Journal article
Silencing the ap65 gene reduces adherence to vaginal epithelial cells by Trichomonas vaginalis
Molecular microbiology, Vol.53(4), pp.1099-1108
08/2004
Handle:
https://hdl.handle.net/2376/107869
PMCID: PMC2562645
PMID: 15306014
Abstract
Host parasitism by
Trichomonas vaginalis
is complex and in part mediated by adherence to vaginal epithelial cells (VECs). Four trichomonad surface proteins bind VECs as adhesins, and AP65 is a major adhesin with sequence identity to an enzyme of the hydrogenosome organelle that is involved in energy generation. In order to perform genetic analysis and assess the role of AP65 in
T. vaginalis
adherence, we silenced expression of
ap65
using antisense RNA. The gene for
ap65
was inserted into the vector pBS-
neo
in sense and antisense orientations to generate plasmids pBS-
neo
S (S) and pBS-
neo
AS (AS), respectively. Trichomonads were then transfected with S and AS plasmids for selection of stable transfectants using Geneticin, and the presence of plasmid in transfectants was confirmed by polymerase chain reaction of the
neo
gene. Reverse transcription polymerase chain reaction and Northern blot analysis showed decreased amounts of
ap65
transcript in AS transfected parasites. Growth kinetics of the antisense-transfected and wild type organisms were similar, suggesting that silencing AP65 did not affect overall energy generation for growth. Immunoblot analysis using monoclonal antibody (mAb) to AP65 of AS transfectants showed decreased amounts of AP65 when compared to wild type or S transfectants. Not unexpectedly, this corresponded to decreased amounts of AP65 bound to VECs in a functional ligand assay. Reduction in parasite surface expression of AP65 was related to lower levels of adherence to VECs by AS-transfectants compared to control organisms. Antisense silencing of
ap65
was not alleviated by growth of trichomonads in high iron, which up-regulates transcription of
ap65
. Our work reaffirms the role for AP65 as an adhesin, and in addition, we demonstrate antisense RNA gene silencing in
T. vaginalis
to study the contribution of specific genes in pathogenesis.
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Details
- Title
- Silencing the ap65 gene reduces adherence to vaginal epithelial cells by Trichomonas vaginalis
- Creators
- V Mundodi - Department of Microbiology, University of Texas Health Science Center, San Antonio, TX, USAA. S Kucknoor - Department of Microbiology, University of Texas Health Science Center, San Antonio, TX, USAD. J Klumpp - Department of Urology, North-western University Medical School, Chicago, IL, USAT.-H Chang - Department of Microbiology, University of Texas Health Science Center, San Antonio, TX, USAJ. F Alderete - Department of Microbiology, University of Texas Health Science Center, San Antonio, TX, USA
- Publication Details
- Molecular microbiology, Vol.53(4), pp.1099-1108
- Academic Unit
- Molecular Biosciences, School of
- Identifiers
- 99900547074601842
- Language
- English
- Resource Type
- Journal article