Fig 1.
Tv is delivered into the mouse male urogenital tract (MUT) using urethral catheterization.
(A) Diagram of catheterization and Tv delivery into the mouse MUT. 108 Nluc expressing parasites suspended in 100 μl RPMI-1640 medium were introduced through the urethral meatus into the urethra via a syringe and polyethylene catheter. Additional mouse penile features such as the glans, penile bone, and the male urogenital mating protuberance (MUMP) are depicted. Mouse penis sketch adapted from a mid-sagittal sectioning of the adult mouse penis [74]. (B) A simplified diagram of a lateral view of the male urogenital system in adult mice modified from Cunha et. al. [75]. Parasites introduced into the mouse MUT via urethral catheterization would be delivered through the urethra (U) to the anterior (AP), dorsal (DP), lateral (LP), and ventral (VP) prostatic lobes. The bladder and seminal vesicles (SV) are also depicted. Histological validation of Tv delivery into the mouse MUT (C) versus the vehicle control. (E) The right panels are a 20X magnification of the left anterior prostate lobe (marked by a circle in the left panels). T. vaginalis parasites are indicated by a blue arrow. (D) 100X magnification of Tv parasite found in infected mouse MUT tissue. Blue arrow indicates the presence of flagella.
Fig 2.
Linearity of luminescence in different Nluc-expressing Tv strains.
T. vaginalis strains LSU 160 MA and P (A) and MSA 1103 (B) nucleofected with pMasterNeo-Nluc plasmid were measured for luminescence at varying amounts of parasites from 0 to 106 parasites (as indicated on the X axis) to determine the linear range of the assay for each strain. Data shown are averages of luminescence signal from 3 technical replicates with standard deviation for each strain.
Fig 3.
Quantifying parasite burden using nanoluciferase-expressing Tv.
(A) Schematic of mouse MUT tissue sample preparation and quantification of Tv parasite burden. Excised mouse MUT tissue is finely minced and suspended in prostate lysis buffer (2 mL/mg tissue). MUT tissue then undergoes additional homogenization via probe sonication to ensure complete tissue dissociation. Insoluble material is pelleted via centrifugation and the supernatant is transferred to a new tube to further quantify luciferase activity and protein concentration using the Nano-Glo luciferase and Bradford assays, respectively. Detailed description found in materials and methods. (B) Mice were inoculated with 108 parasites (n = 3) or RPMI vehicle control (n = 3) and promptly euthanized. Samples were processed and analyzed by Nluc and Bradford assays to quantify parasite load. Data is shown as average luminescence signal/μg protein of the sample.
Fig 4.
Tv found within the murine MUT tissue 72 hours post-inoculation.
Quantification of Nluc-expressing MSA 1103 parasites in infected mouse MUT tissues 48 hrs or 72 hrs post-inoculation (as indicated on the X axis). Data are based on 12 infected mice per time point per strain and are averages of luminescence/μg protein of the sample with standard deviation. Data are representative of 3 independent experiments.
Fig 5.
Viable parasites are recovered from mouse MUT tissue 72 hours post-inoculation.
108 Tv parasites were introduced into the mouse urogenital tract and the infected tissue was then excised at 0 hr, 48 hrs, and 72 hrs post-inoculation (as indicated on the X axis). Tissue was minced, dissociated using Accutase cell detachment solution, resuspended in completed modified TYM media and counted via hemocytometer. Data shown are the total number of parasites per sample with standard deviation and were carried out using 3 mice per timepoint.
Fig 6.
Tv adherence to host cells in vitro correlates with an increased parasite burden in the MUT.
Quantification of Nluc-expressing LSU 160 more adherent (MA) (A) or parental (P) (B) parasites in mouse MUT tissues at 48 hrs and 72 hrs post-inoculation (as indicated on the X-axis). 11–14 mice were used for the MA strain at each time point. 9–12 mice were used for the P strain at each time point. Data shown are averages of luminescence/μg protein of the sample with standard deviation and are representative of 3 independent experiments. (C) Comparison of LSU160 MA (▲) and LSU160 P (●) parasite load quantifications. Data is expressed as fold change in luminescence in the mouse urogenital tract with standard deviation normalized to their respective 0h post-inoculation luminescent signals.
Fig 7.
Co-inoculation of parasite secreted EVs with Tv increases the parasite burden in the mouse urogenital tract.
Quantification of Nluc-expressing MSA 1103 parasites co-inoculated with either 50 μg/μL EVs (▢; Tv+EV) or parasites only (●; Tv) in the mouse MUT tissues at 48 and 72 hrs post-inoculation (as indicated on the X-axis). Ten mice were inoculated at each time point per condition. Data shown are averages of luminescence/μg protein of the sample with standard deviation normalized to 0h post-inoculation luminescent signals. Data are representative of 3 independent experiments.