Targeting metabolic pathways proteins of Orientia tsutsugamushi using combined hierarchical approach to combat scrub typhus

Orientia tsutsugamushi (Ott) is a causative agent of chigger‐borne zoonosis, scrub typhus which is life threatening and highly pervasive illness in humans. In this report, we have mined and classified the proteins involved in pathways unique to Ott by using high‐throughput computational techniques. The 12 metabolic pathways were found to be unique to the pathogen. Forty‐six proteins were reported to be essential for the pathogen's survival and non‐homologous to the humans. The proteins were categorized into different classes, ie, enzymes, transporters, DNA‐binding, secretory, and outer membrane proteins. Further, in silico analysis of 46 proteins showed that 25 proteins were suitable therapeutic targets with known druggable properties. The structural modeling of B3CSG3 (MurA) protein was carried out and catalytic site essential for its functioning was analyzed. Virtual screening of chemical compounds was performed against modeled structure. The docking study by AutodockVina reported compound from PubChem with CID: 16036947 as best and potential inhibitor by means of docking score and binding affinity. The reliability and stability of the MurA‐16036947 complex were confirmed with molecular dynamics simulation. The report will provide insight to understand the mechanism of pathogenesis of Ott and instigate the development of effective treatment strategies against this disease.     

Genetic typing of the 56-kDa type-specific antigen gene of contemporary Orientia tsutsugamushi isolates causing human scrub typhus at two sites in north-eastern and western Thailand

Orientia tsutsugamushi is the causative agent of scrub typhus, a major cause of febrile illness in the rural areas of Southeast Asia. Twenty-three strains of O. tsutsugamushi were isolated from patients with scrub typhus in north-east (Udorn Thani province) and western Thailand (Tak province) between 2003 and 2005. The isolates were characterized by sequencing the entire ORF of the 56-kDa-type-specific antigen gene, followed by phylogenetic analysis. The majority (15/23) of isolates clustered with the Karp-type strain, six with a Gilliam-type strain and one each with the TA716- and TA763-type strains. Overall, there was considerable diversity in sequence, comparable to that seen in strains from across the rest of the scrub typhus-endemic world. There was no significant difference in the distributions of strains between the two provinces (P=0.08, Fisher's exact) nor a temporal change in distribution with year of isolation (P=0.80, Fisher's exact). Within this diversity there were also examples of isolates with identical 56-kDa genotypes that were cultured from patients from the same geographical areas.     

Virulence in Mice of Orientia tsutsugamushi Isolated from Patients in a New Endemic Area in Japan

Four strains of Orientia tsutsugamushi (KN-1, KN-2, KN-3 and GJ-1) isolated from patients in an area of Gifu Prefecture, Japan, in which tsutsugamushi disease is newly endemic, were examined for their virulence in mice. Among these, KN-1 (identified as Kawasaki type), GJ-1 (identified as Kuroki type) and KN-2 strains were found to be non-lethal for BALB/c mice as well as CH3/HeJ mice, even with high doses (10⁶ × being the 50% mouse infectious dose). On the other hand, the KN-3 strain was found to be sufficiently virulent to kill BALB/c mice. Among the prototype strains (Gilliam, Karp and Kato), the Karp and Kato strains exhibited high virulence to mice, while the Gilliam strain killed only a susceptible strain of mouse. BALB/c mice infected with KN-1 and KN-2 strains showed significant splenomegaly and moderate ascites accumulation in the first week of infection, while these symptoms became prominent during the second week of infection using KN-3, Karp and Kato strains. After infection with the GJ-1 strain, these symptoms were not observed. Antibody responses induced by infections with highly virulent strains were lower than that with low or intermediate virulent strains.     

Chemokine and cytokine production in susceptible C3H/HeN mice and resistant BALB/c mice during Orientia tsutsugamushi infection

To understand the pathogenesis of scrub typhus, we examined chemokine and cytokine production in susceptible (C3H/HeN) and resistant (BALB/c) mice after infection with O. tsutsugamushi Gilliam. C3H/HeN mice produced high levels of chemokines macrophage inflammatory proteins 1 alpha (MIP-1 alpha ), MIP-2, monocyte chemoattractant protein 1 (MCP-1), and cytokines gamma-interferon (IFN-gamma ), interleukin-12 (IL-12), IL-10, and tumor necrosis factor alpha (TNF-alpha ) in response to O. tsutsugamushi infection, compared to BALB/c mice. Chemokine profiles in infected mice correlated well with the kinetics of inflammatory cell infiltration. Hyperproduction of chemokines and cytokines was observed in another susceptible-infection model (BALB/c-Karp). These results suggest that hyperproduction of chemokines and cytokines are associated with susceptibility during O. tsutsugamushi infection.     

我国辽宁，吉林地区恙虫病东方体分离株的PCR分型

为鉴定我国东北地区恙虫病东方体(Ot)型别,本文应用Ot外膜主要蛋白型特异抗原56ku构建的群、型引物,采用PCR技术对分离自辽宁、吉林地区的6株恙虫病东方体目的基因进行分析研究,并与Gilliam,Karp,Kato国际参考株进行比较.结果表明,辽宁、吉林恙虫病东方体分离株至少存在Gilliam和Karp2种型别.     

Scrub typhus caused by Shimokoshi type Orientia tsutsugamushi showing variant 56‐kDa type‐specific antigen gene sequence in Tohoku region,Japan

In 2018, a patient was diagnosed with Shimokoshi type scrub typhus in Yamagata Prefecture, Japan. The causative pathogen was likely a variant type because 43 (8.3%) of 521 deduced amino acid sequences of the 56‐kDa type‐specific antigen (TSA) were different from those of the Shimokoshi prototype strain. The patient's paired sera showed low antibody titers against the Shimokoshi prototype strain. Two cases of scrub typhus reported in the Tohoku region during 2011–2012 also involved the same 56‐kDa TSA gene sequence. These findings suggest the presence of diversity in Shimokoshi type Orientia tsutsugamushi, which may impede the laboratory diagnosis of scrub typhus.     

Prokaryotic expression and immunogenicity of 56-kDa protein of Orientia tsutsugamushi strain Karp

To express the 56-kDa protein of O. tsutsugamushi strain Karp, this protein gene was cloned into pET30a(+) before transforming into host bacteria, E. coli Rossetta. Specificity of the recombinant protein was assessed by ELISA using rabbit sera against common members of the order Rickettsiae and 10 other pathogenic bacteria. After IPTG induction, SDS-PAGE analysis of isolated protein demonstrated a band at approximately 46-kDa. Western blot and mass spectrometry analysis proved that the recombinant protein was expressed successfully. Specificity analysis demonstrated that all sera were negative, except sera against O. tsutsugamushi strains TA763, TH1817 and Kato, B. quintana, A. phagocytophilum, E. chaffeensis and B. bacilliformis. The purified protein was used to immunize BALB/c mice and polyclonal antisera were harvested. By examination of IFA and ELISA, the highest titer of the polyclonal antibodies reaches 1:1600. The recombinant 56-kDa protein in the study is valuable for developing a simple and rapid diagnostic test and vaccine for O. tsutsugamushi.     

Identification of potential hosts and vectors of scrub typhus and tick-borne spotted fever group rickettsiae in eastern Taiwan

Scrub typhus and tick-borne spotted fever group (SFG) rickettsioses are transmitted by chiggers (larval trombiculid mites) and hard ticks, respectively. We assessed exposure to these disease vectors by extensively sampling both chiggers and ticks and their small mammal hosts in eastern Taiwan during 2007 and 2008. The striped field mouse Apodemus agrarius Pallas (Rodentia: Muridae) was the most common of the small mammals (36.1% of 1393 captures) and presented the highest rate of infestation with both chiggers (47.8% of 110 760) and ticks (78.1% of 1431). Leptotrombidium imphalum Vercammen-Grandjean & Langston (Trombidiformes: Trombiculidae) and immature Rhipicephalus haemaphysaloides Supino (Ixodida: Ixodidae) were the most abundant chiggers (84.5%) and ticks (>99%) identified, respectively. Immunofluorescent antibody assay revealed high seropositive rates of rodents against Orientia tsutsugamushi Hyashi (Rickettsiales: Rickettsiaceae), the aetiological agent of scrub typhus (70.0% of 437 rodents), and tick-borne SFG rickettsiae (91.9% of 418 rodents). The current study represents a first step towards elucidating the potential hosts and vectors in the enzootic transmission of O. tsutsugamushi and tick-borne SFG rickettsiae in Taiwan. Further studies should focus on characterizing pathogens in L. imphalum and R. haemaphysaloides, as well as the proclivity of both vectors to humans. Uncovering the main hosts of adult ticks is also critical for the prevention of SFG rickettsial infections.     

Epidemiological characteristics of tsutsugamushi disease in Oita Prefecture, Japan: Yearly and monthly occurrences of its infections and serotypes of its causative agent, Orientia tsutsugamushi, during 1984–2005

Using indirect immunofluorescence assay, we examined the sera of 561 patients from November 1984 to February 2005 to determine the incidence of tsutsugamushi disease (scrub typhus) in Oita Prefecture, Japan. The results obtained were positive in 384 individuals (68.4%). Municipalities where patients were presumed to have been infected with Orientia tsutsugamushi were Taketa City (41.7%), Oyama Town (13.5%), and Ogi Town (8.3%). Infections occurred most often in October, November, and December. A small number of cases occurred from January to May. The serotypes Kuroki (47.5%), Kawasaki (42.5%), and Karp (10.0%) were detected by genetic analysis of O. tsutsugamushi DNA extracted from the blood of 120 patients. The gene sequences of the Kuroki type were highly homologous to that of the Nishino strain. The gene sequences of the Kawasaki type were identical to that of the Kawasaki strain. The gene sequence of the Karp type was highly homologous to that of the JP-2 type. To determine the distribution of vector mites, 558 wild rodents were captured and 72 010 mites attached to these rodents were collected from 1982 to 1998. Six genera and 16 species of trombiculid mites were collected. Leptotrombidium pallidum and L. scutellare , which are known to be mite vectors for tsutsugamushi disease, accounted for 20.5% and 5.9%, respectively, of all trombiculid mites collected. The geographical distribution of cases roughly coincided with the distribution of L. scutellare . In Oita Prefecture, L. scutellare is presumed to primarily transmit tsutsugamushi disease. In addition, our results also suggest that L. pallidum transmits the Karp type of the causative rickettsia in some municipalities.     

Inhibition of Orientia tsutsugamushi infection by a truncated recombinant 56‐kDa outer membrane protein

Aims: The objective of this study was to evaluate recombinant 56‐kDa outer membrane protein as a potential inhibitor to infection from Orientia tsutsugamushi. Methods and Results: The 56‐kDa protein was cloned and expressed in an Escherichia coli system, and the degree of target cell attachment to immobilized 56‐kDa protein was measured in a cell adhesion assay. The results showed that the 56‐kDa protein has an ability to attach HeLa cells. Furthermore, treatment of target cells with a truncated 56‐kDa 1–418 (amino acid residues) protein inhibited target cell infection by O. tsutsugamushi, demonstrated with an indirect immunofluorescence antibody assay. Conclusions: The truncated 56‐kDa protein (a.a. 1–418) plays an important role in O. tsutsugamushi infection, and the 56‐kDa protein could be useful and effective in the inhibition of O. tsutsugamushi attachment and infection. Significance and Impact of the Study: The attachment of the 56‐kDa protein to target cells was directly determined by in vitro adherence test, and the invasion of target cells by O. tsutsugamushi was inhibited by treating the target cells with a truncated 56‐kDa protein.     

Phylogenetic analysis of Orientia tsutsugamushi strains based on the sequence homologies of 56-kDa type-specific antigen genes

Close and distant relationship among 31 strains of Orientia tsutsugamushi (20, two, one and eight strains were isolated in Japan, Korea, China and southeast Asia, respectively) were clarified using phylogenetic analyses based on homologies of 56-kDa type-specific antigen genes. Isolates in Japan, Korea and China were located in eight separate clusters in the phylogenetic tree, and each was designated as JG (Japanese Gilliam type), JP-1 and JP-2 (Japanese Karp 1 and 2 types), Kato, Kawasaki, Kuroki, Shimokoshi and LX-1 types. All isolates originated in southeast Asia, including the prototype Gilliam and Karp strains isolated in Burma and New Guinea, respectively, were distantly located in the phylogenetic tree from those isolates in Japan, Korea and China, indicating that strains of O. tsutsugamushi distributed in northeastern and southeastern Asia are different types.     

Decreased prevalence of Orientia tsutsugamushi in trombiculid mites and wild rodents in the Primorye region, Far East Russia

The isolation of Orientia tsutsugamushi was attempted from 249 rodents and approximately 14,000 trombiculid mites captured in the Primorye region, Far East Russia in 1993 and 1994, where high infection rates were recorded in both rodents and mites in the 1960s. However, no rickettsia was isolated from the samples. Low antibody titers against O. tsutsugamushi were detected in 7.1% of the rodents. These results indicate that the prevalence of O. tsutsugamushi in the Primorye region has decreased considerably in the past 30 years.     

Activation of mitogen-activated protein kinases is involved in the induction of interferon β gene in macrophages infected with Orientia tsutsugamushi

We investigated the role of MAPK in IFN-β gene expression in macrophages after infection with Orientia tsutsugamushi . ERK1/2 became phosphorylated in Orientia -stimulated macrophages. Selective inhibition of ERK1/2 and p38 MAPK could all significantly reduce Orientia- stimulated IFN-β mRNA expression. Orientia inactivation by heat abolished IFN-β mRNA induction only, whereas cytochalasin D treatment completely blocked both IFN-β and chemokine expression, suggesting requirement of cellular internalization by viable bacteria for IFN-β gene induction. In conclusion, our data indicate that MAPK pathways are required to induce maximal IFN-β gene expression in macrophages during Orientia infection.     

家禽主要组织相容性复合体的研究进展

随着家禽基因组计划的开展,对家禽的主要组织相容性复合体（MHC）的研究取得了较大的进展。关于家禽MHC的各部分基因研究正在逐步深入,并且完成了MHC部分测序和染色体定位工作。 本文介绍近些年来对家禽MHC的基因结构和作用、与抗体的作用以及相关的基因组研究所取得的进展。人、小鼠及其他动物的相关研究结果将对家禽MHC研究的发展产生重要的影响。 Abstract:As the development of poultry genome project,it has been acquired many advances in the study of poultry MHC.At present,we have achieved some MHC sequences 、 locations of MHC on chromosome and some MHC gene functions.This article give a detailed introduction about gene structure of poultry MHC and its role in immune reaction、relation with antibody and advances in poultry genome about MHC.With the development of related research,how to use the result of the study more efficiently become more and more important to the poultry MHC study.     

Biochemical determination of natural tumor-associated T-cell epitopes

The knowledge of tumor-associated T-cell epitopes is important for the understanding of tumor biology and the development of cancer vaccines. We describe here a biochemical approach for the identification of tumor-associated T-cell epitopes. Peptides are extracted from immunoaffinity isolated MHC class I molecules of tumor cells and separated by HPLC. The HPLC fractions are then tested for biological activity of the peptides which are then sequenced by mass spectrometry. The tumor association of the identified T-cell epitopes is confirmed using synthetic analogs and T-cells of cancer patients.     

The evolution of MHC diversity: evidence of intralocus gene conversion and recombination in a single-locus system

Gene conversion, the unidirectional exchange of genetic material between homologous sequences, is thought to strongly influence patterns of genetic diversity. The high diversity of major histocompatibility complex (MHC) genes in many species is thought to reflect a long history of gene conversion events both within and among loci. Theoretical work suggests that intra- and interlocus gene conversion leave characteristic signatures of nucleotide diversity, but empirical studies of MHC variation have rarely been able to analyze the effects of conversion events in isolation, due to the presence of multiple gene copies in most species. The potbellied seahorse (Hippocampus abdominalis), a species with a single copy of the MH class II beta-chain gene (MHIIb), provides an ideal system in which to explore predictions on the effects of intralocus gene conversion on patterns of genetic diversity. The genetic diversity of the MHIIb peptide binding region (PBR) is high in the seahorse, similar to other vertebrate species. In contrast, the remainder of the gene shows a total absence of synonymous variation and low levels of intronic sequence diversity, concentrated in 3 short repetitive regions and 1-12 SNPs per intron. The distribution of substitutions across the gene results in a patchwork pattern of shared polymorphism between otherwise divergent sequences. The pattern of nucleotide diversity observed in the seahorse MHIIb gene is congruent with theoretical expectations for intralocus gene conversion, indicating that this evolutionary mechanism has played an important role in MHC gene evolution, contributing to both the high diversity in the PBR and the low diversity outside this region. Neutral variation at this locus may be further reduced due to biases in nucleotide composition and functional constraints.     

鸡MHC与传染性疾病遗传抗性的相关性研究进展

鸡是我国主要的家禽品种,抗病分子育种在鸡的疾病尤其是传染病控制中有着重要地位,抗性基因选择是其技术关键。鸡主要组织相容性复合体(MHC)基因具有高度多态性,与多种传染性疾病抗性紧密相关,受到家禽育种专家的高度关注。文章介绍国外有关鸡MHC与传染性疾病抗性的相关性及抗性基因研究进展,并展望其在鸡抗病育种中的应用前景。     

扬子鳄种群MHC Ⅱ类B基因第3外元多态性分析

分析了取自安徽宣城野生种群、安徽省扬子鳄繁殖研究中心和浙江长兴养殖种群的14条扬子鳄MHCⅡ类B基因第3外元的多态性。在这些扬子鳄样本中共检测到34个单倍型,每个亚种群内检测到的单倍型数量分别为15,9和10个,与其他一些动物如哺乳动物和鲤科鱼类相比,扬子鳄MHCⅡ类B基因第3外元多态性较高。另外,非同义替换率显著小于同义替换率,这可能表明扬子鳄种群MHCⅡ类B基因第3外元的多态性不是由平衡选择保持的。Tajima的中性检测拒绝了扬子鳄MHCⅡ类B基因第3外元多态性是由遗传漂变引起的零假设。D=-0．401也暗示了扬子鳄种群中存在较多的稀有变异。同时我们也分析了核苷酸多样性和系统发生关系,结果表明扬子鳄的3个种群遗传多样性无显著性差异。     

Ab initio prediction of peptide-MHC binding geometry for diverse class I MHC allotypes

Since determining the crystallographic structure of all peptide-MHC complexes is infeasible, an accurate prediction of the conformation is a critical computational problem. These models can be useful for determining binding energetics, predicting the structures of specific ternary complexes with T-cell receptors, and designing new molecules interacting with these complexes. The main difficulties are (1) adequate sampling of the large number of conformational degrees of freedom for the flexible peptide, (2) predicting subtle changes in the MHC interface geometry upon binding, and (3) building models for numerous MHC allotypes without known structures. Whereas previous studies have approached the sampling problem by dividing the conformational variables into different sets and predicting them separately, we have refined the Biased-Probability Monte Carlo docking protocol in internal coordinates to optimize a physical energy function for all peptide variables simultaneously. We also imitated the induced fit by docking into a more permissive smooth grid representation of the MHC followed by refinement and reranking using an all-atom MHC model. Our method was tested by a comparison of the results of cross-docking 14 peptides into HLA-A*0201 and 9 peptides into H-2K(b) as well as docking peptides into homology models for five different HLA allotypes with a comprehensive set of experimental structures. The surprisingly accurate prediction (0.75 A backbone RMSD) for cross-docking of a highly flexible decapeptide, dissimilar to the original bound peptide, as well as docking predictions using homology models for two allotypes with low average backbone RMSDs of less than 1.0 A illustrate the method's effectiveness. Finally, energy terms calculated using the predicted structures were combined with supervised learning on a large data set to classify peptides as either HLA-A*0201 binders or nonbinders. In contrast with sequence-based prediction methods, this model was also able to predict the binding affinity for peptides to a different MHC allotype (H-2K(b)), not used for training, with comparable prediction accuracy.