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

IMPORTANCE: Obligate intracellular bacteria, or those only capable of growth inside other living cells, have limited opportunities for horizontal gene transfer with other microbes due to their isolated replicative niche. The human pathogen Ot, an obligate intracellular bacterium causing scrub typhus, encodes an unusually high copy number of a ~40 gene mobile genetic element that typically facilitates genetic transfer across microbes. This proliferated element is heavily degraded in Ot and previously assumed to be inactive. Here, we conducted a detailed analysis of this element in eight Ot strains and discovered two strains with at least one intact copy. This implies that the element is still capable of moving across Ot populations and suggests that the genome of this bacterium may be even more dynamic than previously appreciated. Our work raises questions about intracellular microbial evolution and sounds an alarm for gene-based efforts focused on diagnosing and combatting scrub typhus.

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 Originally, the heavily degraded Ot RAGEs led to speculation that they are remnants of historical ICEs that are no longer active.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.

The obligate intracellular bacterium Orientia tsutsugamushi differentiates into a developmentally distinct extracellular state.

Orientia tsutsugamushi (Ot) is an obligate intracellular bacterium in the family Rickettsiaceae that causes scrub typhus, a severe mite-borne human disease. Its mechanism of cell exit is unusual amongst Rickettsiaceae, as Ot buds off the surface of infected cells enveloped in plasma membrane. Here, we show that Ot bacteria that have budded out of host cells are in a distinct developmental stage compared with intracellular bacteria. We refer to these two stages as intracellular and extracellular bacteria (IB and EB, respectively). These two forms differ in physical properties: IB is both round and elongated, and EB is round. Additionally, IB has higher levels of peptidoglycan and is physically robust compared with EB. The two bacterial forms differentially express proteins involved in bacterial physiology and host-pathogen interactions, specifically those involved in bacterial dormancy and stress response, and outer membrane autotransporter proteins ScaA and ScaC. Whilst both populations are infectious, entry of IB Ot is sensitive to inhibitors of both clathrin-mediated endocytosis and macropinocytosis, whereas entry of EB Ot is only sensitive to a macropinocytosis inhibitor. Our identification and detailed characterization of two developmental forms of Ot significantly advances our understanding of the intracellular lifecycle of an important human pathogen.

Dual RNA-seq of Orientia tsutsugamushi informs on host-pathogen interactions for this neglected intracellular human pathogen.

Studying emerging or neglected pathogens is often challenging due to insufficient information and absence of genetic tools. Dual RNA-seq provides insights into host-pathogen interactions, and is particularly informative for intracellular organisms. Here we apply dual RNA-seq to Orientia tsutsugamushi (Ot), an obligate intracellular bacterium that causes 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 to study the transcriptional architecture of Ot, we find evidence for wide-spread post-transcriptional antisense regulation. Comparing the host response to two clinical isolates, we identify distinct immune response networks for each strain, leading to predictions of relative virulence that are validated 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.

Clickable methionine as a universal probe for labelling intracellular bacteria.

Despite their clinical and biological importance, the cell biology of obligate intracellular bacteria is less well understood than that of many free-living model organisms. One reason for this is that they are mostly genetically intractable. As a consequence, it is not possible to engineer strains expressing fluorescent proteins and therefore fluorescence light microscopy - a key tool in host-pathogen cell biology studies - is difficult. Strain diversity also limits the universality of antibody-based immunofluorescence approaches. Here, we have developed a universal labelling protocol for intracellular bacteria based on a clickable methionine analog. Whilst we have applied this to obligate intracellular bacteria, we expect it to be useful for labelling free living bacteria as well as other intracellular pathogens.

Evidence for a peptidoglycan-like structure in Orientia tsutsugamushi.

Bacterial cell walls are composed of the large cross-linked macromolecule peptidoglycan, which maintains cell shape and is responsible for resisting osmotic stresses. This is a highly conserved structure and the target of numerous antibiotics. Obligate intracellular bacteria are an unusual group of organisms that have evolved to replicate exclusively within the cytoplasm or vacuole of a eukaryotic cell. They tend to have reduced amounts of peptidoglycan, likely due to the fact that their growth and division takes place within an osmotically protected environment, and also due to a drive to reduce activation of the host immune response. Of the two major groups of obligate intracellular bacteria, the cell wall has been much more extensively studied in the Chlamydiales than the Rickettsiales. Here, we present the first detailed analysis of the cell envelope of an important but neglected member of the Rickettsiales, Orientia tsutsugamushi. This bacterium was previously reported to completely lack peptidoglycan, but here we present evidence supporting the existence of a peptidoglycan-like structure in Orientia, as well as an outer membrane containing a network of cross-linked proteins, which together confer cell envelope stability. We find striking similarities to the unrelated Chlamydiales, suggesting convergent adaptation to an obligate intracellular lifestyle.

Agrobacterium tumefaciens estC, Encoding an Enzyme Containing Esterase Activity, Is Regulated by EstR, a Regulator in the MarR Family.

Analysis of the A. tumefaciens genome revealed estC, which encodes an esterase located next to its transcriptional regulator estR, a regulator of esterase in the MarR family. Inactivation of estC results in a small increase in the resistance to organic hydroperoxides, whereas a high level of expression of estC from an expression vector leads to a reduction in the resistance to organic hydroperoxides and menadione. The estC gene is transcribed divergently from its regulator, estR. Expression analysis showed that only high concentrations of cumene hydroperoxide (CHP, 1 mM) induced expression of both genes in an EstR-dependent manner. The EstR protein acts as a CHP sensor and a transcriptional repressor of both genes. EstR specifically binds to the operator sites OI and OII overlapping the promoter elements of estC and estR. This binding is responsible for transcription repression of both genes. Exposure to organic hydroperoxide results in oxidation of the sensing cysteine (Cys16) residue of EstR, leading to a release of the oxidized repressor from the operator sites, thereby allowing transcription and high levels of expression of both genes. The estC is the first organic hydroperoxide-inducible esterase-encoding gene in alphaproteobacteria.

Live imaging of the genetically intractable obligate intracellular bacteria Orientia tsutsugamushi using a panel of fluorescent dyes.

Our understanding of the molecular mechanisms of bacterial infection and pathogenesis are disproportionally derived from a small number of well-characterised species and strains. One reason for this is the enormous time and resources required to develop a new organism into experimental system that can be interrogated at the molecular level, in particular with regards to the development of genetic tools. Live cell imaging by fluorescence microscopy is a powerful technique to study biological processes such as bacterial motility, host cell invasion, and bacterial growth and division. In the absence of genetic tools that enable exogenous expression of fluorescent proteins, fluorescent chemical probes can be used to label and track living cells. A large number of fluorescent chemical probes are commercially available, but these have overwhelmingly been applied to the study of eukaryotic cell systems. Here, we present a methodical analysis of four different classes of probes, which can be used to delineate the cytoplasm, nucleic acids, cell membrane or peptidoglycan of living bacterial cells. We have tested these in the context of the important but neglected human pathogen Orientia tsutsugamushi but expect that the methodology would be broadly applicable to other bacterial species.

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

BACKGROUND: Scrub 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. METHODOLOGY: A 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 RESULTS: Development 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. CONCLUSIONS: Here 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.

Regulation by SoxR of mfsA, Which Encodes a Major Facilitator Protein Involved in Paraquat Resistance in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia MfsA (Smlt1083) is an efflux pump in the major facilitator superfamily (MFS). Deletion of mfsA renders the strain more susceptible to paraquat, but no alteration in the susceptibility levels of other oxidants is observed. The expression of mfsA is inducible upon challenge with redox cycling/superoxide-generating drug (paraquat, menadione and plumbagin) treatments and is directly regulated by SoxR, which is a transcription regulator and sensor of superoxide-generating agents. Analysis of mfsA expression patterns in wild-type and a soxR mutant suggests that oxidized SoxR functions as a transcription activator of the gene. soxR (smlt1084) is located in a head-to-head fashion with mfsA, and these genes share the -10 motif of their promoter sequences. Purified SoxR specifically binds to the putative mfsA promoter motifs that contain a region that is highly homologous to the consensus SoxR binding site, and mutation of the SoxR binding site abolishes binding of purified SoxR protein. The SoxR box is located between the putative -35 and -10 promoter motifs of mfsA; and this position is typical for a promoter in which SoxR acts as a transcriptional activator. At the soxR promoter, the SoxR binding site covers the transcription start site of the soxR transcript; thus, binding of SoxR auto-represses its own transcription. Taken together, our results reveal for the first time that mfsA is a novel member of the SoxR regulon and that SoxR binds and directly regulates its expression.