Antisense RNA decreases AP33 gene expression and cytoadherence by T. vaginalis

V Mundodi; A S Kucknoor; J F Alderete

doi:10.1186/1471-2180-7-64

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Antisense RNA decreases AP33 gene expression and cytoadherence by T. vaginalis

Journal article

Open access

Peer reviewed

Antisense RNA decreases AP33 gene expression and cytoadherence by T. vaginalis

V Mundodi, A S Kucknoor and J F Alderete

BMC microbiology, Vol.7(1), pp.64-64

07/03/2007

DOI: https://doi.org/10.1186/1471-2180-7-64

Handle:

https://hdl.handle.net/2376/104544

PMCID: PMC1929106

PMID: 17608941

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Abstract

Trichomonas vaginalis - pathogenicity

Protozoan Proteins - antagonists & inhibitors

Humans

Molecular Sequence Data

RNA, Messenger - analysis

Immunoblotting

Trichomonas vaginalis - chemistry

Protozoan Proteins - genetics

Epithelial Cells - parasitology

Transfection

Membrane Proteins - physiology

Plasmids - genetics

DNA, Protozoan - chemistry

RNA, Protozoan - analysis

Cell Line

Cell Survival

Membrane Proteins - genetics

Down-Regulation

Trichomonas vaginalis - genetics

Reverse Transcriptase Polymerase Chain Reaction

Protozoan Proteins - biosynthesis

Protozoan Proteins - physiology

Sequence Analysis, DNA

Membrane Proteins - biosynthesis

Animals

Membrane Proteins - antagonists & inhibitors

Cell Adhesion - physiology

RNA, Antisense - metabolism

Trichomonas vaginalis - growth & development

DNA, Protozoan - genetics

Host parasitism by Trichomonas vaginalis is complex. Adherence to vaginal epithelial cells (VECs) is mediated by surface proteins. We showed before that antisense down-regulation of expression of adhesin AP65 decreased amounts of protein, which lowered levels of T. vaginalis adherence to VECs. We now perform antisense down-regulation of expression of the ap33 gene to evaluate and confirm a role for AP33 in adherence by T. vaginalis. We also used an established transfection system for heterologous expression of AP33 in T. foetus as an additional confirmatory approach. We successfully select stable trichomonads with sense (S) and antisense (AS) plasmids. RT-PCR confirmed decreased amounts of ap33 mRNA in AS-transfected parasites, and decreased amounts of AP33 had no effect on growth and viability when compared to wild-type (wt) trichomonads. Immunoblots of proteins from AS-transfectants gave significant decreased amounts of functional AP33 capable of binding to host cells compared to wt- and S-transfected trichomonads. As expected, AS-transfectants had lower levels of adherence to VECs, which was related to reduction in surface expression of AP33. Stable expression of T. vaginalis AP33::HA fusion in T. foetus was confirmed by immunoblots and fluorescence. The episomally-expressed surface AP33::HA fusion increased adherence of trichomonads to human VECs, which was abrogated with anti-AP33 serum. These results using both antisense inhibition of gene expression and AP33 synthesis and the heterologous expression of AP33 in T. foetus confirms a role for this protein as an adhesin in T. vaginalis.

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Title: Antisense RNA decreases AP33 gene expression and cytoadherence by T. vaginalis
Creators: V Mundodi - Department of Microbiology, University of Texas Health Science Center, San Antonio, TX 78229-3900, USA. mundodi@uthscsa.edu
A S Kucknoor
J F Alderete
Publication Details: BMC microbiology, Vol.7(1), pp.64-64
Academic Unit: Molecular Biosciences, School of
Publisher: England
Grant note: R21 AI043940 / NIAID NIH HHS AI43940 / NIAID NIH HHS R01 AI043940 / NIAID NIH HHS
Identifiers: 99900547055501842
Language: English
Resource Type: Journal article