Investigation on the infection of spotted fever group Rickettsiae and Rickettsia mooseri of wild rodents in the field of plague foci in Western Yunnan / 中华地方病学杂志

Objective:To investigate the infection of spotted fever group Rickettsiae (SFGR) and Rickettsia mooseri ( R.mooseri) of wild rodents in the field of plague foci in Western Yunnan. Methods:The DNA of liver samples of 2 512 wild rodents captured from the plague foci in Lianghe County, Jianchuan County and Yulong County in Western Yunnan from 2015 to 2016 was extracted by magnetic bead method, and the heat shock protein groEL gene primers were used for nested PCR amplification. Gene sequence splicing and Blast homology comparison were performed using DNAStar 7.1 software and GenBank of the National Center for Biotechnology Information (NCBI) of the United States, respectively, and DNAStar 7.1 and MEGA 6.0 softwares were used to construct phylogenetic trees.Results:The wild rodents infected with SFGR were Mus pahari, Rattus steini, Crocidura attenuata and Suncus murinus (one for each), with a total infection rate of 0.16% (4/2 512); no R.mooseri infection was detected. The SFGR infection rates of wild rodents in the plague foci of Lianghe County and Jianchuan County were 0.49% (3/611) and 0.10% (1/1 029), respectively; no SFGR infection was detected in the wild rodents in the plague foci of Yulong County. The homology analysis showed that the homology between SFGR positive samples and reference sequences was 95.45%-100.00%; some of the groEL gene sequences were highly similar among the four positive samples, and the homology was 89.60%-97.40%. Sequence evolution analysis showed that the sequences of three SFGR positive samples from the plague focus in Lianghe County were clustered in the same branch, and the homology reached 94.40%-97.40%; one positive sample sequence from the plague focus in Jianchuan County was clustered in one branch. Conclusion:SFGR infection rate of wild rodents in the field of plague foci in Western Yunnan is low, and no R.mooseri infection is found.

Accurate identification of atypical Staphylococcus chromogenes plasma-clotting strains causing bovine mastitis / Identificação acurada de cepas atípicas de Staphylococcus chromogenes que coagulam o plasma envolvidas com mastite bovina

ABSTRACT: We compared the potential of routine techniques used for the identification of Staphylococcus species, aiming to evaluate their accuracy in the detection of 43 Staphylococcus chromogenes strains isolated from bovine mastitis that, despite being a coagulase-negative species, are able to clot plasma. These strains could be mistakenly suspected to be S. aureus and lead to an unappropriated treatment of the disease. MALDI-TOF, PCR-RFLP of the chaperonine gene groEL, and sequencing of the 16S rRNA and elongation factor Tu gene tuf were employed. Results from the four methods were coincident for only half of the strains because of the low accuracy of the groEL PCR-RFLP (51.2% accuracy). Even though all the sequencing results were identical, the high accuracy of the MALDI-TOF results (97.7% accuracy, with only one strain misidentified) encourage the use of this technique, since it does not require laborious sample preparation, being fast and simple to perform.

RESUMO: Nós comparamos o potencial de técnicas rotineiras utilizadas para a identificação de espécies de Staphylococcus, com o objetivo de avaliar a acurácia delas na detecção de 43 isolados de Staphylococcus chromogenes envolvidos com mastite bovina que, apesar de ser uma espécie coagulase-negativa, são capazes de coagular o plasma. Essas cepas poderiam ser erroneamente suspeitas de serem S. aureus e levarem a um tratamento não adequado da doença. MALDI-TOF, PCR-RFLP do gene da chaperonina groEL e sequenciamento do gene do rRNA 16S e do gene do fator de elongação Tu, tuf, foram avaliados. Os resultados dos quatro métodos foram coincidentes para apenas metade das cepas, devido à baixa precisão da PCR-RFLP com groEL (51,2% de acurácia). Apesar de todos os resultados do sequenciamento serem idênticos, a alta precisão dos resultados do MALDI-TOF (97,7% de acurácia, com apenas uma cepa identificada incorretamente) encoraja o uso dessa técnica, pois, não requer preparação laboriosa de amostras, sendo rápida e simples de executar.

Design and analytical validation of a duplex PCR for Ehrlichia and Rickettsia detection in ticks / Diseño y validación analítica de una PCR dúplex para la detección de Ehrlichia y Rickettsia en garrapatas / Projeto e validação analítica de uma PCR duplex para detecção de Ehrlichia e Rickettsia em carrapatos

Abstract Background: Ehrlichia and Rickettsia are two major rickettsial genera transmitted by ticks that affect a number of wild and domestic animal species and human populations around the world. Objective: To design and validate a duplex PCR for Ehrlichia and Rickettsia in ticks. Methods: Assay validation included testing for sensitivity, specificity, reproducibility, and robustness of the PCR. The groEL and 23sr RNA genes were used for Ehrlichia and Rickettsia, respectively. Results: The limit of detection was one hundred gene copies per 50 μL of reaction for Ehrlichia spp, and one gene copy of Rickettsia per 50 μL of reaction. In general, the primers of the test only amplified in silico those bacterial agents for which they were originally designed, with the exception of the primers for Rickettsia that also amplified Methylocystis sp. The test was reproducible (intermediate precision) 96.7% of the times for both agents. The test was robust enough to tolerate concentration changes of all reagents with the exception of Taq DNA polymerase. Conclusions: The validation results indicated that this PCR is useful for detection in both bacterial genera and it is a good candidate for diagnostic validation.

Resumen Antecedentes: Ehrlichia spp. y Rickettsia spp. son dos de los principales géneros rickettsiales transmitidos por garrapatas que afectan a animales silvestres, domésticos y humanos alrededor del mundo. Objetivo: Diseñar y validar una prueba PCR dúplex para Ehrlichia y Rickettsia en garrapatas. Métodos: La validación de la prueba incluyó ensayos de sensibilidad, especificidad, reproducibilidad y robustez. En la PCR se usó groEL y ARNr 23S como genes blanco para Ehrlichia y Rickettsia, respectivamente. Resultados: El límite de detección fue de 100 copias del gen por 50 μL de reacción para Ehrlichia spp y una copia del gen de Rickettsia por 50 μL de reacción. En general, los cebadores de la prueba solo amplificaron in silico los agentes bacterianos para los cuales fueron originalmente diseñados, con la excepción de los cebadores de Rickettsia que también amplificaron Methylocystis sp. La prueba fue reproducible (precisión intermedia) en un 96.7% de las veces para ambos agentes. La prueba fue suficientemente robusta como para tolerar cambios de concentración de los diferentes reactivos, con excepción de la Taq DNA polimerasa. Conclusión: Los resultados de validación indican que la PCR es útil para detectar ambos géneros bacterianos y podria usarse para validación diagnostica.

Resumo Antecedentes: Ehrlichia e Rickettsia são dois dos principais gêneros de rickettsias transmitidos por carrapatos que infectam tanto animais selvagens quanto animais domésticos e até homens em todo o mundo. Objetivo: O objetivo principal foi elaborar e validar uma PCR duplex para Ehrlichia e Rickettsia em carrapatos. Métodos: A validação incluiu testes de sensibilidade, especificidade, reprodução e robustez. Para o PCR, utilizamos os genes groEl e 23Sr-RNA para Ehrlichia e Rickettsia, respectivamente. Resultados: O limite de detecção foi de 100 cópias de genes por 50 ml de reação para Erliquia spp e uma cópia de gene de Rickettsia por 50 ml de reação. Em geral, os iniciadores dos testes amplificaram em modelos computacionais os agentes bacterianos para os quais eles foram projetados, exceto os primers de Rickettsia que também amplificou Methylocystis sp. Os testes foram reproduzíveis (precisão intermediária) 96,7% para ambos os agentes e foram também robustos para tolerar mudanças de concentração em todos os reagentes, exceto o reagente Taq DNA polymerase. Conclusões: Os resultados da validação indicaram que o PCR é útil para detecção em ambos os gêneros bacterianos, portanto, um bom exame para validação diagnóstica.

Identification of predominant T-and B-combined antigenic epitopes in outer membrane protein GroEL of Helicobacter pylori / 中华微生物学和免疫学杂志

Objective To screen and identify the predominant T-and B-cell (T-B) combined antigenic epitopes in outer membrane protein GroEL of Helicobacter pylori (H.pylori).Methods The entire groEL gene of H.pylori strain NCTC11637 was amplified by PCR,and then a prokaryotic expression system for groEL gene was constructed.The expressed target recombinant protein rGroEL was analyzed by SDSPAGE and purified by Ni-NTA affinity chromatography.Laser confocal microscopy was applied to determine the distribution of GroEL in H.pyloristrains of NCTC11637 and SS1.The outer membrane protein (OMP) samples were extracted from the two strains using Triton X-114 method.GroEL in OMP samples was detected by Western blot assay.Special bioinformatic softwares were used to predict T-B combined antigenic epitopes on GroEL molecule,and phage display systems for every T-B combined antigenic epitope were established.Western blot assay and ELISA were used to detect the immunoreactivity of recombinant T-B combined antigenic epitope-containing phage PⅢ proteins and artificially synthesized T-B combined antigenic epitope peptides,respectively.Results The groEL genes from H.pylori strains of NCTC11637 and SS1 showed 97.68％ ～99.63％ identities in their nucleotide and amino acid sequences.The established prokaryotic expression system could efficiently express soluble rGroEL.GroEL located on the outer membrane of H.pylori strains of NCTC11637 and SS1.Among the six major predicted T-B combined epitopes (GroEL168,GroEL258,GroEL288,GroEL365,GroEL396 and GroEL438),GroEL168 showed the strongest positive immunoblotting signal.The results of ELISA showed that the immunoreactivity of GroEL168 was also the strongest (P＜0.01),followed by GroEL258 and GroEL288 (P＜0.05).Conclusion GroEL is an outer membrane protein of H.pylori.GroEL168 being the predominant T-B combined antigenic epitope of GroEL can be used to develop multiple antigenic peptide vaccines of H.pylori.

Fusobacterium nucleatum GroEL signaling via Toll-like receptor 4 in human microvascular endothelial cells

The GroEL heat-shock protein from Fusobacterium nucleatum, a periodontopathogen, activates risk factors for atherosclerosis in human microvascular endothelial cells (HMEC-1) and ApoE-/- mice. In this study, we analyzed the signaling pathways by which F. nucleatum GroEL induces the proinflammatory factors in HMEC-1 cells known to be risk factors associated with the development of atherosclerosis and identified the cellular receptor used by GroEL. The MAPK and NF-kappaB signaling pathways were found to be activated by GroEL to induce the expression of interleukin-8 (IL-8), monocyte chemoattractant protein 1 (MCP-1), intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), E-selectin, and tissue factor (TF). These effects were inhibited by a TLR4 knockdown. Our results thus indicate that TLR4 is a key receptor that mediates the interaction of F. nucleatum GroEL with HMEC-1 cells and subsequently induces an inflammatory response via the MAPK and NF-kappaB pathways.

Recombinant expression of Helicobacter pylori GroEL protein and immunoprotection of the expression product in mice / 中华微生物学和免疫学杂志

Objective To determine the sequence diversity in groEL gene of Helicobacter pylori isolates,and the antigenicity,immunoreactivity and immunoprotection of recombinant expressed GroEL (rGroEL) against H.pylori-infection in mice.Methods The sequences of groEL genes from H.pylori isolates were detected by PCR and sequencing.A prokaryotic expression system of H.pylori groEL gene using pET42a and E.coli BL21DE3 was generated.SDS-PAGE and Gel Image Analyzer were used to measure the yield of rGroEL and Ni-NTA affinity chromatography was applied to extract the expressed rGroEL.The antigenicity and immunoreactivity of rGroEL were examined using ELISA and Western blot assay.The membrane location of GroEL was determined by ELISA using whole cells of H.pylorias the coated antigen.The immunoprotection of rGroEL was detected in H.pylori-infected mouse model.Results All the 35 tested H.pylori isolates possess the groEL gene with 99.4％ to 100％ sequence identities.The yield of rGroEL expression occupied 56％ of the total bacterial proteins and the extracted rGroEL showed a single band in gel after SDSPAGE.rGroEL could induce the production of high-titer IgG in rabbits or mice.The lgG against whole cell of H.pylori(Hp-IgG) and rGroEL-IgG could recognize as well as combine with rGroEL protein.GroEL is a superficial protein antigen of H.pylori.The immunization with 50,100 or 200 μg rGroEL prevented 50.0％ (6/12),75.0％ (9/12) or 91.7％ (11/12) mice from infection of H.pylori strain SS1.The immunoprotective rate ( 91.7％ ) due to the immunization of 200 μg rG roEL was significantly higher than that ( 50.0％ ) of 50 μg rGroEL ( P＜0.05 ).Conclusion The GroEL protein is a sequence-conserved,immunogenic and immunoprotective superficial antigen of H.pylori,which can be used as the immunogen candidate for developing genetically engineering vaccines.

Increased expression and purification of soluble iron-regulatory protein 1 from Escherichia coli co-expressing chaperonins GroES and GroEL

Iron is an essential metal for all living organisms. However, iron homeostasis needs to be tightly controlled since iron can mediate the production of reactive oxygen species, which can damage cell components and compromise the integrity and/or cause DNA mutations, ultimately leading to cancer. In eukaryotes, iron-regulatory protein 1 (IRP1) plays a central role in the control of intracellular iron homeostasis. This occurs by interaction of IRP1 with iron-responsive element regions at 5' of ferritin mRNA and 3' of transferrin mRNA which, respectively, represses translation and increases mRNA stability. We have expressed IRP1 using the plasmid pT7-His-hIRP1, which codifies for human IRP1 attached to an NH2-terminal 6-His tag. IRP1 was expressed in Escherichia coli using the strategy of co-expressing chaperonins GroES and GroEL, in order to circumvent inclusion body formation and increase the yield of soluble protein. The protein co-expressed with these chaperonins was obtained mostly in the soluble form, which greatly increased the efficiency of protein purification. Metal affinity and FPLC ion exchange chromatography were used in order to obtain highly purified IRP1. Purified protein was biologically active, as assessed by electrophoretic mobility shift assay, and could be converted to the cytoplasmic aconitase form. These results corroborate previous studies, which suggest the use of folding catalysts as a powerful strategy to increase protein solubility when expressing heterologous proteins in E. coli.

Rapid Identification of Rickettsiae using the Real-Time PCR

In this study, new real-time PCR method based on the groEL gene was developed and investigated. Four spotted fever group (SFG) strains, four typhus group (TG) strains, and four scrub typhus group (STG) strains were easily differentiated as a distinct entity. This PCR assay was applied to detect Rickettsia DNA from 100 ticks. Twelve Haemaphysalis longicornis ticks were found positive and identified as spotted fever group Rickettsia. This real-time PCR method could simultaneously perform the rapid identification of rickettsiae and the differential diagnosis of SFG, TG, and STG in a single reaction.

Prevalence of Spotted Fever Group Rickettsia from Haemaphysalis Ticks in Chungju Province

A total of 190 ticks collected from the Chungju area of Korea was examined for the presence of Spotted Fever Group(SFG) Rickettsia using a PCR assay. Twenty-five (13.2%) Haemaphysalis ticks were found positive of the groEL gene of SFG Rickettsia. The prevalence rate of R. japonica in these 25 Haemaphysalis ticks was 72% (18 out of 25 SFG Rickettsia). The prevalence rate of R. conorii and new SFG rickettsia in these 25 Haemaphysalis ticks was 4% (1 out of 25 SFG Rickettsia) and 24% (6 out of 25 SFG Rickettsia), respectively. These results suggest that R. japonica was the highest infection frequency among in Haemaphysalis ticks SFG Rickettsia, and that R. conorii and new SFG Rickettsia are also present in the Chungju area.

groEL Gene Analysis of Borrelia afzelii Isolated in Korea

Eleven Borrelia afzelii strains, isolated from Ixodes nipponensis and Apodemus agrarius in Korea, were characterized by groEL gene analysis. Results from previous studies suggested that the groEL gene, which encodes the 60-kDa heat shock protein GroEL, was useful for the differentiation of B. burgdorferi sensu lato. The B. afzelii isolates could be divided into two groups by the phylogenetic tree constructed by UPGMA method and Tsp509 I PCR-RFLP analysis. The result suggested that the groEL gene is useful for identification and characterization of B. burgdorferi sensu lato though a short DNA fragment (310 bp) of the gene was sequenced and compared each other, and that Korean B. afzelii strains are heterogeneous genotypically.

groEL Gene Analysis of Borrelia afzelii Isolated in Korea

Eleven Borrelia afzelii strains, isolated from Ixodes nipponensis and Apodemus agrarius in Korea, were characterized by groEL gene analysis. Results from previous studies suggested that the groEL gene, which encodes the 60-kDa heat shock protein GroEL, was useful for the differentiation of B. burgdorferi sensu lato. The B. afzelii isolates could be divided into two groups by the phylogenetic tree constructed by UPGMA method and Tsp509 I PCR-RFLP analysis. The result suggested that the groEL gene is useful for identification and characterization of B. burgdorferi sensu lato though a short DNA fragment (310 bp) of the gene was sequenced and compared each other, and that Korean B. afzelii strains are heterogeneous genotypically.

Development of Rickettsia Specific Nested PCR Method Based on groEL Gene Sequences

To detect Rickettsia, we have developed a nested PCR method amplifying the groEL gene. Rickettsia strains were successfully amplified by this PCR method but the microorganisms causing other febrile diseases, such as Orientia tsutsugamushi, Coxiella burnetii, Ehrlichia sennetsu, Borrelia burgdorferi sensu lato, Borrelia hermsii, and Leptospira interrogans were not amplified. This PCR assay was applied to detect Rickettsia DNA from 100 ticks. Sixteen Haemaphysalis longicornis ticks were positive by this PCR assay. These results suggest that the new nested PCR method might be sensitive and useful for discrimination between Rickettsia and other febrile disease-causing microorganisms.

Studies on the Prokaryotic Expression Conditions of groEL from Endosymbionts in Bemisia tabaci / 微生物学通报

The study on prokaryotic expression conditions of groEL from endosymbiont in B. tabaci was conducted.The results showed that:The optimal IPTG content for inducing prokaryotic expression of groEL was 100?mol/L. When it was induced by IPTG at 35℃,higher GroEL product would be acquired.The optimal induced culturing time for prokaryotic expression of groEL was 4～5 hours.Higher inoculated quantity would benefit prokaryotic expression of groEL. A little NH~+_(4) content would improved prokaryotic expression of groEL, but prokaryotic expression of groEL would be inhibit by Ca~(2+)、Fe~(3+)、K~(+ )and Mg~(2+).When low content glucose was added to the LB medium,it would benefit prokaryotic expression of groEL.Prokaryotic expression of groEL would be inhibit by high content glucose.