Structural details and composition of Trichomonas vaginalis lipophosphoglycan in relevance to the epithelial immune function.

Trichomonas vaginalis causes the most common non-viral sexually transmitted infection linked to increased risk of premature birth, cervical cancer and HIV. This study defines molecular domains of the parasite surface glycoconjugate lipophosphoglycan (LPG) with distinct functions in the host immunoinflammatory response. The ceramide phospho-inositol glycan core (CPI-GC) released by mild acid had Mr of approximately 8,700 Da determined by MALDI-TOF MS. Rha, GlcN, Gal and Xyl and small amounts of GalN and Glc were found in CPI-GC. N-acetyllactosamine repeats were identified by endo-beta-galactosidase treatment followed by MALDI-MS and MS/MS and capLC/ESI-MS/MS analyses. Mild acid hydrolysis led to products rich in internal deoxyhexose residues. The CPI-GC induced chemokine production, NF-kappaB and extracellular signal-regulated kinase (ERK)1/2 activation in human cervicovaginal epithelial cells, but neither the released saccharide components nor the lipid-devoid LPG showed these activities. These results suggest a dominant role for CPI-GC in the pathogenic epithelial response to trichomoniasis.

Characterization of a cysteine protease from Tritrichomonas foetus that induces host-cell apoptosis.

Tritrichomonas foetus is a serious veterinary pathogen, causing bovine trichomoniasis and affecting cattle herds world-wide, resulting in inflammation of the genital tract, infertility and huge economic losses. The parasite secretes a cysteine protease (CP8), which induces cytotoxicity and apoptosis in bovine vaginal and uterine epithelial cells. Mallinson et al. [D.J. Mallinson, J. Livingstone, K.M. Appleton, S.J. Lees, G.H. Coombs, M.J. North, Microbiology 1995, 141 (12) 3077-3085.] originally reported a partial DNA sequence of T. foetus CP8 based on PCR cloning of T. foetus genomic DNA. Here we report the biochemical properties of the CP8 enzyme. Kinetic properties and the substrate specificity profile of T. foetus CP8 were studied using positional scanning synthetic combinatorial libraries and Michaelis-Menten kinetic analysis of three synthetic fluorogenic substrates. The preferred substrate Z-Leu-Arg-MCA prevented host-cell death/apoptosis induced by CP8. In addition, the DNA sequence was completed by 3' and 5' rapid amplification of cDNA ends (RACE) and the full-length amino acid sequence was obtained.

In vitro cytopathic effects of a cysteine protease of Tritrichomonas foetus on cultured bovine uterine epithelial cells.

OBJECTIVE: To evaluate the cytopathic effects of Tritrichomonas foetus and a purified cysteine protease (ie, CP30) of T foetus on cultured bovine uterine epithelial cells (BUECs) in vitro. SAMPLE POPULATION: 10 reproductive tracts were obtained from late-term bovine fetuses at a commercial abattoir. PROCEDURE: An in vitro culture system of BUECs was developed to study the cytopathic effects of T foetus and purified CP30 of T foetus on host cells. Cytotoxicity of T foetus or CP30 on exposed BUECs was determined. Fluorescence microscopy and flow cytometry analyses were used to detect apoptosis. A fluorometric assay was used to detect BUEC caspase 3 activation. The CP inhibitor E-64 and a caspase inhibitor were used to inhibit apoptosis. RESULTS: Cytopathic effects were observed in BUECs treated with parasites or CP30 and were concentration and time dependent. The BUECs underwent apoptosis in the presence of parasites or CP30. The specific CP inhibitor E-64 abolished the induction of apoptosis in BUECs by CP30. The caspase inhibitor reduced the amount of apoptosis in BUECs. CONCLUSIONS AND CLINICAL RELEVANCE: T foetus and its CP30 induce apoptosis in cultured BUECs in vitro. Induction of apoptosis by CP30 is correlated with protease activity. Endometrial cell death as a result of a T foetus infection is likely to be more important in mediating infertility than a direct effect on the conceptus. Provoking an apoptotic reaction in the host may mitigate an inflammatory reaction or immune response and therefore favor survival of the parasite in a chronic infection.

Induction of human host cell apoptosis by Trichomonas vaginalis cysteine proteases is modulated by parasite exposure to iron.

Trichomonas vaginalis is an understudied parasitic organism whose mechanisms of pathogenesis remain unclear. The adherence to host cells, the induction of host cell cytotoxicity and protease activity are all, however, thought to be contributing factors towards the development of the disease. T. vaginalis CP30 is an extracellular fraction containing four cysteine proteases, CP2, CP3, CP4 and CPT that induce apoptosis in primary human vaginal epithelial cells (HVECs) [Sommer U, Costello CE, Hayes GR, Beach DH, Gilbert RO, Lucas JJ, Singh BN. Identification of Trichomonas vaginalis cysteine proteases that induce apoptosis in human vaginal epithelial cells. J Biol Chem 2005; 280: 23853-60]. We now show that CP30, and the induction of HVEC apoptosis are modulated by iron availability in the parasite growth medium. Growth of parasites under high iron conditions results in a decrease in levels of CP30 found in an extracellular soluble fraction (SF), a concomitant decline in protease activity, and a decreased ability of SF to induce host cell death. Conversely, iron restriction leads to an increase in CP30 levels, an increase in CP30 protease activity, and an increased ability to induce HVEC cell death. Iron-loaded lactoferrin, but not transferrin is an effective iron source for parasites. We hypothesize that CP30 induction of host cell apoptosis is crucial for the development of a persistent infection, and that iron plays a determining role in parasite pathogenesis.

Trichomonas vaginalis lipophosphoglycan triggers a selective upregulation of cytokines by human female reproductive tract epithelial cells.

Trichomonas vaginalis is one of the most common nonviral sexually transmitted human infections and, worldwide, has been linked to increased incidence of human immunodeficiency virus type 1 transmission, preterm delivery, low birth weight, cervical cancer, and vaginitis. The molecular pathways that are important in initiating host inflammatory and immune responses to T. vaginalis are poorly understood. Here we report interactions of human cervicovaginal epithelial cells with the most abundant cell surface glycoconjugate of the parasite, the T. vaginalis lipophosphoglycan (LPG). Purified LPG mediated the adhesion of parasites to human vaginal epithelial cells in a dose-dependent manner. Furthermore, T. vaginalis LPG (but not LPG from Tritrichomonas foetus, the causative agent of bovine trichomoniasis) induced a selective upregulation of chemotactic cytokines by human endocervical, ectocervical, and vaginal epithelial cells, which do not express Toll-like receptor 4/MD2. The T. vaginalis LPG triggered interleukin 8 (IL-8), which promotes the adhesion and transmigration of neutrophils across the endothelium, and macrophage inflammatory protein 3alpha, which is a chemoattractant for immune cells and is essential for dendritic cell maturation. These effects were dose dependent and sustained in the absence of cytotoxicity and IL-1beta release and utilized, at least in part, a signaling pathway independent from the Toll-like/IL-1 receptor adaptor protein MyD88.

Identification of Trichomonas vaginalis cysteine proteases that induce apoptosis in human vaginal epithelial cells.

A secreted cysteine protease (CP) fraction from Trichomonas vaginalis is shown here to induce apoptosis in human vaginal epithelial cells (HVEC) and is analyzed by mass spectrometry. The trichomonad parasite T. vaginalis causes one of the most common non-viral sexually transmitted infection in humans, trichomoniasis. The parasite as well as a secreted cysteine protease (CP) fraction, isolated by affinity chromatography followed by Bio-Gel P-60 column chromatography, are shown to induce HVEC apoptosis, as demonstrated by the Cell Death Detection ELISA(PLUS) assay and annexin V-fluorescein isothiocyanate flow cytometry analyses. Initiation of apoptosis is correlated with protease activity because the specific CP inhibitor E-64 inhibits both activities. SDS-PAGE analysis of the CP fraction reveals triplet bands around 30 kDa, and matrix-assisted laser desorption ionization time-of-flight MS indicates two closely associated peaks of molecular mass 23.6 and 23.8 kDa. Mass spectral peptide sequencing of the proteolytically digested CPs results in matches to previously reported cDNA clones, CP2, CP3, and CP4 (Mallinson, D. J., Lockwood, B. C., Coombs, G. H., and North, M. J. (1994) Microbiology 140, 2725-2735), as well as another sequence with high homology to CP4 (www.tigr.org). These last two species are the most abundant components of the CP fraction. The present results, suggesting that CP-induced programmed cell death may be involved in the pathogenesis of T. vaginalis infection in vivo, may have important implications for therapeutic intervention.