename: A
authors.initials: A
authors.lastname: Gangjee
mesh_terms: D015394:Molecular Structure; D045363:Pneumocystis carinii / Q000187:drug effects / Q000472:pathogenicity*; D017433:Protein Structure, Secondary; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics*; D014295:Trimethoprim / Q000737:chemistry / Q000494:pharmacology; D014296:Trimethoprim Resistance / Q000235:genetics / Q000502:physiology*
chemical_list: D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase
references.citation: J Infect Dis. 1998 Sep;178(3):700-6
references.pmid: 9728538
references.citation: J Clin Microbiol. 2005 Mar;43(3):1443-4
references.pmid: 15750128
references.citation: J Infect Dis. 1999 Dec;180(6):1969-78
references.pmid: 10558954
references.citation: J Med Chem. 2003 Nov 6;46(23):5074-82
references.pmid: 14584957
references.citation: Antimicrob Agents Chemother. 2001 Apr;45(4):1104-8
references.pmid: 11257022
references.citation: Structure. 1994 Oct 15;2(10):915-24
references.pmid: 7866743
references.citation: J Med Microbiol. 2002 Jun;51(6):510-515
references.pmid: 12018659
references.citation: Antimicrob Agents Chemother. 2005 Feb;49(2):741-8
references.pmid: 15673759
references.citation: FEBS J. 2007 May;274(10):2661-71
references.pmid: 17451440
references.citation: J Clin Microbiol. 2011 May;49(5):1810-5
references.pmid: 21389160
references.citation: J Infect Dis. 2009 Nov 15;200(10):1616-22
references.pmid: 19795979
references.citation: J Antimicrob Chemother. 2006 Dec;58(6):1246-9
references.pmid: 17038348
references.citation: Clin Infect Dis. 2010 Aug 1;51(3):259-65
references.pmid: 20572759
references.citation: Med Mycol. 2008 Jun;46(4):389-92
references.pmid: 18415849
references.citation: Clin Infect Dis. 2010 Aug 15;51(4):e28-33
references.pmid: 20604718
references.citation: Biochemistry. 1999 Apr 6;38(14):4303-12
references.pmid: 10194348
references.citation: Nihon Ishinkin Gakkai Zasshi. 2009;50(2):67-73
references.pmid: 19430180
references.citation: Antimicrob Agents Chemother. 2013 Jun;57(6):2669-77
references.pmid: 23545530
references.citation: Biochemistry. 2009 Mar 3;48(8):1702-11
references.pmid: 19196009
references.citation: J Mol Biol. 1997 Feb 14;266(1):23-30
references.pmid: 9054967
references.citation: Arch Biochem Biophys. 1993 Sep;305(2):499-508
references.pmid: 8373187
references.citation: Antimicrob Agents Chemother. 2004 Nov;48(11):4301-5
references.pmid: 15504856
references.citation: Diagn Microbiol Infect Dis. 2012 Mar;72(3):204-13
references.pmid: 22321995