Biology subjects

Yamagishi, J.

Publications and source records attributed to Yamagishi, J..

4 recordsLinked to original sources

bioRxiv2024

ves1α genes expression is the major determinant of Babesia bovis-infected erythrocytes cytoadhesion to endothelial cells

Babesia bovis causes the most pathogenic form of babesiosis in cattle, resulting in high mortality in naive adults. This parasite invades red blood cells (RBCs) within the bovine hosts where they multiply and produce clinical disease. Babesia bovis exports numerous proteins into invaded RBCs changing its properties. Thus, the infected RBCs (iRBCs) are capable to cytoadhere in the microvasculature of internal organs and brain, leading to respiratory distress, neurologic signs, and mortality. Variant Erythrocyte Surface Antigen 1 (VESA1) is one of those exported proteins by B. bovis which represents a major virulence factor due to its central role in immune evasion by antigenic variation and intravascular parasite sequestration. VESA1 is a heterodimer protein encoded by ves1 and ves1{beta} multigene family and localized on the ridges, the focal point for cytoadhesion. To gain further insights into the molecular mechanisms of cytoadhesion of B. bovis, we panned the parasites with bovine brain microvasculature endothelial cells, which resulted in obtaining several clones with different cytoadherence abilities. The transcriptome analysis of 2 high and 2 low cytoadherent clones revealed that ves1 sequences were diversified, likely resulting from genomic recombination. On the other hand, ves1{beta} sequences were almost identical among these 4 clones. Insertion and expression of ves1 of a clone with high binding into ef-1 locus of a low binging clone increased cytoadherence confirming the role of ves1 suggested by our transcriptome data. Whole genome sequencing of cytoadherent clones revealed active locus of ves1 on chromosome 2. These results suggest that VESA1a proteins encoded by ves1 genes determine the cytoadherence specificity and/or cytoadherence strength of B. bovis and they are in the active site for recombination. Author summaryBabesia bovis is an apicomplexan intraerythrocytic protozoan parasite which causes the most pathogenic form of babesiosis in cattle. This pathogenicity is the result of parasite multiplication and cytoadherence of infected red blood cells (iRBCs) in the microvasculature of brain and internal organs and is mediated by B. bovis surface exposed ligand, Variant Erythrocyte Surface Antigen 1 (VESA1). Here using parasite panning assay, transcriptomics, and genetic tools, we showed that VESA1a is the main determinant of B. bovis cytoadhesion. VESA1 are large hypervariable proteins (>100kDa) consisting of VESA1a and VESA1b subunits encoded by ves1 and ves1{beta} multigene family. Panning B. bovis with bovine brain endothelial cells resulted in obtaining cytoadherent parasite clones with different binding abilities. Comparative transcriptome analysis revealed diversification of ves1 sequences. Insertion and expression of ves1 of a clone with high-binding ability in the genome of a low-binding clone increased cytoadherence confirming the role of ves1. Mapping RNA-seq on the genome of cytoadherent clones revealed the locus of active transcription and this locus was suggested to be the active site for recombination which promoted the production of variants of ves1 with different binding abilities. Altogether, our results provide new insights into B. bovis cytoadhesion and VESA1 biology.

microbiology↗

bioRxiv2024

Critical role of Babesia bovis spherical body protein 3 in ridge formation on infected red blood cells

Babesia bovis, an apicomplexan intraerythrocytic protozoan parasite, causes serious economic loss to cattle industries around the world. Infection with this parasite leads to accumulation of infected red blood cells (iRBCs) in the brain microvasculature that results in severe clinical complications known as cerebral babesiosis. Throughout its growth within iRBCs, the parasite exports various proteins to the iRBCs that lead to the formation of protrusions known as "ridges" on the surface of iRBCs, which serve as sites for cytoadhesion to endothelial cells. Spherical body proteins (SBPs; proteins secreted from spherical bodies, which are organelles specific to Piroplasmida) are exported into iRBCs, and four proteins (SBP1-4) have been reported to date. In this study, we elucidated the function of SBP3 using an inducible gene knockdown (KD) system. Localization of SBP3 was assessed by immunofluorescence assay, and only partial colocalization was detected between SBP3 and SBP4 inside the iRBCs. In contrast, colocalization was observed with VESA-1, which is a major parasite ligand responsible for the cytoadhesion. Immunoelectron microscopy confirmed localization of SBP3 at the ridges. SBP3 KD was performed using the glmS system, and effective KD was confirmed by Western blotting, immunofluorescence assay, and RNA-seq analysis. The SBP3 KD parasites showed severe growth defect suggesting its importance for parasite survival in the iRBCs. VESA-1 on the surface of iRBCs was scarcely detected in SBP3 KD parasites, whereas SBP4 was still detected in the iRBCs. Moreover, abolition of ridges on the iRBCs and reduction of iRBCs cytoadhesion to the bovine brain endothelial cells were observed in SBP3 KD parasites. Immunoprecipitation followed by mass spectrometry analysis detected the host Band 3 multiprotein complex, suggesting an association of SBP3 with iRBC cytoskeleton proteins. Taken together, this study revealed the vital role of SBP3 in ridge formation and its significance in the pathogenesis of cerebral babesiosis. Author summaryBabesia bovis causes a high-mortality complication called cerebral babesiosis in cattle, similar to cerebral malaria in humans. Both complications are caused by the cytoadhesion of infected red blood cells (iRBCs) to the host brain endothelial cells. These parasites export numerous proteins to the host iRBCs and produce protrusions on the iRBCs that are called ridges for B. bovis and knobs for Plasmodium falciparum. Ridges and knobs play an important role in cytoadhesion as they are the sites of adherence; however, the molecules responsible for ridge formation remain unknown. In this study, we showed that SBP3 is a crucial protein for ridge formation. The SBP3 knockdown parasites showed severe growth defects and abolition of ridges on the iRBCs, and cytoadhesion of iRBCs to the bovine brain endothelial cells was significantly reduced. An immunoprecipitation experiment suggested an association of SBP3 with the host Band 3 multiprotein complex. Although there is no similarity in amino acid sequences, we suggest SBP3 to be a functional ortholog of KAHRP in P. falciparum. In summary, our results shed light on the molecular mechanism of ridge formation and the pathogenesis of B. bovis.

microbiology↗

bioRxiv2023

Combination therapy with oral antiviral and anti-inflammatory drugs improves the efficacy of delayed treatment in severe COVID-19

Pulmonary infection with SARS-CoV-2 stimulates host immune responses and can also result in the progression of dysregulated and critical inflammation. Throughout the pandemic, the management and treatment of COVID-19 has been continuously updated with a range of antiviral drugs and immunomodulators. Monotherapy with oral antivirals has proven to be effective in the treatment of COVID-19. However, the treatment should be initiated in the early stages of infection to ensure beneficial therapeutic outcomes, and there is still room for further consideration on therapeutic strategies using antivirals. Here, we show that the oral antiviral ensitrelvir combined with the anti-inflammatory corticosteroid methylprednisolone has higher therapeutic effects and better outcomes in a delayed dosing model of SARS-CoV-2 infected hamsters compared to the monotherapy with ensitrelvir or methylprednisolone alone. Combination therapy with these drugs improved respiratory conditions and the development of pneumonia in hamsters even when the treatment was started after 2 days post infection. The combination therapy led to a differential histological and transcriptomic pattern in comparison to either of the monotherapies, with reduced lung damage and down-regulated expressions of genes involved in inflammatory response. Furthermore, we found that the combination treatment is effective in infection with both highly pathogenic delta and circulating omicron variants. Our results demonstrate the advantage of combination therapy with antiviral and corticosteroid drugs in COVID-19 treatment. Since both drugs are available as oral medications, this combination therapy could provide a clinical and potent therapeutic option for COVID-19.

microbiology↗

bioRxiv2018

A single test approach for accurate and sensitive detection and taxonomic characterization of Trypanosomes by comprehensive analysis of ITS1 amplicons

The World Health Organization has targeted stopping the transmission of Human African Trypanosomiasis by 2030. To achieve this, better tools are urgently required to identify and monitor Trypanosome infections in human, animals, and tsetse fly vectors. This study presents a single test approach for detection and identification of Trypanosomes and their comprehensive characterization at species and sub-group level. Our method uses newly designed ITS1 PCR primers (a widely used method for detection of African Trypanosomes, amplifying the ITS1 region of ribosomal RNA genes) coupled to Illumina sequencing of the amplicon. The protocol is based on the widely used Illuminas 16s bacterial metagenomic analysis procedure that makes use of multiplex PCR and dual indexing. We analyzed wild tsetse flies collected from Zambia and Zimbabwe. Our results show that the traditional method for Trypanosome species detection based on band size comparisons on a gel is unable to distinguish between T. vivax and T. godfreyi accurately. Additionally, this approach shows increased sensitivity of detection at species level. Through phylogenetic analysis, we identified Trypanosomes at species and sub-group level without the need for any additional tests. Our results show T. congolense Kilifi sub-group is more closely related to T. simiae than to other T. congolense sub-groups. This agrees with previous studies using satellite DNA and 18s RNA analysis. While current classification does not list any sub-groups for T. vivax and T. godfreyi, we observed distinct subgroups for these species. Interestingly, sequences matching T. congolense Tsavo (now classified as T. simiae Tsavo) clusters distinctly from the rest of the T. simiae Tsavo sequences suggesting that the Nannomonas group is more divergent than currently thought thus the need for a better classification criteria. This approach has the potential for refining classification of Trypanosomes and provide detailed molecular epidemiology information useful for surveillance and transmission control efforts.\n\nAuthor summaryDetection of Trypanosomes in the tsetse flies plays an important role in the control of African trypanosomiasis by providing information on circulating Trypanosome species in a given area. We have developed a method that combines multiplex PCR and next-generation sequencing for Trypanosome species detection. The method is based on the widely used bacterial metagenomic analysis protocol and uses a modular, two-step PCR process followed by sequencing of all amplicons in a single run, making sequencing of amplicons more efficient and cost-effective when dealing with large sample sizes. As part of this approach, we designed novel primers for amplifying the ITS1 region of the Trypanosome rRNA gene that is more sensitive than conventional primers. Identification of Trypanosome species is based on BLAST searches against the constantly updated NCBIs nt database, which facilitates the identification of Trypanosome subgroups. Our approach is more accurate than traditional gel-based analysis and shows how the latter is prone to misidentification. It is also sensitive and is able to discriminate between subgroups within Trypanosome species. Applied as an epidemiological tool, it has the potential to provide new, comprehensive and more accurate information on vector-pathogen-host interconnections which are key in the control and management of African trypanosomiasis.

epidemiology↗