Remodeling of protein and mRNA expression in Leishmania mexicana induced by deletion of glucose transporter genes.
Glucose is a major nutrient in the insect vector stage of Leishmania parasites. Glucose transporter null mutants of Leishmania mexicana exhibit profound phenotypic changes in both insect stage promastigotes and mammalian host stage amastigotes that reside within phagolysosomes of host macrophages. Some of these phenotypic changes could be either mediated or attenuated by changes in gene expression that accompany deletion of the glucose transporter genes. To search for changes in protein expression, the profile of proteins detected on two-dimensional gels was compared for wild type and glucose transporter null mutant promastigotes. A total of 50 spots whose intensities changed significantly and consistently in multiple experiments were detected, suggesting that a cohort of proteins is altered in expression levels in the null mutant parasites. Following identification of proteins by mass spectrometry, 3 such regulated proteins were chosen for more detailed analysis: mitochondrial aldehyde dehydrogenase, ribokinase, and hexokinase. Immunoblots employing antisera against these enzymes confirmed that their levels were upregulated, both in glucose transporter null mutants and in wild type parasites starved for glucose. Quantitative reverse transcriptase PCR (qRT-PCR) revealed that the levels of mRNAs encoding these enzymes were also enhanced. Global expression profiling using microarrays revealed a limited number of additional changes, although the sensitivity of the microarrays to detect modest changes in amplitude was less than that of two-dimensional gels. Hence, there is likely to be a network of proteins whose expression levels are altered by genetic ablation of glucose transporters, and much of this regulation may be reflected by changes in the levels of the cognate mRNAs. Some of these changes in protein expression may reflect an adaptive response of the parasites to limitation of glucose.
Feng
Xiuhong
X
Department of Molecular Microbiology and Immunology, Oregon Health & Science University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97239, USA.
Feistel
Torben
T
Buffalo
Cosmo
C
McCormack
Ashley
A
Kruvand
Elizabeth
E
Rodriguez-Contreras
Dayana
D
Akopyants
Natalia S
NS
Umasankar
P K
PK
David
Larry
L
Jardim
Armando
A
Beverley
Stephen M
SM
Landfear
Scott M
SM
D015180:Electrophoresis, Gel, Two-Dimensional; D017353:Gene Deletion; D020869:Gene Expression Profiling; D015151:Immunoblotting; D007894:Leishmania mexicana / Q000235:genetics* / Q000378:metabolism*; D013058:Mass Spectrometry; D046228:Microarray Analysis; D009004:Monosaccharide Transport Proteins / Q000172:deficiency*; D020543:Proteome / Q000032:analysis*; D015800:Protozoan Proteins / Q000032:analysis*; D012333:RNA, Messenger / Q000096:biosynthesis; D016053:RNA, Protozoan / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D009004:Monosaccharide Transport Proteins; D020543:Proteome; D015800:Protozoan Proteins; D012333:RNA, Messenger; D016053:RNA, Protozoan; C115490:glucose transporter, Leishmania