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Trichomonas vaginalis

Trichomonas vaginalis is the most common sexually-transmitted protozoan parasite. It is an extracellular parasite, a facultative anaerobe and lacks mitochondria (it is amitochondriate) and peroxisomes.

Trichomonas vaginalis has a unique organelle called the hydrogenosome. It resides in the urogenital tract and is transmitted via sexual intercourse. It lacks a cyst stage, meaning it cannot survive outside the host. Its trophozoite has one anterior nucleus, five flagella (four at one end and one along the cell), an axostyle, unique cytoskeletal proteins and a hydrogenosome.

Trichomonas is the most common infectious protozoan in Europe and North America.

Worldwide there are over 300 million cases of non-viral sexually-transmitted infections reported annually: 170 million by Trichomonas; 89 million by Chlamydia; 62 million by gonorrhea; and 12 million by syphilis.

Trichomonas vaginalis infects both men and women. Symptoms are variable.

In men it is often asymptomatic but may cause irritation, prostatitis and urethritis. In women it may cause vaginitis and pregnancy problems (preterm delivery; low birth weight; and increased infant mortality). Long-term untreated infections are associated with infertility in men and women.

Trichomonas vaginalis & HIV

Trichomonas vaginalis causes 
. In an HIV-negative person, this gives HIV ready access of white blood cells.

A similar mechanism is responsible for increased susceptibility to cervical and prostate cancer. In an HIV-positive person, the hemorrhages leak the virus and causes a higher concentration of HIV in the infected area. Epidemiological
Trichomonas vaginalis.

Life cycle of Trichomonas vaginalis

Diagnostic Stage Trophozoite is present in vaginal and prostatic secretions and urine.
Division Multiplies by longitudinal binary fission.
Infective Stage Trophozoite in vagina or orifice of urethra.

Trophozoite virulence factors

Trichomonas vaginalis virulence factors are poorly defined.
  1. Cell-cell and cell-matrix interaction proteins. Trichomonas vaginalis trophozoites are extracellular and must therefore bind host cell surfaces somehow.
  2. Extracellular cysteine proteases and other proteases.
  3. Pore-forming proteins.
  4. Terminal N-acetylglucosamine (GlcNAc) and Galactose (Gal). Trichomonas vaginalis' surface has a high-density coating of a lipophosphoglycan (LPG) (~2.7x106 per cell).

Is LPG A Virulence Factor? Part I

To determine if LPG is a virulence factor, the adherence and cytotoxicity of Trichomonas vaginalis LPG mutants is assayed.
Mutagenesis Chemical mutagenesis was performed with ethyl methanesulfonate (EMS). Transposons failed.
Selection Incubate mutagenized populations with the lectin ricin. Ricin binds to &beta1,4 galactose residues in LPG. Centrifugation removes agglutinated (LPG+) cells and remaining cells are LPGx
Study LPGx mutants were fluorescently stained with ricin to verify their mutation. Remaining polysaccharides are purified and assayed for adherence and cytotoxicity to vaginal epithelium.
Complement Complementation experiments identify the mutant gene encoding the LPG.

Is LPG A Virulence Factor? Part II

Binding LPG mutants did not bind RCA120 nor wheat germ agglutinin, indicating an inability to bind Gal and GlcNAc, respectively.
Adhesion &

Wild-type and LPG-mutant adhesion/cytotoxicity to vaginal epithelial cells was studied in vitro.

  1. Ectocervical cells grown to ~70-80% confluency as monolayers.
  2. Parasites are labeled with cell-tracker blue and added to ectocervical cells at 37°C and 5% CO2
  3. T. vaginalis infections proceed for indicated time points before analyses.

LPG mutants were dramatically ineffective at adhesion (number of parasites adhered) and cytotoxicity (lactate dehydrogenase release).

OK. LPG Is A Virulence Factor. What Sugars Are Crucial?

Are there host cell receptors that interact with LPG? To answer this question, identify possible host cell lectins that acts as adhesion factors for T. vaginalis, using wild-type and LPG mutant parasites for comparison.

WT LPG In wild-type cells, LPG monosaccharide composition is primarily four sugars: ~30% rhamnose; ~15% xylose; ~25% galactose; ~25% GlcNAc.
LPGx Mut LPG mutants with altered virulence have significantly reduced levels of GlcNAc and galactose, while rhamnose and xylose are largely unaffected.
WT LPG++ Certain wild-type parasites are highly adherent. Their LPG has a high proportion of galactose.

Does Trichomonas vaginalis LPG Bind Galectin-1?

Incubate Incubate galectin-1 with Trichomonas vaginalis in presence of non-competing (sucrose) or competing (lactose) sugars.
Separate Separate and collect unbound galectin-1 by centrifugation (Sample U).
Elute Elute parasite-bound galectin-1 and collect via centrifugation (Sample E).
Parasites Collect remaining parasites (Sample P)
Blot Western blot the three samples and stain with a galectin-1 antibody.

Galectin-1 is present in all three samples incubated with a non-competing sugar. This is because some galectin-1 remained unbound (Sample U), plenty was bound to the parasite (Sample E) and a small amount of bound organisms had remained in the un-eluted portion (Sample P).

When incubated with a competing sugar, galectin-1 is only present in Sample U. This is because some galectin-1 remained unbound (Sample U), and the only binding that did occur was between galectin-1 and lactose; no galectin-1 bound the parasites (Samples E and P).

This experiment was repeated with LPGx mutant Trichomonas vaginalis and these mutants did not bind galectin-1. Furthermore, wild-type LPG can compete parasite binding to LPG (yieleded results as when lactose was used as a competing sugar) but mutant LPG cannot compete parasite binding.

Additional experiments corroborate that host galectin-1 binds to parasite LPG. Increasing galectin-1 levels in host cells increases parasite binding. RNAi knockouts of galectin-1 have hugely reduced parasite binding.

T. vaginalis LPG Conclusions, Part I

LPG is a parasite adherence factor that binds host cell surface galectin-1. LPGx mutants have reduced adherence and cytotoxicity to cervical epithelial cells. LPGx sugar composition has reduced galactose and GlcNAc (glucosamine). Wild-type Trichomonas vaginalis mutants bind host galectin-1 in a sugar-specific manner. LPGwild-type can compete galectin-1 binding to Trichomonas vaginalis, but not LPGx.

T. vaginalis LPG Conclusions, Part II

Binding between the host and the parasite is enhanced when the host cell surface is saturated with galectin-1 capable of dimerizing; this indicates that galectin-1 and LPG cross-link. Binding is reduced when galectin-1 is knocked down via RNAi, but binding is restored when galectin-1 levels are replenished. This demonstrates that parasite LPG adherences to epithelial cells via a carbohydrate-mediated binding to host cell galectin-1

Searching the Genome for Protein Virulence Factors

The genome is large (~160 Mb) and highly (~65%) repetitive. There are likely ~60,000 genes present. It has 59 repeat families, including retro/transposons and viral-like genes. Of interest are massively expanded gene families. There are 927 protein kinases, 658 BspA-like genes and 328 small GTPases. Moderate expansion is also common, including a family of 24 SAPLIPs.

SAPLIPs & Hemolysis

Some Trichomonas vaginalis strains are hemolytic while others are not. Trichomonas vaginalis Strain G3 lyses ~60% of RBC in 90 minutes, while Strain SS22 is non-hemolytic. Examining SAPLIP expression was informative: hemolytic Strain G3 upregulated SAPLIP 10B by a magnitude of ~20, while non-hemolytic Strain SS22 did not upregulate SAPLIP 10B (it ↑↑ SAPLIP 12 instead).

Any Surface Proteins? Tetraspanins!

Biotinylation Surface proteins were biotinylated. Biotin can't permeate the membrane and thus labels surface proteins. Biotin binds terminal amine groups.
Washing Cells were lysed and run over a streptavidin column. Straptavidin binds biotin tightly and specifically even amidst harsh washes to remove unbound contaminants.
Elution Proteins bound to biotin (in turn bound to streptatividin) can be eluted by a change in pH.
MudPIT MudPIT separates populations of proteins.

TSP-1,6,8 were found to be highly abundant on pathogenic strain surfaces. In animals, TSP is involved in proliferation, adhesion, migration, fusion and signal transduction.

IFA TSP-1,6,8 were tagged with IFA to very their surface expression pattern.

TSP-1,8 are found on the plasma membrane in microdomains. TSP-6 is a flagellar protein. IFA tagging was repeated in the presence of ectocervical cells. TSP-1 was upregulated after 4 hours. TSP-8 was upregulated after 30 minutes (dropping off after six hours). TSP-6 showed a bimodal curve with expression heightened at 15-30min and again at 4hrs; furthermore, TSP-6 relocalized to intracellular vacuoles from 0.5-1hrs and migrated again to flagella at 3-6hrs. Since adhesion to host cells usually occurs within 30 minutes, TSP-6 is the only one possibly involved.

A Closer Look at TSP-6

A mutant tetraspanin (TSP6ΔCt) was engineered that did not localize to the flagella, and instead targeted the plasma membrane and vacuoles. Localization did not change in presence of host cells. To test if this impacted parasite migration, a matrigel assay was performed.

ParasitesParasites were placed in wells at one end of a matrigel.
MediaAn extract rich in ECM proteins was placed at the opposite end.
IncubateThe matrigel was incubated at 37°C.
MeasuringMigrated cells were counted.

Trichomonas vaginalis migration was reduced by ~40% in cells transfected with TSP6ΔCt relative to those transfected with TSP6.

T. vaginalis TSP-6 Conclusions, Part I

Trichomonas vaginalis surface proteins and secreted proteins, such as SAPLIPs, are likely involved in pathogenesis. Studies identified abundant T. vaginalis membrane tetraspanins: two (TSP-1,8) were transmembrane; a third (TSP-6) was mostly flagellar. In the presence of vaginal epithelial cells, TSP-6 migrates from flagella to intracellular vesicles and back to flagella.

T. vaginalis TSP-6 Conclusions, Part II

Truncation of TSP-6's C-terminus results in loss of flagella localization and a decrease in Trichomonas vaginalis migration -- it is likely involved in parasite migration through host cell extracellular matrix. TSP-6’s role in pathogenesis is unclear, but it appears to be involved in sensing host cells, perhaps leading to outside-in signaling, and also in cellular migration.

Trichomonas vaginalisComments