Conference Agenda

We describe a comparative and evolutionary analysis of the new Trichomonas vaginalis reference genome with the first genomes of two other human parasites, T. tenax and Pentatrichomonas hominis, and four avian Trichomonas species, including the closest T. vaginalis relative T. stableri [2]. Long-read assembly of six chromosome-size T. vaginalis scaffolds vastly improves on the fragmented 2007 [1] assembly, enabling accurate annotation of protein-coding and RNA genes, and highly repetitive multigene families and transposable elements (TEs). We report trichomonad genome sizes, gene content, synteny, and multigene families; resolve phylogenetic ambiguities; and find evidence to support at least two host switches in Trichomonas evolution. We assessed TE abundance across genomes, its role in expansion of human Trichomonas genomes relative to avian, its relation to TE expression, and classified >4600 long virus-like Maverick TEs that constitute >40% of the T. vaginalis genome. Our evolutionary analyses further report: 1) evidence that the increase in repeat sequences (TEs and multi-gene families) was driven by relaxed selection and genetic drift; 2) the functional roles of T. vaginalis gene families that expanded the most and convergently expanded in T. tenax; and 3) single-copy genes putatively important for the transition of the parasite from avian to human host

The protozoan parasite Histomonas meleagridis is the causative agent of histomonosis, an important poultry disease emphasized by the absence of any licensed product for prophylaxis and treatment. The interplay between the parasite and its bacterial environment highlights a unique interaction in medicine and infection biology. Clonal monoxenic cultures used to establish high-quality genome sequence data from virulent and attenuated strains, originating from the same parental lineage of H. meleagridis, enabled the link between genomic variations and attenuation. Furthermore, comparative proteomic and surfaceome studies on in vitro propagated parasites revealed molecular data associated with virulence and in vitro attenuation. An intricate relationship between H. meleagridis and bacteria was revealed, demonstrating that the parasite itself substantially influenced co-cultivated bacteria's proteome pattern. Recently, we performed an immunoproteome analysis to identify the key antigens involved in the antibody response to H. meleagridis. Immunogenic proteins were isolated by immunoprecipitation employing sera from chickens and turkeys with different infection background. Comparative analysis of immunogenic proteins using a virulent and an attenuated antigenic preparation demonstrated minor variations depending on the host. Most differential immunogenic proteins were detected with either chicken or turkey sera, underlying a specific immune reaction of each host.

Trichomonas vaginalis, a flagellated protozoan parasite, thrives predominantly in the anaerobic environment of the human genitourinary tract. Previous research has established that the genus Trichomonasvirus, a double-stranded ribonucleic acid (dsRNA) virus belonging to the Totiviridae family, can specifically infect T. vaginalis. Within the Trichomonasvirus family, there are five distinct species, referred to as Trichomonas vaginalis virus 1 through Trichomonas vaginalis virus 5. Notably, a single T. vaginalis isolate can be infected by multiple Trichomonasvirus species simultaneously. While many studies suggest that Trichomonasvirus affects the virulence, physiology or drug resistance of T. vaginalis, there are also numerous studies that hold opposing views. A consensus remains elusive due to disparate findings leading to varying conclusions. As a result, we postulate that there may exist heterogeneity in Trichomonasvirus species distribution among cells within the same T. vaginalis isolate. To investigate this phenomenon, we utilized single-cell RNA sequencing (scRNA-seq) alongside an anti-dsRNA antibody-based immunofluorescent assay on the T. vaginalis isolate, ATCC 30236, as a model to study the inconsistent distribution of Trichomonasvirus. Importantly, our scRNA-seq data not only indicate the presence of heterogeneity in virus distribution but also have the potential to reveal subpopulations within the population derived from a single isolate.

Several studies have reported that Trichomonas vaginalis, the causative agent of trichomoniasis, can cause persistent infections in the human reproductive tract. In addition to the parasite’s trophozoite stage, a nonmotile pseudocyst stage has also been described, which may be a factor in such persistent infections. Methods to induce the development of pseudocysts have been described including culturing parasites (i) in iron-depleted media, (ii) at cold temperatures, and in acidic media that is physiologically appropriate to reproductive-age vaginas. Testing all three methods, we determined that incubating at cold temperatures is not physiologically relevant, and culturing in acidic media leads to parasite death, but iron-depletion yields live, persistent pseudocysts for up to a week. To determine gene expression patterns and putative marker genes associated with pseudocysts, we generated RNA-Seq data from triplicate cultures of T. vaginalis generated under iron-depletion. The data revealed hundreds of significantly differentially expressed genes in pseudocysts that are distinct from trophozoites in each daily dataset with 189 downregulated and 214 upregulated protein coding genes found across the first four days. The functions of these genes shed light on the structural and metabolic upheaval required to form pseudocysts and provide several promising candidate marker genes.

Bovine trichomonosis, caused by Tritrichomonas foetus, is a venereal disease that impairs cattle reproduction. Traditional vaccines use whole inactivated T. foetus, requiring extensive culturing. This study explores reverse vaccinology by sequencing Australian T. foetus strains to identify putative vaccine candidates against bovine trichomonosis.

In 2020-2021, T. foetus was detected across northern Australia using qPCR, and two isolates (TfOz5 and TfOz-N36) were purified from preputial samples from Queensland and Northern Territory. Genomic DNA was extracted via cold alkaline lysis, sequenced using SQK-LSK110 kit in MinION Mk1C for 48 hours using R9.4.1 flow cells. Base calling used Guppy 6.5.7 with super-high accuracy and reads under 20 Kbp with an average Q-score under 12 were filtered out using NanoFilt. Genome assembly was conducted with Flye, and ab initio gene prediction was performed with Augustus.

The genome assembly of the TfOz5 strain resulted in 194 contigs (110 Mb, N50 of 1.49 Mb), while TfOz-N36 had 368 contigs (105 Mb, N50 of 0.48 Mb). The isolates share 99.2% similarity and represent a 10 to 100-fold improvement in contig reduction compared to previous T. foetus genomes. Augustus predicted 28,480 genes and further bioinformatic analyses will screen for vaccine candidates using reverse vaccinology approaches.

Trichomonads infect a broad range of hosts, including wild and farmed animals and humans. The vast majority of studies focus on human infecting species, and Trichomonas vaginalis in particular. Here we will illustrate, and argue for the importance of expanding, comparative studies of Trichomonads across a broad range of hosts to gain new insights into the molecular and cellular basis of the complex interactions between Trichomonads, the host microbiota and host cells. Animals hosting Trichomonads are also important as sources of food and as nodes in terrestrial ecosystems. Hence increasing our knowledge of Trichomonads is relevant in a number of contexts including medical and veterinary sciences. A selection of examples will be covered to illustrate these points and aim to stimulate: (i) collaborations across disciplines including microbial ecology and evolutionary biology and more generally (ii) a One Health approach for the Trichomonads.