Biology subjects

Shiratori, M.

Publications and source records attributed to Shiratori, M..

7 recordsLinked to original sources

bioRxiv2026

Stop codon readthrough in Trichomonas is a mechanism for gene expression regulation and expanding protein function

Trichomonas vaginalis is the causative agent of trichomoniasis, a common sexually transmitted infection among women of reproductive and peri-menopausal age. The parasite has an unusually large genome, rich in complex repeats, including a vast repertoire of transposable elements and multi-copy gene families. Since very few T. vaginalis genes have introns, gene expression is usually straightforward, with ribosomal translational machinery proceeding from a start codon to the next in-frame stop codon of an unspliced poly(A)denylated mRNA. However, our previous studies raised the possibility of T. vaginalis gene expression involving stop codon readthrough (SCR), where transcription through in-frame stop codons produces longer-than-predicted mRNAs that translate to fully functional proteins. Here, we leverage long-read RNA-seq and new chromosome-scale assemblies of two T. vaginalis strains and two avian sister species to investigate and characterize ~1,400 long, mature mRNAs that contain more than one predicted protein-coding gene transcribed from what we call '' RT genes '', composites of adjacent predicted genes. We first identify RT genes in a second T. vaginalis strain and in close relatives T. vaginalis-like and T. stableri, indicating that this phenomenon is conserved among Trichomonas species and strains. Second, we find transcripts of RT genes to be more abundant by many orders of magnitude than monocistronic genes. Third, we found the distance between predicted genes within RT genes to be significantly shorter than between adjacent independent predicted genes. Fourth, functional annotation revealed that RT genes encode at least 50 distinct protein functions, suggesting that this unusual transcriptional mechanism has a role in an array of biological processes in Trichomonas. Our results from two Trichomonas species suggest that SCR is an important mechanism controlling gene expression and the diversity of protein function in this parasite.

molecular biology↗

bioRxiv2026

Trichomonas vaginalis pseudocysts are a quiescent cell stage characterized by a differentially regulated transcriptome and intact membranes

Trichomonas vaginalis is the causative agent of trichomoniasis, the most common non-viral sexually transmitted infection (STI). Despite its prevalence, low levels of public knowledge and research funding and the absence of T. vaginalis screening or control programs have led to its categorization as a "neglected" STI. Unlike other STIs in the USA, prevalence increases with age, peaking among individuals in their 40s. Both a motile trophozoite stage and a non-motile pseudocyst state have been described for the parasite, although it is debated whether the latter is a quiescent stage or a degenerate form on its way to cell death. Here we characterize the T. vaginalis pseudocyst by flow cytometry, membrane integrity assays, transcriptomics, and reversion studies. Pseudocysts were induced by culturing trophozoites in acidic media or in iron depleted media, with a variety of resulting survival rates. Flow cytometry studies showed that pseudocysts have intact cell membranes and express phosphatidylserine on their cell surface. Fluorescence-activated cell sorting studies also identified distinct sub-populations of parasites, revealing the importance of using pure live pseudocyst cultures in reversion studies. Pseudocysts were transcriptionally active for several days and had consistent subsets of genes with increased expression compared to trophozoites, although decreased transcription of genes involved in metabolism. Comparative transcriptomics of pseudocysts and trophozoites of two T. vaginalis strains revealed distinct cell states. Combined, our results provide evidence that the pseudocyst cell state is a stress-induced quiescent stage of T. vaginalis that can remain viable for days, with implications for a role in persistent infections. Author SummaryThe sexually transmitted parasite Trichomonas vaginalis has two well-known cell forms: a free-swimming, flagellated trophozoite and an amoeboid form adhered to host epithelia. A third morphology, the pseudocyst, has been described, but it is unclear whether this is a quiescent stage capable of facilitating persistent infections, or a degenerate form indicating cell death. Here we describe experiments revealing that pseudocysts have overall decreased gene expression compared to trophozoites but exhibit stage-specific gene transcription and plasma membrane integrity for days. Moreover, pseudocysts maintain externalized phosphatidylserine for multiple days. Taken together, our results suggest that pseudocysts are a viable cell stage in T. vaginalis distinct from cell death. Further research into pseudocysts and particularly their interactions with host immune cells is needed to reveal what role they may play in T. vaginalis pathogenesis and persistent or asymptomatic infections.

microbiology↗

bioRxiv2026

Adaptive immune responses to vaccination reflect social status at first exposure in female rhesus macaques

Social gradients are consistently associated with variation in health outcomes, including infectious disease. However, distinguishing between social gradients in antigen exposure versus social gradients in susceptibility remains challenging. Here, we use a nonhuman primate model for chronic social stress to investigate how social status affects the influenza vaccine-induced adaptive immune response. We first manipulated the social status of female rhesus macaques and then tested the response to influenza antigens in naive individuals and after secondary exposure. Higher social status at the time of first exposure, but not at the time of secondary exposure, predicted stronger antibody responses to both exposures. Social status also drove gene expression differences in adaptive immune pathways, and genes that predicted the magnitude of the antibody response overlapped with those linked to social status. Thus, social gradients shape the adaptive immune response in a temporally dependent manner, with particular sensitivity at the time of initial antigen exposure.

genomics↗

bioRxiv2024

Redefining the spliceosomal introns of the sexually transmitted parasite Trichomonas vaginalis and its close relatives in columbid birds

Trichomonas vaginalis infects the urogenital tract of men and women and causes the sexually transmitted infection trichomoniasis. Since the publication of its draft genome in 2007, the genome has drawn attention for several reasons, including its unusually large size, massive expansion of gene families, and high repeat content. The fragmented nature of the draft assembly made it challenging to obtain accurate metrics of features, such as spliceosomal introns. The number of introns identified varied over the years, ranging from 41 when first characterized in 2005, to 32 in 2018 when the repertoire was revised. In both cases, the results suggested that more introns could be present in the genome. In this study, we exploited our new T. vaginalis G3 chromosome-scale assembly and annotation and high-coverage transcriptome datasets to provide a definitive analysis of the complete repertoire of spliceosomal introns in the species. We developed a custom pipeline that distinguishes true splicing events from chimeric alignments by utilizing the extended motifs required by the splicing machinery and experimentally verified the results using transcript evidence. We identified a total of 63 active introns and 34 putative "inactive" intron sequences in T. vaginalis, enabling an analysis of their length distribution, extended consensus motifs, intron phase distribution (including an unexpected expansion of UTR introns), and functional annotation. Notably, we found that the shortest intron in T. vaginalis, at only 23 nucleotides in size, is one of the shortest introns known to date. We tested our pipeline on a chromosome-scale assembly of the bird parasite Trichomonas stableri, the closest known relative to T. vaginalis. Our results revealed some conservation of the main features (total intron count, sequence, length distribution, and motifs) of these two closely related species, although differences in their functional annotation and duplication suggest more specialized splicing machinery in T. vaginalis.

genetics↗

bioRxiv2024

In-gel refolding allows fluorescence detection of fully denatured GFPs after SDS-PAGE

Green fluorescent proteins (GFPs) have been widely used as fusion tags, especially to visualize subcellular localization and dynamics of the fused partner proteins. Also, GFPs serve as fluorescent tags in size-exclusion chromatography and native-PAGE, facilitating the evaluation of expression levels and quality of the expressed fusion proteins. However, the fluorescent detection of GFPs is generally incompatible with denaturing SDS-polyacrylamide gel electrophoresis (PAGE), where the samples are heat-denatured before loading. Accordingly, detecting GFP-fused proteins after SDS-PAGE usually relies on western blotting with anti-GFP antibodies. To enable in-gel fluorescence detection of SDS-PAGE-separated GFPs, some protocols employ mild denaturing conditions to keep the GFPs intact. However, such mild denaturation sometimes results in partial denaturation of the proteins and irregular electrophoretic mobility that is not proportional to their molecular weights. Here, we demonstrate that the fully denatured GFPs can be refolded within the gel by cyclodextrin-mediated removal of SDS in the presence of 20% methanol, enabling the in-gel fluorescence detection of the GFP-fused proteins. The protocol is compatible with subsequent total protein staining and western blotting. Although future studies are needed to clarify the scope and generality, the technique developed here would provide a simple, time- and cost-effective alternative to the immunodetection of GFPs. HighlightsO_LIFully denatured GFPs could be in-gel refolded to enable fluorescence detection. C_LIO_LI? cyclodextrin effectively removed SDS to assist in the in-gel refolding of GFPs. C_LIO_LIGFPs amenable for in-gel refolding include GFPuv, EGFP, turboGFP, and sfGFP. C_LIO_LICompatible with total protein staining and western blotting. C_LIO_LIThe in-gel refolding procedure was successfully applied to two turboGFP-fusion proteins. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/615947v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@19fe9b7org.highwire.dtl.DTLVardef@1a927a8org.highwire.dtl.DTLVardef@6aa42borg.highwire.dtl.DTLVardef@1400b78_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2023

A female-biased gene expression signature of dominance in cooperatively breeding meerkats

Dominance is a primary determinant of social dynamics and resource access in social animals. Recent studies show that dominance is also reflected in the gene regulatory profiles of peripheral immune cells. However, the strength and direction of this relationship differs across the species and sex combinations investigated, potentially due to variation in the predictors and energetic consequences of dominant status. To test this possibility, we investigated the association between social status and gene expression in the blood of wild meerkats (Suricata suricatta; n=113 unique individuals), including in response to lipopolysaccharide, Gardiquimod, and glucocorticoid stimulation. Meerkats are cooperatively breeding social carnivores in which breeding females physically outcompete other females to suppress reproduction, resulting in high reproductive skew. They therefore present an opportunity to disentangle the effects of social dominance from those of sex per se. We identify a sex-specific signature of dominance, including 1,045 differentially expressed genes in females but none in males. Dominant females exhibit elevated activity in innate immune pathways and an exacerbated response to LPS challenge. In this respect, female meerkats resemble wild male baboons (for which similar data are available), where physical competition is also central to determining rank hierarchies and mating effort is high. However, they differ from female primates in which social status is nepotistically determined. Our results support the hypothesis that the gene regulatory signature of social status depends on the determinants and energetic costs of social dominance. They also support potential life history trade-offs between investment in reproduction versus somatic maintenance.

animal behavior and cognition↗

bioRxiv2023

Reply to Barton et al: signatures of natural selection during the Black Death

Barton et al.1 raise several statistical concerns regarding our original analyses2 that highlight the challenge of inferring natural selection using ancient genomic data. We show here that these concerns have limited impact on our original conclusions. Specifically, we recover the same signature of enrichment for high FST values at the immune loci relative to putatively neutral sites after switching the allele frequency estimation method to a maximum likelihood approach, filtering to only consider known human variants, and down-sampling our data to the same mean coverage across sites. Furthermore, using permutations, we show that the rs2549794 variant near ERAP2 continues to emerge as the strongest candidate for selection (p = 1.2x10-5), falling below the Bonferroni-corrected significance threshold recommended by Barton et al. Importantly, the evidence for selection on ERAP2 is further supported by functional data demonstrating the impact of the ERAP2 genotype on the immune response to Y. pestis and by epidemiological data from an independent group showing that the putatively selected allele during the Black Death protects against severe respiratory infection in contemporary populations.

genomics↗