Biology subjects

Chiarelli, T. J.

Publications and source records attributed to Chiarelli, T. J..

6 recordsLinked to original sources

bioRxiv2026

Transformation and allelic exchange in Orientia tsutsugamushi

Orientia tsutsugamushi is a mite-transmitted obligate intracellular bacterium that causes the potentially deadly zoonosis, scrub typhus. The absence of genetic tools for Orientia have limited studies of the microbe-host interactions that underlie scrub typhus. To address this gap, we developed a protocol for transforming and achieving allelic exchange in O. tsutsugamushi str. Ikeda. From evaluating multiple cell lines and antibiotics, we found that contact-inhibited EA.hy926 human endothelial-like cells best supported Orientia replication and that chloramphenicol was an effective selection marker. We engineered a homologous recombination cassette encoding a codon-modified version of the O. tsutsugamushi ank13 gene (OTT_RS04140) (CMank13) and its promoter alongside genes for mScarlet-I and chloramphenicol acetyltransferase under control of the O. tsutsugamushi tsa22-up and tsa56-down promoters, respectively. A PCR product encompassing the cassette and chromosomal flanking regions was transformed into O. tsutsugamushi via electroporation or CaCl2, the latter of which better preserved bacterial and host cell viability. EA.hy926 cells inoculated with transformed O. tsutsugamushi were grown in glass-bottom plates in the presence of chloramphenicol and imaged by live-cell microscopy to identify cultures containing mScarlet-I positive bacteria, which could be maintained in perpetuity. Chromosomal integration of the CMank13 cassette and loss of wild-type ank13 were verified by PCR and nanopore sequencing. This report establishes platforms for genetically manipulating O. tsutsugamushi and building additional genetic tools to investigate this globally significant pathogen. IMPORTANCEOrientia tsutsugamushi causes scrub typhus, a globally emerging rickettsiosis that can have a high mortality rate and has been a known human disease since the fourth century. Of the genera of obligate intracellular bacterial pathogens that cause human disease, Orientia is the only one for which genetic tools have not been developed. This has limited understanding of O. tsutsugamushi-host dynamics that drive the bacteriums pathobiology and hindered development of novel treatment or protection strategies against scrub typhus. Here, we successfully transformed and achieved allelic exchange in O. tsutsugamushi. Transgenic bacteria were selected via antibiotic resistance, validated by PCR and nanopore sequencing, and visualized by immunofluorescence and live-cell fluorescence imaging. Our report includes detailed descriptions of empirically determined host cell cultivation, multiplicity of infection, transformation, and selection conditions to provide a foundation on which other researchers can build. Overall, this work begins to establish a genetic toolbox for O. tsutsugamushi.

genetics↗

bioRxiv2026

Small-molecule inhibition of the Orientia tsutsugamushi deubiquitylating enzyme OtDUB impairs bacterial reproduction

Scrub typhus is a potentially fatal infectious disease caused by the obligate intracellular bacterium Orientia tsutsugamushi. While antibiotic treatment is generally effective, it requires extended treatment, and drug resistance and treatment failures have emerged. O. tsutsugamushi encodes a deubiquitylating enzyme, OtDUB, which interferes with host ubiquitin-dependent pathways. OtDUB cleaves ubiquitin from various substrates, but whether this activity can be selectively targeted by small molecules is unknown. Here we have screened a chemically diverse small-molecule library using a fluorescence-based deubiquitylation assay to identify potential inhibitors of OtDUB. Two compounds, gentisic acid and amiloride hydrochloride, inhibited OtDUB activity at low dosage, with little effect on the related Wolbachia CidB or yeast Ulp1 enzymes. Computational docking predicted the compounds engage regions near the OtDUB catalytic pocket, suggesting a competitive mode of inhibition; this was supported by enzyme kinetic analyses. Neither compound caused detectable cytotoxicity in mammalian cells. Amiloride hydrochloride treatment reduced both total cellular deubiquitylating activity and the O. tsutsugamushi bacterial load in infected cells. While the identified compounds are not optimized inhibitors, they establish that bacterial pathogen-encoded deubiquitylating enzymes can be targeted by small molecules. Overall, our results provide a framework for using selective inhibitors as tools to study DUB function in genetically intractable intracellular bacteria and as potential treatments for scrub typhus.

biochemistry↗

bioRxiv2024

The chlamydial transcriptional regulator Euo is a key switch in cell form developmental progression but is not involved in the committed step to the formation of the infectious form.

Bacteria in the genus Chlamydia are a significant health burden world wide. They infect a wide range of vertebrate animals including humans and domesticated animals. In humans, C. psittaci can cause zoonotic pneumonia while C. pneumoniae causes a variety of respiratory infections. Infections with C. trachomatis cause ocular or genital infections. All chlamydial species are obligate intracellular parasites of eukaryotic cells and are dependent on a complex infection cycle that depends on transitions between specific cell forms. This cycle consists of cell forms specialized for host cell invasion, the Elementary Body (EB), and a form specialized for intracellular replication, the Reticulate Body (RB). In addition to the EB and RB there is a transitionary cell form that mediates the transformation between the RB and the EB, the Intermediate Body (IB). In this study we ectopically expressed the regulatory protein Euo and showed that high levels of expression resulted in reversible arrest of the development cycle. The arrested chlamydial cells were trapped phenotypically at an early IB stage of the cycle. These cells had exited the cell cycle but had not shifted gene expression from RB-like to IB/EB-like. This arrested state was dependent on continued expression of Euo. When ectopic expression was reversed, Euo levels dropped in the arrested cells which led to the repression of native Euo expression and the resumption of the developmental cycle. Our data are consistent with a model where Euo expression levels impact IB maturation to the infectious EB but not the production of the IB form. ImportanceBacterial species in the Chlamydiales order infect a variety of vertebrate animals and are a global health concern. They cause various diseases in humans, including genitital and respiratory infections. The bacteria are obligate intracellular parasites that rely on a complex infectious cycle involving multiple cell forms. All species share the same life cycle, transitioning through different states to form the infectious elementary body (EB) to spread infections to new hosts. The Euo gene, encoding a DNA binding protein, is involved in regulating this cycle. This study showed that ectopic expression of Euo halted the cycle at an early stage. This arrest depended on continued Euo expression. When Euo expression was reversed, the developmental cycle resumed. Additionally, this study suggests that high levels of Euo expression affects the formation of the infectious EB, but not the production of the cell form committed to EB formation.

microbiology↗

bioRxiv2024

The T3SS structural and effector genes of Chlamydia trachomatis are expressed in distinct phenotypic cell forms.

Bacteria in the chlamydiales order are obligate intracellular parasites of eukaryotic cells. Within this order, the genus Chlamydia contains the causative agents of a number of clinically important infections of humans. Biovars of C. trachomatis are the causative agents of trachoma, the leading cause of preventable blindness worldwide, as well as sexually transmitted infections with the potential to cause pelvic inflammatory disease and infertility. Irrespective of the resulting disease, all chlamydial species share the same obligate intracellular life cycle and developmental cell forms. They are reliant on an infectious cycle consisting of at least three phenotypically distinct cell forms termed the reticulate body (RB), the intermediate body (IB) and the elementary body (EB). The EB is infectious but does not replicate. The RB replicates in the host cell but is non-infectious, while the IB is an intermediate form that transitions to the EB form. In this study, we ectopically expressed the transcriptional repressor Euo, the two nucleoid-associated proteins HctA and HctB, and the two component sensor kinase CtcB in the RB. Transcriptional analysis using RNA-seq, differential expression clustering and fluorescence in situ hybridization analysis show that the chlamydial developmental cycle is driven by three distinct regulons corresponding to the RB, IB or EB cell forms. Moreover, we show that the genes for the T3SS were cell type restricted, suggesting defined functional roles for the T3SS in specific cell forms. ImportanceChlamydia trachomatis, a sexually transmitted bacterial infection, poses a significant global health threat, causing over 100 million infections annually and leading to complications like ectopic pregnancy and infertility. This study investigates the gene expression patterns of Chlamydia trachomatis during its unique life cycle within human cells. As an obligate intracellular parasite, C. trachomatis transitions through distinct developmental stages - one for infection and dissemination, another for replication, and a third for transitioning back to the infectious form. By analyzing gene expression profiles at each stage, we identified key genes involved in these processes. Interestingly, our research also reveals the presence of two separate T3SS (Type III Secretion System) translocons expressed in distinct stages, suggesting their crucial roles in specific functions during the infection cycle.

microbiology↗

bioRxiv2021

Translational gene expression control in Chlamydia trachomatis.

The human pathogen Chlamydia trachomatis proceeds through a multi phenotypic developmental cycle with each cell form specialized for different roles in pathogenesis. Understanding the mechanisms regulating this complex cycle has historically been hampered by limited genetic tools. In an effort to address this issue, we developed a translational control system to regulate gene expression in Chlamydia using a synthetic riboswitch. Here we demonstrate that translational control via a riboswitch can be used in combination with a wide range of promoters in C. trachomatis. The synthetic riboswitch E, inducible with theophylline, was used to replace the ribosome binding site of the synthetic promoter T5-lac, the native chlamydial promoter of the pgp4 plasmid gene and an anhydrotetracycline responsive promoter. In all cases the riboswitch inhibited translation, and high levels of protein expression was induced with theophylline. Combining the Tet transcriptional inducible promoter with the translational control of the riboswitch resulted in strong repression and allowed for the cloning and expression of the potent chlamydial regulatory protein, HctB. The ability to control the timing and strength of gene expression independently from promoter specificity is a new and important tool for studying chlamydial regulatory and virulence genes.

microbiology↗

bioRxiv2020

Cell Type Development in Chlamydia trachomatis Follows a Program Intrinsic to the Reticulate Body.

The obligate intracellular bacterial pathogen Chlamydia trachomatis (Ctr) is reliant on an unusual developmental cycle consisting of two cell forms termed the elementary body (EB) and the reticulate body (RB). The EB is infectious and utilizes a type III secretion system and preformed effector proteins during invasion, but does not replicate. The RB replicates in the host cell but is non-infectious. This developmental cycle is central to chlamydial pathogenesis. In this study we developed mathematical models of the chlamydial developmental cycle that account for potential factors influencing the timing of RB to EB cell type switching during infection. Our models predicted that two broad categories of regulatory signals for RB to EB development could be differentiated experimentally; an "intrinsic" cell autonomous program inherent to each RB or an "extrinsic" environmental signal to which RBs respond. To experimentally differentiate between these hypotheses, we tracked the expression of Ctr developmental specific promoters using fluorescent reporters and live cell imaging. These experiments indicated that EB production was not influenced by increased MOI or by superinfection, suggesting the cycle follows an intrinsic program that is not influenced by environmental factors. Additionally, live cell imaging of these promoter constructs revealed that EB development is a multistep process linked to RB growth rate and cell division. The formation of EBs followed a cell type gene expression progression with the promoters for euo and ihtA active in RBs, while the promoter for hctA was active in early EBs/intermediate cells and finally the promoters for the true late genes, hctB, scc2, and tarp active in the maturing EB. ImportanceChlamydia trachomatis is an obligate intracellular bacteria that can cause trachoma, cervicitis, urethritis, salpingitis, and pelvic inflammatory disease. To establish infection in host cells Chlamydia must complete a multi cell type developmental cycle. The developmental cycle consists of two specialized cells; the EB which mediates infection of new cells and the RB which replicates and eventually produces more EB cells to mediate the next round of infection. By developing and testing mathematical models to discriminate between two competing hypotheses for the nature of the signal controlling RB to EB cell type switching. We demonstrate that RB to EB development follows a cell autonomous program that does not respond to environmental cues. Additionally, we show that RB to EB development is a function of cell growth and cell division. This study serves to further our understanding of the chlamydial developmental cycle that is central to the bacteriums pathogenesis.

microbiology↗