Dissecting the Developmental Life Cycle and Developing Genetic Tools for Understanding the Biology of <i>Orientia tsutsugamushi</i>

Orientia tsutsugamushi is an obligate intracellular bacterium that is spread by mites and causes a life-threatening human disease, scrub typhus. This disease affects at least one million people annually with a high risk of mortality if not treated promptly. Orientia is poorly understood compared to many other pathogens due to genetic intractability. This bacterium only propagates and replicates within host cells. they are predominantly found in endothelial, dendritic, and monocyte/macrophage cells, and stay inside infected cells for seven days or longer before exiting by a budding mechanism. The developmental differentiation of the intracellular infection cycle of Orientia has not been studied previously. My thesis research initially focused on how Orientia differentiates into distinct subpopulations during the infection cycle, how distinct subpopulations of Orientia affect the infection in host cells, and different protein profiles of bacteria in distinct stages during the infection cycle. My research demonstrates that O. tsutsugamushi differentiates into five distinct subpopulations: early entry, pre-replicative, replicative, maturation, and extracellular, representing a new model for developmental differentiation in the intracellular cycle of Orientia tsutsugamushi. Each subpopulation relates to different degrees of metabolic activity, replication, infectivity, including morphology, presence of marker gene, and subcellular localization. The transition between the subpopulations likely results from an integration of signals from the host cell environment and the bacteria which leads to morphological and physiological changes through the regulation of genes and proteins. This work allows us to understand the fundamentals of bacterial development and the regulation mechanism of differentiation of Orientia tsutsugamushi, which could be beneficial for the improvement of diagnosis and treatment. Genetic tools have not been developed previously for Orientia, but genetic manipulation in the most closely related bacteria, rickettsial species, has been successfully established in the last decade. My work on genetic manipulation of Orientia is described in the second part of this thesis. The transposon mutagenesis vector and nucleotide analog (BNA) were used to manipulate the genome of Orientia. Unfortunately, the transposon mutagenesis system failed to integrate into the Orientia genome, whereas BNA was able to down regulate the expression of the targeted protein (TSA56). Using a nucleotide analog to target a specific gene is the first step to manipulate the genome of Orientia, and this has led to the study of other targeted genes involving virulence and pathogenicity. Due to the genetic intractability of Orientia that limits the molecular dissection of bacteria pathogenesis, virulence, and host-pathogen interaction, sequencing technologies provide a promising way to understand the molecular processes of the disease. This motivated the third part of this thesis, in which dual RNA-seq was applied to two clinical isolates grown in cultured HUVEC cells. This study revealed the transcriptomic profile of Orientia and identified the different immune response networks in response to each strain. Differential activation of the immune response in cultured cells between the two strains was shown to correlate with differences in virulence as measured in a mouse model of infection. These findings will help to understand the mechanism of bacterial pathogenesis in Orientia tsutsugamushi and may be used for characterization of other genetically intractable bacterial pathogens

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

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

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

List of primers

<p>List of primers</p

Gel mobility shift assays and DNase I footprinting of EstR bound to the <i>estR-estC</i> promoters.

<p>A, Gel mobility shift assays of the [³²P]-labeled 314-bp DNA fragment spanning the <i>estR</i> and <i>estC</i> promoters with purified EstR protein (0–125 nM) were performed. The complexes were separated on native PAGE. The specificity of the binding was validated by adding the cold DNA fragment (CD) to the binding mixture or adding bovine serum albumin (BSA) to the reaction instead of the EstR protein. CHP represents the bound complexes treated with 1 mM CHP. B and F indicate bound and free probes, respectively. B, DNase I protection assays were performed with the [³²P]- labeled <i>estR-estC</i> promoter fragment and purified EstR protein at the indicated concentrations. The digested and protected DNA fragments were separated on 8% denaturing DNA sequencing gels beside the sequence ladders (G, A, T, C) generated from pGEM-3Zf (+) using the pUC/M13 forward primer. The numbers on the left side are the size in base pairs of the bands. C, The depicted sequence shows the <i>estR-estC</i> promoter region. +1 and Met indicate the transcription and translation start sites. The -10 and -35 regions are underlined. RBS represents the ribosome binding site. The sequences corresponding to the sites of OI and OII protection are shaded. Arrows indicate palindromic sequences. Small letters above the sequence line represent the putative EstR binding box derived from site OI protection. Identical nucleotides are marked by asterisks.</p

Multiple alignments of Atu5211 (EstR) and Atu5212 (EstC).

<p>A, The deduced amino acid sequence of Atu5211 (EstR) was aligned with OhrR from <i>Xanthomonas campestris</i> pv. phaseoli (OhrR_Xcp), <i>A</i>. <i>tumefaciens</i> (OhrR_Atu) and <i>Bacillus subtilis</i> (OhrR_Bsu). Cysteine residues are marked by arrow heads. B, A phylogenetic tree constructed from the amino acid sequences of transcriptional regulators belonging to MarR super family. EstR_Atu, <i>A</i>. <i>tumefaciens</i> EstR; OhrR_Xcp, <i>Xanthomonas campestris</i> pv. phaseoli OhrR (AAK62673); OhrR_Bsu, <i>Bacillus subtilis</i> OhrR (NP_389198); OhrR_Sco, <i>Streptomyces coelicolor</i> OhrR (CAB87337); OhrR_Atu, <i>Agrobacterium tumefaciens</i> OhrR (AAK86653); ExpG_Sme, <i>Sinorhizobium meliloti</i> ExpG (CAB01941); MepR_Sau, <i>Staphylococcus aureus</i> MepR (AAU95767), GraR_Rhi, <i>Rhizobium sp</i> GraR (BAF44528); TcaR_Sau, <i>Staphylococcus aureus</i> TcaR (AAG23887); ScoC_Bsu, <i>Bacillus subtilis</i> ScoC (NP_388880); PecS_Ech, <i>Erwinia chrysanthemi</i> PecS (CAA52427); HucR_Dra, <i>Deinococcus radiodurans</i> HucR (NP_294883); MexR_Pae, <i>Pseudomonas aeruginosa</i> MexR (AAO40258); MarR_Eco, <i>Escherichia coli</i> MarR (ABE11597); SlyA_Sty, <i>Salmonella typhimurium</i> SlyA (NP_460407); SlyA_Efa, <i>Enterococcus faecalis</i> SlyA (NP_816617); MobR_Cte, <i>Comamonas testosterone</i> MobR (BAF34929); BadR_Rpa, <i>Rhodopseudomonas palustris</i> BadR (NP_946008); MoaI_Kae, <i>Klebsiella aerogenes</i> MoaI (BAA09790); and HpcR_Eco, <i>Escherichia coli</i> HpcR (AAB25801). C, The deduced amino acid sequence of Atu5212 (EstC) was aligned with EstC from <i>Burkholderia gladioli</i> EstC [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168791#pone.0168791.ref038" target="_blank">38</a>]. The Ser-His-Asp catalytic triad residues are indicated by asterisks. The bar above the sequences represents the esterase-lipase active domain. Alignment was performed using ClustalW [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168791#pone.0168791.ref032" target="_blank">32</a>].</p

Phenotypic analysis of <i>A</i>. <i>tumefaciens</i> NTL4 and derivatives.

<p>The oxidant resistance levels of <i>A</i>. <i>tumefaciens</i> NTL4, the <i>estC</i> mutant, the complemented <i>estC</i> mutant (<i>estC</i>/pEstC), NTL4 harboring pEstC (NTL4/pEstC), the <i>estR</i> mutant, and the complemented <i>estR</i> mutant (<i>estR</i>/pEstR) were determined using plate sensitivity assays as described in the Methods. The concentrations of oxidant used are 0.25 mM CHP, 1.2 mM BHP, 0.35 mM H₂O₂, and 0.55 mM MD. The survival colonies were counted after 24 h incubation at 30°C. The surviving fraction is defined as the number of colony forming units (CFU) on plates containing oxidant divided by the number of CFU on plates without oxidant.</p

Esterase activity in <i>A</i>. <i>tumefaciens</i> NTL4 and derivatives.

<p>The esterase activity in NTL4 and the <i>estC</i> mutant strains harboring the pBBR1MCS-5 vector control (pBBR), pEstC or pEstC_S101A was assayed in crude lysates prepared from exponential phase cultures. A unit of esterase activity is defined as the amount of enzyme capable of hydrolyzing <i>p</i>-nitrophenyl butyrate to generate 1 μmol of <i>p</i>-nitrophenol at 25°C. Asterisks indicate a significant difference (<i>P</i> < 0.05) from NTL4 or the <i>estC</i> mutant harboring vector control.</p

Gene organization of the <i>estC</i> locus in various bacteria.

<p>The arrow indicates the orientation of the transcription. The number in the arrows represents percentage of identity of each sequence to the corresponding <i>A</i>. <i>tumefaciens</i> genes. The asterisk indicates a truncated gene. <i>scd</i>, short chain dehydrogenase; <i>zdh</i>, zinc dehydrogenase; <i>cdh</i>, choline dehydrogenase; and <i>gst</i>, glutathione S-transferase.</p