Improved Quantification, Propagation, Purification and Storage of the Obligate Intracellular Human Pathogen Orientia tsutsugamushi

Abstract

BackgroundScrub typhus is a leading cause of serious febrile illness in rural Southeast Asia. The causative agent, Orientia tsutsugamushi, is an obligate intracellular bacterium that is transmitted to humans by the bite of a Leptotrombidium mite. Research into the basic mechanisms of cell biology and pathogenicity of O. tsutsugamushi has lagged behind that of other important human pathogens. One reason for this is that O. tsutsugamushi is an obligate intracellular bacterium that can only be cultured in mammalian cells and that requires specific methodologies for propagation and analysis. Here, we have performed a body of work designed to improve methods for quantification, propagation, purification and long-term storage of this important but neglected human pathogen. These results will be useful to other researchers working on O. tsutsugamushi and also other obligate intracellular pathogens such as those in the Rickettsiales and Chlamydiales families.MethodologyA clinical isolate of O. tsutsugamushi was grown in cultured mouse embryonic fibroblast (L929) cells. Bacterial growth was measured using an O. tsutsugamushi-specific qPCR assay. Conditions leading to improvements in viability and growth were monitored in terms of the effect on bacterial cell number after growth in cultured mammalian cells.Key resultsDevelopment of a standardised growth assay to quantify bacterial replication and viability in vitro.Quantitative comparison of different DNA extraction methods.Quantification of the effect on growth of FBS concentration, daunorubicin supplementation, media composition, host cell confluence at infection and frequency of media replacement.Optimisation of bacterial purification including a comparison of host cell lysis methods, purification temperature, bacterial yield calculations and bacterial pelleting at different centrifugation speeds.Quantification of bacterial viability loss after long term storage and freezing under a range of conditions including different freezing buffers and different rates of freezing.ConclusionsHere we present a standardised method for comparing the viability of O. tsutsugamushi after purification, treatment and propagation under various conditions. Taken together, we present a body of data to support improved techniques for propagation, purification and storage of this organism. This data will be useful both for improving clinical isolation rates as well as performing in vitro cell biology experiments.

Orientia tsutsugamushi Modulates Endoplasmic Reticulum-Associated Degradation To Benefit Its Growth

“…Strikingly, YFP-TCR␣ levels were relatively unchanged in cells that had been infected for 24 h and cycloheximide treated but were The timing of O. tsutsugamushi-mediated ERAD inhibition is critical for bacterial growth. O. tsutsugamushi population growth during the first 24 to 48 h of infection is minimal, but this is followed by logarithmic expansion (9). It is reasonable to presume that the bacterial demand for amino acids is initially low during the first 24 h but increases when the pathogen population prepares for/enters log-phase growth that initiates at or after 48 h. Coincident with such timing, O. tsutsugamushi induces the UPR and simultaneously inhibits ERAD during the first 48 h, which would stall production of ERAD-derived amino acids.…”

“…It is reasonable to presume that the bacterial demand for amino acids is initially low during the first 24 h but increases when the pathogen population prepares for/enters log-phase growth that initiates at or after 48 h. Coincident with such timing, O. tsutsugamushi induces the UPR and simultaneously inhibits ERAD during the first 48 h, which would stall production of ERAD-derived amino acids. By 72 h, when ERAD is no longer inhibited and amino acids generated by the degradative process would be present, the bacterium grows expansively (9). Moreover, amino acid supplementation restores O. tsutsugamushi growth in TUDCA-treated cells, but this complementation effect does not occur until 72 h. We therefore hypothesized that O. tsutsugamushi transiently inhibits ERAD during the first 24 h, when the pathogen does not require a large pool of amino acids, but it utilizes ERAD-derived amino acids to benefit its growth beginning between 48 and 72 h.…”

Evidence for a peptidoglycan‐like structure in Orientia tsutsugamushi

“…S7). This morphology may reflect a disruption in the cell envelope structure, which would be consistent with our previous report that the viability of isolated O. tsutsugamushi was higher when stored in SPG compared with PBS or water (Giengkam et al ., ).…”

“…Cell culture was performed using DMEM media with 10% FBS at 378C (uninfected cells) or 358C (infected cells) and 5% CO 2 . For routine propagation bacteria were grown in 25 or 75 cm 2 culture flasks as described previously (Giengkam et al, 2015). All experiments were performed using bacteria harvested from the intracellular fraction of infected cells, 7 days after infection.…”

“…Mass spectrometry analysis was performed on biomass extracted from between 15 and 45 ml infected cells, which had been infected at an MOI of 10:1 to 1000:1. Bacteria were grown in L929 cells for 7 days, then purified using mechanical lysis of host cells and filtering with a 2 mm filter (Giengkam et al, 2015). The sample was pelleted and washed in 600 ml of 300 mM ice-cold sucrose two times.…”