The Role of Small Extracellular Vesicles in Viral-Protozoan Symbiosis: Lessons From Trichomonasvirus in an Isogenic Host Parasite Model

Abstract

The protozoan parasite Trichomonas vaginalis (TV), exclusively adapted to the human genital tract, is one of the most common sexually transmitted pathogens. Adding to the complexity of the host-pathogen interactions, the parasite harbors TV-specific endosymbiont viruses (Trichomonasvirus, TVV). It was reported that small extracellular vesicles (sEVs) released by TV play a role in host immunity; however, the role of the viral endosymbiosis in this process remained unknown. We hypothesized that the virus may offer evolutionary benefit to its protozoan host at least in part by altering the immunomodulatory properties of sEVs spreading from the site of infection to non-infected immune effector cells. We infected human vaginal epithelial cells, the natural host of the parasite, with TV natively harboring TVV and an isogenic derivative of the parasite cured from the viral infection. sEVs were isolated from vaginal cell culture 24 h post TV infection and from medium where the isogenic TV strains were cultured in the absence of the human host. sEVs from TVV-negative but not TVV-positive parasites cultured alone caused NF-κB activation and increase of IL-8 and RANTES expression by uterine endocervical cells, which provide innate immune defense at the gate to the upper reproductive tract. Similarly, mononuclear leukocytes increased their IL-8, IL-6 and TNF-α output in response to sEVs from virus-negative, but not isogenic virus-positive parasites, the latter exosomes being immunosuppressive in comparison to TV medium control. The same phenomenon of suppressed immunity induced by the TVV-positive compared to TVV-negative phenotype was seen when stimulating the leukocytes with sEVs originating from infected vaginal cultures. In addition, the sEVs from the TVV-positive infection phenotype suppressed immune signaling of a toll-like receptor ligand derived from mycoplasma, another frequent TV symbiont. Quantitative comparative proteome analysis of the secreted sEVs from virus-positive versus virus-negative TV revealed differential expression of two functionally uncharacterized proteins and five proteins involved in Zn binding, protein binding, electron transfer, transferase and catalytic activities. These data support the concept that symbiosis with viruses may provide benefit to the protozoan parasite by exploiting sEVs as a vehicle for inter-cellular communications and modifying their protein cargo to suppress host immune activation.

Keywords: T. vaginalis; Trichomonasvirus; cytokines; exosomes; extracellular vesicles; immune modulation; proteomics.

Figure 1

TVV infection status modifies the immunostimulatory properties of TV sEVs in bystander cervical cells. Endocervical cells were treated with control no sEV, sEVs derived from TV medium alone, 347V+, or 347V- for 24 h. (A) NFkB activity was measured by a luciferase reporter assay and shown as fold change from control. (B) IL-8 and (C) RANTES protein levels by MSD assays. Data represent two independent experiments performed in triplicate and plotted as mean ± standard deviation. ****p < 0.0001 represent sEVs different from control. ⁺⁺⁺⁺p < 0.0001 represent 347V- different from 347V+.

Figure 2

sEVs from virus-negative TV but not virus-positive parasites induce multi-cytokine response in immune cells. PBMCs were treated with control no sEV, sEVs derived from TV medium, 347V+, and 347V- TV strains for 24 h. (A) IL-8, (B) IL-6, and (C) TNF-α protein levels were measured by MSD assays. Graphs represent data from PBMC donor X in duplicate and plotted as mean ± standard error of mean. Results are shown as protein levels normalized to cell viability. *p < 0.05; **p < 0.01; and ***p < 0.001 represent sEVs different from control. ⁺p < 0.05 and ⁺⁺⁺p < 0.001 represent 347V- different from 347V+.

Figure 3

sEVs from TV-infected vaginal cells regulate cellular immunity in a manner dependent on the viral status of the parasite. PBMCs were treated with control no sEV, sEVs derived from vaginal cells (Vk cells), Vk+347V+, Vk+347V-, Vk+UR1, and Vk+Poly(I:C) with or without MALP-2 for 24 h. (A) IL-8 and (B) IL-10 protein levels were measured by MSD assay. (C) MTT assays were performed to confirm cell viability after 24 h treatment relative to control. Graphs represent data from PBMC donor A in duplicate and plotted as mean ± standard error of mean. *p < 0.05; **p < 0.01; and ***p < 0.001 represent sEVs different from control. ^$p < 0.05; ^$$p < 0.01; and ^$$$$p < 0.0001 represent comparisons different to Vk cells sEVs. +p < 0.05; ++p < 0.01; and ⁺⁺⁺⁺p < 0.0001 represent comparisons different to Vk+347V- sEVs.

Figure 4

TV sEVs cluster separately from background medium control. Principal component analysis was used to compare sEVs on the log2-transformed abundance values of all proteins identified. Triangles represent background medium (BKG), filled circles represent virus-negative TV phenotype (PH1), unfilled circles represent virus-positive TV phenotype (PH2).

Figure 5

Functional categorization of TV sEV proteome. The identified TV sEV proteins were sorted into functional groups for molecular function with gene ontology (GO) terms and for protein class (PC) using PANTHER classification system (pantherdb.org).

Figure 6

Differentially expressed sEV proteins between virus-negative TV vs. virus-positive TV. Differential expression analysis compared log2-transformed abundance values of all sEV proteins identified between virus-negative TV vs. virus-positive TV. Dashed line represents p value = 0.05. X-axis represents log2-transformed fold change (FC). Y-axis represents -log2 transformed P value.

Figure 7

Relative abundance of the differentially expressed sEV proteins. Differential expression analysis compared log2-transformed abundance values of all sEV proteins identified between virus-negative TV vs. virus-positive TV. Graphs represent relative abundance values of the 7 significantly differentially expressed (p < 0.05) proteins between three virus-negative TV strains vs. three virus-positive TV strains and plotted as mean ± standard error of mean. The isogenic strains 347V+ and 347V- are noted with a red asterisk within each phenotype. For each protein, gene ontology (GO) terms for molecular function are annotated.