Biology subjects

Sunyakumthorn, P.

Publications and source records attributed to Sunyakumthorn, P..

4 recordsLinked to original sources

bioRxiv2026

Gentamicin Treatment in a Murine Infection Model Suggests Extracellular Orientia tsutsugamushi Contributes to Systemic Dissemination

Orientia tsutsugamushi, an obligate intracellular bacterial pathogen, infects multiple cell types and disseminates via the blood and lymphatic system to affect various organs in human and other mammalian hosts. This bacterium replicates exclusively within living cells; however, it also exists transiently in a metabolically inactive extracellular form. While infected leukocytes are known contributors to the systemic spread of the bacterium, the role of the inactive extracellular form in dissemination has been unclear. In this study, we demonstrate that extracellular bacteria are present in the cell-free plasma fraction of infected human and murine blood. Furthermore, treatment with gentamicin, a membrane-impermeable antibiotic, significantly reduces systemic bacterial growth in a murine infection model. These findings suggest a potential role for the extracellular form of the bacterium in the spread of infection and raise the possibility of exploring the use of gentamicin to augment conventional antibiotic therapy during scrub typhus infection.

microbiology↗

bioRxiv2025

Comparative virulence analysis of seven diverse strains of Orientia tsutsugamushi reveals a multifaceted and complex interplay of virulence factors responsible for disease.

Orientia tsutsugamushi is an obligate intracellular bacterium found in Leptotrombidium mites that causes the human disease scrub typhus. A distinguishing feature of O. tsutsugamushi is its extensive strain diversity, yet differences in virulence between strains are not well defined nor well understood. We sought to determine the bacterial drivers of pathogenicity by comparing murine infections using seven strains combined with epidemiological human data to rank each strain in terms of relative virulence. Murine cytokine expression data revealed that the two most virulent strains, Ikeda and Kato, induced higher levels of IL-6, IL-10, IFN-{gamma} and MCP-1 than other strains, consistent with increased levels of these cytokines in severe human scrub typhus patients. We sought to identify the mechanistic basis of the observed differential virulence between strains by comparing their genomes, in vitro growth properties and cytokine/chemokine induction in host cells. We found that there was no single gene or gene group that correlated with virulence, and no clear pattern of in vitro growth rate that predicted disease. However, microscopy-based analysis of the intracellular infection cycle revealed that the only fully avirulent strain in our study, TA686, differed from all the virulent strains in its subcellular localisation and expression of its surface protein ScaC. We conclude that drivers of pathogenicity in Orientia tsutsugamushi are distributed throughout the genome, likely in the large and varying arsenal of effector proteins encoded by different strains, and that these interact in complex ways to induce differing immune responses and thus differing disease outcomes in mammalian hosts. Author SummaryScrub typhus is a vector-borne human disease caused by the bacterium Orientia tsutsugmushi and spread by mites. There are numerous different strains of this bacterium with some causing more severe disease in humans than others, and some that do not cause any illness at all. The factors driving these differences are not yet understood, and gaining insight into them could aid in vaccine development and help predict the severity of disease caused by new isolates. To better determine the mechanistic basis of pathogenicity in scrub typhus, we carried out experiments in which we compared seven diverse strains for virulence in animals. We measured their ability to cause disease in mice, so that we could reliably classify them as virulent or avirulent in this model. We then analysed various genomic and biological aspects to identify disease markers in both mice and humans. We report here that there is no single factor that predicts whether a strain will be pathogenic or not, but that disease in scrub typhus is a complex process resulting from the activity of multiple bacterial genes working together to drive different immune responses in the host, resulting in either clearance of the bacteria from the host, or escalating disease. Future work exploring the relationship of bacterial effector proteins will help to disentangle this complex relationship in mechanistic detail.

microbiology↗

bioRxiv2022

A whole blood intracellular cytokine assay optimised for field site studies demonstrates polyfunctionality of CD4+ T cells in acute scrub typhus

BackgroundAssessment of cellular immune responses by combining intracellular cytokine staining and immunophenotyping using flow cytometry enables the simultaneous measurement of T cell phenotype and effector function in response to pathogens and vaccines. The use of whole blood samples rather than peripheral blood mononuclear cells avoids both the need for immediate processing and loss of functional antigen presenting cells due to processing and cryopreservation. Using whole blood provides the possibility to stimulate peripheral T cells in situ, and is more suitable for studies where sample volume is limited, such as those involving children, the elderly and critically ill patients. The aim of this study was to provide a robust tool for the assessment of antigen-specific T cell responses in a field site setting with limited resources. Methodology/Principle FindingsWe optimised a flow cytometry-based whole blood intracellular cytokine assay (WBA) with respect to duration of antigen stimulation and intracellular protein retention time. We demonstrate the ability of the WBA to capture polyfunctional T cell responses in the context of acute scrub typhus infection, by measuring IFN-{gamma}, TNF and IL-2 in CD4+ and CD8+ T cells in response to the causative agent O. tsutsugamushi (OT). Using an optimised OT antigen preparation, we demonstrate the presence of polyfunctional antigen-specific memory CD4+ T cells in the blood of scrub typhus patients. Conclusions/SignificanceIn conclusion, this flow cytometry-based WBA is well-suited for use at field study sites, and enables the assessment of polyfunctional T cell responses to infectious agents and vaccines through delineation of antigen-specific cytokine secretion at the single cell level. Author summaryScrub typhus is an acute febrile illness caused by the bite of infected chigger mites transmitting O. tsutsugamushi, a gramnegative obligate intracellular bacteria in the family Rickettsiaceae. The disease progression and clinical manifestations are closely associated with host immune responses. T cell responses are in strong relation with immune protection against scrub typhus. As there is limited knowledge on specific T cell roles against scrub typhus, we optimized a flow-cytometer based protocol to assess O. tsutsugumushi-specific T cell responses in whole blood samples, which is suitable for studies in limited resource settings. This method requires low blood volumes, but enables multiparametric immunophenotyping assessments. The optimized WBA protocol could be a useful tool for comprehensive immunopathological studies in scrub typhus, and other infectious diseases as well as vaccine studies in areas with limited availability of specialized equipment, while reliably capturing the complexity of the immune response.

immunology↗

bioRxiv2019

Dual RNA-seq provides insight into the biology of the neglected intracellular human pathogen Orientia tsutsugamushi

Emerging and neglected diseases pose challenges as their biology is frequently poorly understood, and genetic tools often do not exist to manipulate the responsible pathogen. Organism agnostic sequencing technologies offer a promising approach to understand the molecular processes underlying these diseases. Here we apply dual RNA-seq to Orientia tsutsugamushi (Ot), an obligate intracellular bacterium and the causative agent of the vector-borne human disease scrub typhus. Half the Ot genome is composed of repetitive DNA, and there is minimal collinearity in gene order between strains. Integrating RNA-seq, comparative genomics, proteomics, and machine learning, we investigated the transcriptional architecture of Ot, including operon structure and non-coding RNAs, and found evidence for wide-spread post-transcriptional antisense regulation. We compared the host response to two clinical isolates and identified distinct immune response networks that are up-regulated in response to each strain, leading to predictions of relative virulence which were confirmed in a mouse infection model. Thus, dual RNA-seq can provide insight into the biology and host-pathogen interactions of a poorly characterized and genetically intractable organism such as Ot.

microbiology↗