Identification, characterization, and expression of a unique secretory lipase from the human pathogen Leishmania donovani.
Lipases have been implicated to be of importance in the life cycle development, virulence, and transmission of a variety of parasitic organisms. Potential functions include the acquisition of host resources for energy metabolism and as simple building blocks for the synthesis of complex parasite lipids important for membrane remodeling and structural purposes. Using a molecular approach, we identified and characterized the structure of an LdLip3-lipase gene from the primitive trypanosomatid pathogen of humans, Leishmania donovani. The LdLip3 encodes a approximately 33 kDa protein, with a well-conserved substrate-binding and catalytic domains characteristic of members of the serine lipase-protein family. Further, we showed that LdLip3 mRNA is constitutively expressed by both the insect vector (i.e., promastigote) and mammalian (i.e., amastigote) life cycle developmental forms of this protozoan parasite. Moreover, a homologous episomal expression system was used to express an HA epitope-tagged LdLip3 chimeric construct (LdLip3::HA) in these parasites. Expression of the LdLip3 chimera was verified in these transfectants by Western blots and indirect immuno-fluorescence analyses. Results of coupled immuno-affinity purification and enzyme activity experiments demonstrated that the LdLip3::HA chimeric protein was secreted/released by transfected L. donovani parasites and that it possessed functional lipase enzyme activity. Taken together these observations suggest that this novel secretory lipase might play essential role(s) in the survival, growth, and development of this important group of human pathogens.
Shakarian
Alison M
AM
The Department of Biology and Biomedical Sciences, Salve Regina University, Newport, RI 02840, USA. shakaria@salve.edu
McGugan
Glen C
GC
Joshi
Manju B
MB
Stromberg
Mary
M
Bowers
Lauren
L
Ganim
Christine
C
Barowski
Jessica
J
Dwyer
Dennis M
DM
D000818:Animals; D017125:Genes, Protozoan; D006801:Humans; D007893:Leishmania donovani / Q000201:enzymology*; D008018:Life Cycle Stages; D008049:Lipase / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection
D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D008049:Lipase