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.     

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.     

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.     

Geographical distribution of Orientia tsutsugamushi strains in chiggers from three provinces in Korea

Chiggers were collected from the central and southern parts of South Korea between April and November, 2009 with the aim of investigating the seasonal and geographical distribution of Or. A total of 1136 chiggers were identified. They included eight species belonging to four genera, as follows: Leptotrombidium scutellare (27.2%, n = 309), L. pallidum (54.6%, n = 621), L. orientale (6.25%, n = 71), L. palpale (1.59%, n = 18), L. zetum (2.0%, n = 23), Euschoengastia koreaensis (1.5%, n = 17), Cheladonta ikaoensis (0.08%, n = 1) and Neotrombicula japonica (1.05%, n = 12). The density of L. pallidum was high from April to May, whereas L. scutallare was not found in spring, being observed from October. Serotype‐specific nested PCR targeting the 56 kDa protein gene and sequencing analysis identified that the strains of 1136 O. tsutsugamushi in the chiggers as Boryong (6.8%), Kanda (0.4%), Oishi (0.3%), Jecheon (0.1%), Youngworl (0.1%) and Wonju (0.1%). Our findings indicate that L. pallidum and L. scutellare are dominant species in Korea and have geographical and seasonal variations.

     

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.     

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.     

Phylogenetic characterization of Orientia tsutsugamushi isolated in Taiwan according to the sequence homologies of 56-kDa type-specific antigen genes

Fourteen strains of Orientia tsutsugamushi isolated in Taiwan were characterized by sequencing 56-kDa type-specific antigen genes and patterns of restriction fragment length polymorphism (RFLP) predicted by a computer program. The strains showed high varieties in sequence homologies and were classified to 10 types by predicted patterns of RFLP. Furthermore, all Taiwan strains were not identical in typing with strains analyzed previously. These results suggest that there are various types of O. tsutsugamushi in Taiwan that are different from those distributed in other countries.     

Global gene expression profile of Orientia tsutsugamushi

Orientia tsutsugamushi, an obligate intracellular bacterium, is the causative agent of Scrub typhus. The control mechanisms for bacterial gene expression are largely unknown. Here, the global gene expression of O. tsutsugamushi within eukaryotic cells was examined using a microarray and proteomic approaches for the first time. These approaches identified 643 genes, corresponding to approximately 30% of the genes encoded in the genome. The majority of expressed genes belonged to several functional categories including protein translation, protein processing/secretion, and replication/repair. We also searched the conserved sequence blocks (CSBs) in the O. tsutsugamushi genome which is unique in that up to 40% of its genome consists of dispersed repeated sequences. Although extensive shuffling of genomic sequences was observed between two different strains, 204 CSBs, covering 48% of the genome, were identified. When combining the data of CSBs and global gene expression, the CSBs correlates well with the location of expressed genes, suggesting the functional conservation between gene expression and genomic location. Finally, we compared the gene expression of the bacteria‐infected fibroblasts and macrophages using microarray analysis. Some major changes were the downregulation of genes involved in translation, protein processing and secretion, which correlated with the reduction in bacterial translation rates and growth within macrophages.     

Sequence analysis of the hypervariable regions of the 56 kDa immunodominant protein genes of Orientia tsutsugamushi strains in Malaysia

The DNA sequences encompassing two hypervariable regions, VD II and III of the 56 kDa immunodominant protein gene of 21 Malaysian strains of Orientia tsutsugamushi were determined. Two strains demonstrated a 100% DNA homology with the Gilliam prototype strain, and one with TH1817 strain and TA678 strain respectively. High percentages of DNA similarity (95-99%) were observed with Karp (4 strains), Gilliam (2 strains), TH1817 (4 strains), TC586 (3 strains) and TA763 (1 strain). The remaining strains demonstrated the highest DNA similarity with TA763 (1 strain, 89%), TA678 (1 strain, 86%) and TA686 (1 strain, 87%). Our study provides additional evidence on the existence and the genetic heterogeneity of TA strains of the Southeast Asia and their closely related strains in Malaysia.     

Identification of B- and T-cell epitopes on HtrA protein of Orientia tsutsugamushi

Orientia tsutsugamushi, a cause of scrub typhus is emerging as an important pathogen in several parts of the tropics. The control of this infection relies on rapid diagnosis, specific treatment, and prevention through vector control. Development of a vaccine for human use would be very important as a public health measure. Antibody and T-cell response have been found to be important in the protection against scrub typhus. This study was undertaken to predict the peptide vaccine that elicits both B- and T-cell immunity. The outer-membrane protein, 47-kDa high-temperature requirement A was used as the target protein for the identification of protective antigen(s). Using BepiPred2 program, the potential B-cell epitope PNSSWGRYGLKMGLR with high conservation among O. tsutsugamushi and the maximum surface exposed residues was identified. Using IEDB, NetMHCpan, and NetCTL programs, T-cell epitopes MLNELTPEL and VTNGIISSK were identified. These peptides were found to have promiscuous class-I major histocompatibility complex (MHC) binding affinity to MHC supertypes and high proteasomal cleavage, transporter associated with antigen processing prediction, and antigenicity scores. In the I-TASSER generated model, the C-score was −0.69 and the estimated TM-score was 0.63 ± 0.14. The location of the epitope in the 3D model was external. Therefore, an antibody to this outer-membrane protein epitope could opsonize the bacterium for clearance by the reticuloendothelial system. The T-cell epitopes would generate T-helper function. The B-cell epitope(s) identified could be evaluated as antigen(s) in immunodiagnostic assays. This cocktail of three peptides would elicit both B- and T-cell immune response with a suitable adjuvant and serve as a vaccine candidate.     

马链球菌马亚种IgG结合蛋白的原核表达和免疫原性

为研究马链球菌马亚种IgG结合蛋白(EAG)免疫原性和保护力,评价其作为马链球菌疫苗抗原的价值。采用PCR法扩增马链球菌马亚种EAG基因,将测序正确的EAG扩增产物与原核表达载体pET-28a(+)连接构建重组质粒,对转化后的大肠杆菌进行诱导表达,用诱导纯化后的重组蛋白作免疫原免疫小鼠,分析重组蛋白免疫小鼠后的抗体水平及对小鼠的免疫保护力。结果表明,诱导后可得到26 kDa的EAG重组蛋白,且该蛋白可与该菌阳性血清发生特异性反应。间接ELISA检测免疫小鼠的抗体效价可达1∶8 100,重组蛋白免疫后对小鼠保护力可达90%。该结果表明,表达的EAG蛋白具有良好的抗原性,可有效提高小鼠的体液免疫水平及免疫保护力。     

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.     

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.     

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.     

狐源犬瘟热病毒CDV-FOX-TA株核蛋白基因的原核表达及鉴定

应用RT-PCR技术扩增出犬瘟热病毒(CDV) 核衣壳(N)蛋白基因抗原性好的高保守基因片段,将其TA克隆至pMD18-T载体中,再利用酶切、连接的方法将测序正确的N基因目的片段亚克隆到原核表达载体pET24b中6×His Tag编码基因的上游,并将该重组质粒转化大肠杆菌Rosetta 2 (DE3)株,经IPTG诱导,N基因融合蛋白获得了高效表达。SDS-PAGE 分析和Western blot 分析的结果显示,表达产物的分子质量为15 kD,与CDV标准阳性血清呈阳性反应,间接ELISA结果也表明重组表达产物具有良好的抗原性,能够有效区分CDV标准阳性与阴性血清,表明大肠杆菌表达的CDV N 蛋白在免疫原性上具有与天然N蛋白同样的特性,可作为检测CDV的间接ELISA包被抗原。     

戊型肝炎病毒IV型衣壳蛋白缺失突变体原核表达与性质

采用PCR技术扩增基因IV型HEV(Hepatitis E Virus,HEV)开放阅读框2(Open Reading Frame 2,ORF2)的缺失突变体(aa384-606),亚克隆到表达载体后,转化到大肠杆菌中进行诱导表达,表达蛋白命名为rP24。SDS-PAGE和免疫印迹实验表明,rP24获得了高效表达,且和单克隆抗体15B2具有强的反应活性。rP24经过包涵体洗涤、溶解复性、离子交换层析和分子筛层析纯化后,免疫印迹实验表明,纯化rP24能与抗HEV ORF2中和单克隆抗体8C11以及HE(Hepatitis E,HE)阳性血清发生很强的免疫反应性,说明rP24上具有构象型中和表位,模拟了HEV衣壳蛋白的空间结构。动态光散色测量结果表明,rP24的平均水化半径为7.48 nm;纯化rP24免疫动物实验表明,rP24具有强的抗原性,小鼠阳转周期短,抗体持续时间长;纯化rP24作为包被抗原检测HE阳性血清和阴性血清,结果显示rP24对HE阳性血清和阴性血清检出率与北京万泰公司的抗HEV-IgG检测试剂盒的检出率一致。这些实验结果说明,具有较好免疫反应性和免疫原性的rP24获得了高效表达,该蛋白模拟了天然病毒衣壳蛋白的中和表位,为进一步研究基因I型和基因IV型HEV感染不同宿主细胞差异的分子机制奠定了基础。     

Isolation of Orientia tsutsugamushi from patients in Shikoku and finding of a strain which grows preferentially at low temperatures

Three strains of Orientia tsutsugamushi were isolated from patients in Anan City, Tokushima Prefecture. The strains were identified as Karp type by analyses of reactivities with type-specific monoclonal antibodies. One strain, Okazaki, was isolated in L cells cultivated at 31 C, but not in cells at 36 C or in mice. This strain showed better growth at 31 C than 36 C. This is the first report of an O. tsutsugamushi strain which grows preferentially at low temperatures.     

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.