Cloning of the Pneumocystis jirovecii trifunctional FAS gene and complementation of its DHPS activity in Escherichia coli.
Pneumocystis pneumonia or PCP is caused by Pneumocystis jirovecii, an obligate parasite of the human lung. In this study P. jirovecii genomic sequence encoding FAS, a trifunctional protein including dihydroneopterin aldolase (DHNA), hydroxymethyldihydropterin pyrophosphokinase (PPPK) and dihydropteroate synthase (DHPS) were identified by PCR amplification from fixed broncheolar lavage samples from patients having Pneumocystis pneumonia. The P. jirovecii trifunctional DHNA-PPPK-DHPS genes (PjFAS) showed a high degree of conservation with the rat Pneumocystis carinii and P. carinii f. sp. macaca sequences. To test the functionality of the PjFAS sequences introns were removed followed by cloning and expression of PjFAS sequences in a DHPS-disrupted Escherichia coli strain. Complementation depended on the presence of N-terminal FAS sequences in addition to a glutathione S- transferase tag to the N-terminus of PjFAS. Functional complementation allowed evaluation of DHPS mutations implicated with sulfa drug resistance.
Iliades
Peter
P
CSIRO Health Sciences and Nutrition, Parkville, Vic., Australia. Peter.Iliades@csiro.au
Walker
Daniel J
DJ
Castelli
Laura
L
Satchell
Jacqueline
J
Meshnick
Steven R
SR
Macreadie
Ian G
IG
D000446:Aldehyde-Lyases / Q000235:genetics; D000595:Amino Acid Sequence; D000890:Anti-Infective Agents / Q000494:pharmacology; D001992:Bronchoalveolar Lavage Fluid / Q000382:microbiology; D017124:Conserved Sequence / Q000235:genetics; D004271:DNA, Fungal / Q000737:chemistry / Q000302:isolation & purification; D004094:Dihydropteroate Synthase / Q000235:genetics; D017854:Diphosphotransferases / Q000235:genetics; D025141:Drug Resistance, Fungal / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D006801:Humans; D007438:Introns / Q000235:genetics; D008969:Molecular Sequence Data; D045363:Pneumocystis carinii / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D011020:Pneumonia, Pneumocystis / Q000382:microbiology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013424:Sulfanilamides / Q000494:pharmacology
D000890:Anti-Infective Agents; D004271:DNA, Fungal; C094979:FasA protein, Pneumocystis; C094980:FasB protein, Pneumocystis; D005656:Fungal Proteins; D011994:Recombinant Proteins; D013424:Sulfanilamides; D004094:Dihydropteroate Synthase; D017854:Diphosphotransferases; C019684:2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase; D000446:Aldehyde-Lyases; C020044:dihydroneopterin aldolase