Identification of Functional Candidates amongst Hypothetical Proteins of Treponema pallidum ssp. pallidum

Syphilis is a globally occurring venereal disease, and its infection is propagated through sexual contact. The causative agent of syphilis, Treponema pallidum ssp. pallidum, a Gram-negative sphirochaete, is an obligate human parasite. Genome of T. pallidum ssp. pallidum SS14 strain (RefSeq NC_010741.1) encodes 1,027 proteins, of which 444 proteins are known as hypothetical proteins (HPs), i.e., proteins of unknown functions. Here, we performed functional annotation of HPs of T. pallidum ssp. pallidum using various database, domain architecture predictors, protein function annotators and clustering tools. We have analyzed the sequences of 444 HPs of T. pallidum ssp. pallidum and subsequently predicted the function of 207 HPs with a high level of confidence. However, functions of 237 HPs are predicted with less accuracy. We found various enzymes, transporters, binding proteins in the annotated group of HPs that may be possible molecular targets, facilitating for the survival of pathogen. Our comprehensive analysis helps to understand the mechanism of pathogenesis to provide many novel potential therapeutic interventions.     

Functional Annotation of Conserved Hypothetical Proteins from Haemophilus influenzae Rd KW20

Haemophilus influenzae is a Gram negative bacterium that belongs to the family Pasteurellaceae, causes bacteremia, pneumonia and acute bacterial meningitis in infants. The emergence of multi-drug resistance H. influenzae strain in clinical isolates demands the development of better/new drugs against this pathogen. Our study combines a number of bioinformatics tools for function predictions of previously not assigned proteins in the genome of H. influenzae. This genome was extensively analyzed and found 1,657 functional proteins in which function of 429 proteins are unknown, termed as hypothetical proteins (HPs). Amino acid sequences of all 429 HPs were extensively annotated and we successfully assigned the function to 296 HPs with high confidence. We also characterized the function of 124 HPs precisely, but with less confidence. We believed that sequence of a protein can be used as a framework to explain known functional properties. Here we have combined the latest versions of protein family databases, protein motifs, intrinsic features from the amino acid sequence, pathway and genome context methods to assign a precise function to hypothetical proteins for which no experimental information is available. We found these HPs belong to various classes of proteins such as enzymes, transporters, carriers, receptors, signal transducers, binding proteins, virulence and other proteins. The outcome of this work will be helpful for a better understanding of the mechanism of pathogenesis and in finding novel therapeutic targets for H. influenzae.     

Metabolic Responses to Orientia tsutsugamushi Infection in a Mouse Model

Tsutsugamushi disease is an infectious disease transmitted to humans through the bite of the Orientia tsutsugamushi-infected chigger mite; however, host-pathogen interactions and the precise mechanisms of damage in O. tsutsugamushi infections have not been fully elucidated. Here, we analyzed the global metabolic effects of O. tsutsugamushi infection on the host using ¹H-NMR and UPLC-Q-TOF mass spectroscopy coupled with multivariate statistical analysis. In addition, the effect of O. tsutsugamushi infection on metabolite concentrations over time was analyzed by two-way ANOVAs. Orthogonal partial least squares-discriminant analysis (OPLS-DA) showed distinct metabolic patterns between control and O. tsutsugamushi-infected mice in liver, spleen, and serum samples. O. tsutsugamushi infection caused decreased energy production and deficiencies in both remethylation sources and glutathione. In addition, O. tsutsugamushi infection accelerated uncommon energy production pathways (i.e., excess fatty acid and protein oxidation) in host body. Infection resulted in an enlarged spleen with distinct phospholipid and amino acid characteristics. This study suggests that metabolite profiling of multiple organ tissues and serum could provide insight into global metabolic changes and mechanisms of pathology in O. tsutsugamushi-infected hosts.     

Functional annotation of putative hypothetical proteins from Candida dubliniensis

An extensive analysis of C. dubliniensis proteomics data showed that ~ 22% protein are conserved hypothetical proteins (HPs) whose function is still not determined precisely. Analysis of gene sequence of HPs provides a platform to establish sequence–function relationships to a more profound understanding of the molecular machinery of organisms at systems level. Here we have combined the latest versions of bioinformatics tools including, protein family, motifs, intrinsic features from the amino acid sequence, sequence–function relationship, pathway analysis, etc. to assign a precise function to HPs for which no any experimental information is available. Our results show that 27 HPs have well defined functions and we categorized them as enzyme, nucleic acid binding, transport protein, etc. Five HPs showed adhesin character that is likely to be essential for the survival of yeast and pathogenesis. We also addressed issues related to the sub-cellular localization and signal peptide identification which provides an idea about its colocalization and function. The outcome of the present study may facilitate better understanding of mechanism of virulence, drug resistance, pathogenesis, adaptability to host, tolerance for host immune response, and drug discovery for treatment of C. dubliniensis infections.     

Dissemination of Orientia tsutsugamushi and Inflammatory Responses in a Murine Model of Scrub Typhus

Central aspects in the pathogenesis of scrub typhus, an infection caused by Orientia (O.) tsutsugamushi, have remained obscure. Its organ and cellular tropism are poorly understood.The purpose of this study was to analyze the kinetics of bacterial dissemination and associated inflammatory responses in infected tissues in an experimental scrub typhus mouse model, following infection with the human pathogenic strain Karp. We provide a thorough analysis of O. tsutsugamushi infection in inbred Balb/c mice using footpad inoculation, which is close to the natural way of infection. By a novel, highly sensitive qPCR targeting the multi copy traD genes, we quantitatively monitored the spread of O. tsutsugamushi Karp from the skin inoculation site via the regional lymph node to the internal target organs. The highest bacterial loads were measured in the lung. Using confocal imaging, we also detected O. tsutsugamushi at the single cell level in the lung and found a predominant macrophage rather than endothelial localization. Immunohistochemical analysis of infiltrates in lung and brain revealed differently composed lesions with specific localizations: iNOS-expressing macrophages were frequent in infiltrative parenchymal noduli, but uncommon in perivascular lesions within these organs. Quantitative analysis of the macrophage response by immunohistochemistry in liver, heart, lung and brain demonstrated an early onset of macrophage activation in the liver. Serum levels of interferon (IFN)-γ were increased during the acute infection, and we showed that IFN-γ contributed to iNOS-dependent bacterial growth control.Our data show that upon inoculation to the skin, O. tsutsugamushi spreads systemically to a large number of organs and gives rise to organ-specific inflammation patterns. The findings suggest an essential role for the lung in the pathogenesis of scrub typhus. The model will allow detailed studies on host-pathogen interaction and provide further insight into the pathogenesis of O. tsutsugamushi infection.     

Orientia tsutsugamushi Subverts Dendritic Cell Functions by Escaping from Autophagy and Impairing Their Migration

Background

Dendritic cells (DCs) are the most potent antigen-presenting cells that link innate and adaptive immune responses, playing a pivotal role in triggering antigen-specific immunity. Antigen uptake by DCs induces maturational changes that include increased surface expression of major histocompatibility complex (MHC) and costimulatory molecules. In addition, DCs actively migrate to regional lymph nodes and activate antigen-specific naive T cells after capturing antigens. We characterize the functional changes of DCs infected with Orientia tsutsugamushi, the causative agent of scrub typhus, since there is limited knowledge of the role played by DCs in O. tsutsugamushi infection.

Methodology/Principal Finding

O. tsutsugamushi efficiently infected bone marrow-derived DCs and induced surface expression of MHC II and costimulatory molecules. In addition, O. tsutsugamushi induced autophagy activation, but actively escaped from this innate defense system. Infected DCs also secreted cytokines and chemokines such as IL-6, IL-12, MCP5, MIP-1α, and RANTES. Furthermore, in vitro migration of DCs in the presence of a CCL19 gradient within a 3D collagen matrix was drastically impaired when infected with O. tsutsugamushi. The infected cells migrated much less efficiently into lymphatic vessels of ear dermis ex vivo when compared to LPS-stimulated DCs. In vivo migration of O. tsutsugamushi-infected DCs to regional lymph nodes was significantly impaired and similar to that of immature DCs. Finally, we found that MAP kinases involved in chemotactic signaling were differentially activated in O. tsutsugamushi-infected DCs.

Conclusion/Significance

These results suggest that O. tsutsugamushi can target DCs to exploit these sentinel cells as replication reservoirs and delay or impair the functional maturation of DCs during the bacterial infection in mammals.     

The Whole-genome Sequencing of the Obligate Intracellular Bacterium Orientia tsutsugamushi Revealed Massive Gene Amplification During Reductive Genome Evolution

Scrub typhus (‘Tsutsugamushi’ disease in Japanese) is a mite-borne infectious disease. The causative agent is Orientia tsutsugamushi, an obligate intracellular bacterium belonging to the family Rickettsiaceae of the subdivision alpha-Proteobacteria. In this study, we determined the complete genome sequence of O. tsutsugamushi strain Ikeda, which comprises a single chromosome of 2 008 987 bp and contains 1967 protein coding sequences (CDSs). The chromosome is much larger than those of other members of Rickettsiaceae, and 46.7% of the sequence was occupied by repetitive sequences derived from an integrative and conjugative element, 10 types of transposable elements, and seven types of short repeats of unknown origins. The massive amplification and degradation of these elements have generated a huge number of repeated genes (1196 CDSs, categorized into 85 families), many of which are pseudogenes (766 CDSs), and also induced intensive genome shuffling. By comparing the gene content with those of other family members of Rickettsiacea, we identified the core gene set of the family Rickettsiaceae and found that, while much more extensive gene loss has taken place among the housekeeping genes of Orientia than those of Rickettsia, O. tsutsugamushi has acquired a large number of foreign genes. The O. tsutsugamushi genome sequence is thus a prominent example of the high plasticity of bacterial genomes, and provides the genetic basis for a better understanding of the biology of O. tsutsugamushi and the pathogenesis of ‘Tsutsugamushi’ disease.Key words: Orientia tsutsugamushi, genome sequencing, obligate intracellular bacterium, repetitive sequence, IS element, integrative and conjugative element, gene amplification, genome reduction     

Phylogenetic clustering of 4 prevalent virulence genes in <Emphasis Type="Italic">Orientia tsutsugamushi</Emphasis> isolates from human patients

The pathogenicity of microbes is involved in many kinds of virulence genes. The relationships between these virulence genes and strains are not clear in Orientia tsutsugamushi yet. In this study, we confirmed the presence of the virulence genes and classified into O. tsutsugamushi isolates using phylogenetic analysis of the virulence genes. We also compared the fatality rates of every isolate via an infection experiment in BALB/c mice using the O. tsutsugamushi isolates, Deajeon03-01, Wonju03-01, and Muju03-01. Moreover, we compared the phylogenetic analysis, in basis with 56 kDa protein sequence which determined from serotype, and virulence genes of O. tsutsugamushi. Our results showed remarkably different fatality rates between Deajeon03-01 and Muju03-01, which are both Boryong strains of O. tsutsugamushi. Also, clustering analyses including these two isolates gave slightly different results depending on whether they were clustered based on virulence genes or on the 56 kDa protein sequences. Consequently, we conclude that fatality rates in O. tsutsugamushi are correlated with differences in both serotypes and virulence genes. We identified some variations within the virulence genes dnaA, virB8, tolR, and trxA among the isolates.     

Differences in clinical features according to Boryoung and Karp genotypes of Orientia tsutsugamushi

Background

Scrub typhus is an infectious disease caused by Orientia tsutsugamushi. The differences in virulence of O. tsutsugamushi prototypes in humans are still unknown. We investigated whether there are any differences in the clinical features of the Boryoung and Karp genotypes.

Methodology/Principal Findings

Patients infected with O. tsutsugamushi, as Boryoung and Karp clusters, who had visited 6 different hospitals in southwestern Korea were prospectively compared for clinical features, complications, laboratory parameters, and treatment responses. Infected patients in the Boryoung cluster had significantly more generalized weakness, eschars, skin rashes, conjunctival injection, high albumin levels, and greater ESR and fibrinogen levels compared to the Karp cluster. The treatment response to current antibiotics was significantly slower in the Karp cluster as compared to the Boryoung cluster.

Conclusion

The frequency of occurrence of eschars and rashes may depend on the genotype of O. tsutsugamushi.     

Proposed vector candidate: Leptotrombidium palpale for Shimokoshi type Orientia tsutsugamushi

To identify the vector species for Shimokoshi type Orientia tsutsugamushi, a survey of larval trombiculid mites was conducted in Yamagata Prefecture, Japan from April to May 2012. In all, 2889 larval trombiculid mites were obtained from 21 Apodemus speciosus rodent hosts, 2600 of which were morphologically classified into eight species in three genera. After screening of O. tsutsugamushi DNA in individual larval trombiculid mites using real‐time PCR targeting the 16S ribosomal RNA gene, serotype‐specific nested PCRs targeting the 56 kDa protein gene were performed, followed by sequencing analysis. As a result, Shimokoshi type O. tsutsugamushi DNA was identified from 3 (1.9%) of 157 Leptotrombidium palpale. This is the first study to identify Shimokoshi type O. tsutsugamushi DNA in L. palpale. The results indicate that L. palpale is a possible vector for Shimokoshi type O. tsutsugamushi.     

Seroepidemiological Survey of Zoonotic Diseases in Small Mammals with PCR Detection of Orientia tsutsugamushi in Chiggers,Gwangju, Korea

Serosurveillance for zoonotic diseases in small mammals and detection of chiggers, the vector of Orientia tsutsugamushi, were conducted from September 2014 to August 2015 in Gwangju Metropolitan Area. Apodemus agrarius was the most commonly collected small mammals (158; 91.8%), followed by Myodes regulus (8; 4.6%), and Crocidura lasiura (6; 3.5%). The highest seroprevalence of small mammals for O. tsutsugamushi (41; 26.3%) was followed by hantaviruses (24; 15.4%), Rickettsia spp. (22; 14.1%), and Leptospira (2; 1.3%). A total of 3,194 chiggers were collected from small mammals, and 1,236 of 3,194 chiggers were identified with 7 species of 3 genera: Leptotrombidium scutellare was the most commonly collected species (585; 47.3%), followed by L. orientale (422; 34.1%), Euchoengastia koreaensis (99; 8.0%), L. palpale (58; 4.7%), L. pallidum (36; 2.9%), Neotrombicula gardellai (28; 2.3%), and L. zetum (8; 0.6%). L. scutellare was the predominant species. Three of 1,236 chigger mites were positive for O. tsutsugamushi by PCR. As a result of phylogenetic analysis, the O. tsutsugamushi strain of chigger mites had sequence homology of 90.1-98.2% with Boryong. This study provides baseline data on the distribution of zoonotic diseases and potential vectors for the development of prevention strategies of vector borne diseases in Gwangju metropolitan area.     

Monitoring Chigger Mites for Orientia tsutsugamushi in Field Small Mammals in Hwaseong-si,Gyeonggi-do,Korea, 2019-2020

Incidence of tsutsugamushi disease (scrub typhus) caused by Orientia tsutsugamushi, is steadily increasing. It is a mite-borne disease transmitted by chigger mites. In this study, the chigger mites were collected from field small mammals in Hwaseong-si (city), Gyeonggi-do (province), Korea, 2019 and 2020. The field small mammals captured were 56 Apodemus agrarius (94.9%) and 3 Crocidura lasiura (5.1%). A total of 7,531 chigger mites were collected from the captured small mammals. Using PCR test, 153 chigger mite pools were examined and 17 pools were reported positive for O. tsutsugamushi. The O. tsutsugamushi were identified to 5 strains; Jecheon strain was most prevalent, followed by Boryong strain. The other strains were OI011, Taguchi, and Shimokoshi. Collectively, these results provide essential regional information on mite-borne tsutsugamushi disease in the Hwaseong-si, and further contribute to bring awareness and rapid diagnosis for the tsutsugamushi disease.     

The intracellular pathogen Orientia tsutsugamushi responsible for scrub typhus induces lipid droplet formation in mouse fibroblasts

Mammalian cells store excess fatty acids in the form of triglycerides within lipid droplets. The intracellular bacterium Orientia tsutsugamush is the causative agent of severe human rickettiosis. We found that O. tsutsugamushi infection induces the formation of lipid droplets in mouse L-929 fibroblasts. In infected cells, a parallel increase in the number of lipid droplets and pathogens was observed. Interestingly, the pathogen-infection induced the accumulation of triglycerides even without external supply of fatty acids. These results suggest that O. tsutsugamushi alters lipid metabolism of host cells to induce lipid droplets.     

Scrub Typhus,a Disease with Increasing Threat in Guangdong,China

There has been a rapid increase in the number of scrub typhus cases in Guangdong Province, China. For this reason, an epidemiologic study was conducted to understand the characteristics of scrub typhus epidemics in Guangdong. From 2006 to 2013, the incidence of human cases increased from 0.4321 to 3.5917 per 100,000 with a bimodal peak in human cases typically occurring between May and November. To detect the prevalence of Orientia tsutsugamushi among suspected human cases and rodents, we performed ELISA tests of IgM/IgG and nested PCR tests on 59 whole blood samples from the suspected cases and 112 spleen samples from the rodents. Suspected cases tested positive for anti-O. tsutsugamushi IgM and IgG 66.1% (39/59) and 50.8% (30/59) of the time, respectively. Additionally, 20.3% (12/59) of blood samples and 13.4% (15/112) of spleen samples were positive for PCR. Phylogenetic analysis revealed that there were four definable clusters among the 27 nucleotide sequences of the 56-kDa antigen genes: 44.4% Karp (12/27), 25.9% Kato (7/27), 22.2% Gilliam (6/27) and 7.4% TA763 (2/27). We concluded many suspected cases may result in diagnostic errors; therefore, it is necessary to perform laboratory tests on suspected cases in hospitals. The high infection rate of O. tsutsugamushi among the limited rodents tested suggested that further rodent sampling throughout the province is necessary to further define high-risk areas. Furthermore, the multiple co-circulating genotypes of O. tsutsugamushi play a key role in the pervasiveness of scrub typhus in the Guangdong area.     

A Hematogenously Disseminated Orientia tsutsugamsushi-Infected Murine Model of Scrub Typhus

Orientia tsutsugamushi, the etiologic agent of scrub typhus, is a mite-borne rickettsia transmitted by the parasitic larval stage of trombiculid mites. Approximately one-third of the world''s population is at risk of infection with Orientia tsutsugamushi, emphasizing its importance in global health. In order to study scrub typhus, Orientia tsutsugamushi Karp strain has been used extensively in mouse studies with various inoculation strategies and little success in inducing disease progression similar to that of human scrub typhus. The objective of this project was to develop a disease model with pathology and target cells similar to those of severe human scrub typhus. This study reports an intravenous infection model of scrub typhus in C57BL/6 mice. This mouse strain was susceptible to intravenous challenge, and lethal infection occurred after intravenous inoculation of 1.25×10⁶ focus (FFU) forming units. Signs of illness in lethally infected mice appeared on day 6 with death occurring ∼6 days later. Immunohistochemical staining for Orientia antigens demonstrated extensive endothelial infection, most notably in the lungs and brain. Histopathological analysis revealed cerebral perivascular, lymphohistiocytic infiltrates, focal hemorrhages, meningoencephalitis, and interstitial pneumonia. Disseminated infection of endothelial cells with Orientia in C57BL/6 mice resulted in pathology resembling that of human scrub typhus. The use of this model will allow detailed characterization of the mechanisms of immunity to and pathogenesis of O. tsutsugamushi infection.     

Development of Recombinase Polymerase Amplification Assays for Detection of Orientia tsutsugamushi or Rickettsia typhi

Sensitive, specific and rapid diagnostic tests for the detection of Orientia tsutsugamushi (O. tsutsugamushi) and Rickettsia typhi (R. typhi), the causative agents of scrub typhus and murine typhus, respectively, are necessary to accurately and promptly diagnose patients and ensure that they receive proper treatment. Recombinase polymerase amplification (RPA) assays using a lateral flow test (RPA-nfo) and real-time fluorescent detection (RPA-exo) were developed targeting the 47-kDa gene of O. tsutsugamushi or 17 kDa gene of R. typhi. The RPA assay was capable of detecting O. tsutsugamushi or R. typhi at levels comparable to that of the quantitative PCR method. Both the RPA-nfo and RPA-exo methods performed similarly with regards to sensitivity when detecting the 17 kDa gene of R. typhi. On the contrary, RPA-exo performed better than RPA-nfo in detecting the 47 kDa gene of O. tsutsugamushi. The clinical performance of the O. tsutsugamushi RPA assay was evaluated using either human patient samples or infected mouse samples. Eight out of ten PCR confirmed positives were determined positive by RPA, and all PCR confirmed negative samples were negative by RPA. Similar results were obtained for R. typhi spiked patient sera. The assays were able to differentiate O. tsutsugamushi and R. typhi from other phylogenetically related bacteria as well as mouse and human DNA. Furthermore, the RPA-nfo reaction was completed in 20 minutes at 37^oC followed by a 10 minute incubation at room temperature for development of an immunochromatographic strip. The RPA-exo reaction was completed in 20 minutes at 39^oC. The implementation of a cross contamination proof cassette to detect the RPA-nfo fluorescent amplicons provided an alternative to regular lateral flow detection strips, which are more prone to cross contamination. The RPA assays provide a highly time-efficient, sensitive and specific alternative to other methods for diagnosing scrub typhus or murine typhus.     

Isolation of a New Orientia tsutsugamushi Serotype

Orientia tsutsugamushi, the etiological agent of scrub typhus, is an antigenically diverse organism and many serologically distinct strains have been identified. The 56 kDa protein of O. tsutsugamushi, a major protein in the outer membrane, has been thought to be responsible for this antigenic variability. A strain of O. tsutsugamushi isolated in Korea cross-reacted with both Gilliam strain-specific and Karp strain-specific monoclonal antibodies. When its 56 kDa protein gene was cloned and analyzed, its sequence showed variation especially between 1,200 and 1,250 bp, showing that this isolate is a new O. tsutsugamushi strain.