Biology subjects

Kullapanich, C.

Publications and source records attributed to Kullapanich, C..

6 recordsLinked to original sources

bioRxiv2025

Developmental Progression and Single-Cell Heterogeneity of Orientia tsutsugamushi during Intracellular Infection

Orientia tsutsugamushi, the causative agent of human scrub typhus disease, is an obligate intracellular bacterium transmitted by mites. It replicates exclusively within eukaryotic cells, inhabiting a range of host cell types in mammals and mites. Previously, we demonstrated that its extracellular, non-replicative form is developmentally distinct from the intracellular replicative form. In this study, we investigated spatial and temporal heterogeneity within the intracellular bacterial population. By quantifying bacterial DNA replication, protein synthesis, cell division, and subcellular localization, we defined four distinct intracellular growth stages: pre-active, lag, log, and maturation. Using microscopy-based single-cell analysis of selected transcripts and proteins, we further revealed transcriptional and translational heterogeneity within microcolonies. These findings highlight the complex, asynchronous nature of intracellular O. tsutsugamushi growth and underscore the importance of single-cell analysis in understanding its pathobiology. ImportanceScrub typhus is a serious, sometimes life-threatening disease caused by the bacterium Orientia tsutsugamushi, which is spread by the bites of tiny mites and is common in many rural parts of Asia. This bacterium can only survive and grow inside living cells, where hundreds of individual bacteria may infect a single host cell. Until now, it was unclear whether all the bacteria inside a cell behaved the same way or if they existed in different stages of growth. In this study, we show that O. tsutsugamushi goes through several distinct stages while growing inside host cells and that not all bacteria are at the same stage at the same time. Even within a single infected cell, some bacteria are actively dividing, while others are preparing to leave the cell. Understanding these stages provides a new framework for exploring how the bacterium develops, spreads, and causes disease -- insights that could help guide future research and improve ways to control or treat scrub typhus.

microbiology↗

bioRxiv2025

High levels of antisense transcription from numerous genes in the obligate intracellular bacterium Orientia tsutsugamushi.

Orientia tsutsugamushi (Ot) is a cytoplasmic, Gram-negative, obligate intracellular bacterium that causes the human disease scrub typhus. It has a highly repetitive genome whereby approximately 50% is composed of multiple copies of a proliferated integrative and conjugative element, the Rickettsial amplified Genetic Element (RAGE). Previous RNA sequencing analysis revealed a high level of antisense transcription in Ot, particularly from RAGE-encoded genes, with 18% of all genes having a sense:antisense ratio <1 and 5% with a ratio <0.1. In our current study, we have confirmed the earlier RNA sequencing findings via PCR-based methods and established that this antisense transcription is a consistent feature across eight distinct Ot strains. Furthermore, we have utilized PCR to monitor differences in sense and antisense expression between intracellular and extracellular bacteria. Our findings lend weight to the hypothesis that antisense transcription in Ot is a regulated mechanism, possibly playing a crucial role in the pathogens lifecycle during infection.

microbiology↗

bioRxiv2025

Comparative Dual RNA-Seq Analysis of Eight Orientia tsutsugamushi Strains in Endothelial Cells Reveals Regulatory Patterns in Bacterial and Host Pathways During Infection

Orientia tsutsugamushi is an obligate intracellular bacterium that causes the mite-borne human disease scrub typhus. The species is characterised by having many strains that differ in their ability to cause disease in murine infection models and in human patients. The genomes of a diverse set of Orientia tsutsugamushi strains have recently been analysed in detail, revealing broadly similar gene content with genomes differing primarily in synteny and in the composition of a large arsenal of predicted secreted effector proteins. Given the similarity in gene content, here we asked whether the observed differences in virulence were driven by genome-wide differences in gene expression of bacterial genes, and differences in the ensuing response of infected host cells. To explore this, we carried out a dual RNA sequencing analysis of eight diverse strains of Orientia tsutsugamushi grown in cultured human endothelial cells. An overall analysis found no clear patterns in the bacterial or host gene expression patterns that correlate with the ability to cause disease. We found that all strains induce a strong type 1 interferon response in endothelial cells, but that within that broad response each strain has a unique fingerprint of induced genes likely leading to different disease outcomes when combined with a complex immune system in vivo. We compared expression levels of orthologous genes between different strains and identified some bacterial pathways with constant expression levels whilst others were more variable, leading to the identification of specific pathways under tight transcriptional control during cellular infection. Together our data show that inter-strain differences in virulence are not defined by expression levels of any one set of bacterial or host genes and also reveal insights into transcriptional regulation of different pathways in Orientia tsutsugamushi.

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↗

bioRxiv2024

The intracellular bacterium Orientia tsutsugamushi hijacks the adaptor protein BICD2 for dynein-based motility

The intracellular bacterium Orientia tsutsugamushi relies on the microtubule cytoskeleton and the motor protein dynein to traffic to the perinuclear region within infected cells. However, it remains unclear how the bacterium is coupled to the dynein machinery and how transport is regulated. Here, we discover that O. tsutsugamushi uses its autotransporter protein ScaC to recruit the dynein adaptor BICD2 to the bacterial surface. We show that ScaC is sufficient to engage dynein-based motility in the absence of other bacterial proteins and that BICD2 is required for efficient movement of O. tsutsugamushi during infection. Using TIRF single-molecule assays, we demonstrate that ScaC induces BICD2 to adopt an open conformation which activates the assembly of dynein-dynactin complexes. Our results reveal a novel role for BICD2 during bacterial infection and provide mechanistic insights into the life cycle of an important human pathogen.

microbiology↗

bioRxiv2023

Orientia tsutsugamushi: analysis of the mobilome of a highly fragmented and repetitive genome reveals ongoing lateral gene transfer in an obligate intracellular bacterium.

The rickettsial human pathogen Orientia tsutsugamushi (Ot) is an obligate intracellular Gram-negative bacterium with one of the most highly fragmented and repetitive genomes of any organism. Around 50% of its [~]2.3 Mb genome is comprised of repetitive DNA that is derived from the highly proliferated Rickettsiales amplified genetic element (RAGE). RAGE is an integrative and conjugative element (ICE) that is present in a single Ot genome in up to 92 copies, most of which are partially or heavily degraded. In this report, we analysed RAGEs in eight fully sequenced Ot genomes and manually curated and reannotated all RAGE-associated genes, including those encoding DNA mobilisation proteins, P-type (vir) and F-type (tra) type IV secretion system (T4SS) components, Ankyrin repeat- and tetratricopeptide repeat-containing effectors, and other piggybacking cargo. Originally, the heavily degraded Ot RAGEs led to speculation that they are remnants of historical ICEs that are no longer active. Our analysis, however, identified two Ot genomes harbouring one or more intact RAGEs with complete F-T4SS genes essential for mediating ICE DNA transfer. As similar ICEs have been identified in unrelated rickettsial species, we assert that RAGEs play an ongoing role in lateral gene transfer within the Rickettsiales. Remarkably, we also identified in several Ot genomes remnants of prophages with no similarity to other rickettsial prophages. Together these findings indicate that, despite their obligate intracellular lifestyle and host range restricted to mites, rodents and humans, Ot genomes are highly dynamic and shaped through ongoing invasions by mobile genetic elements and viruses.

microbiology↗