Jin Wang

Genetics of Metabolic Variations between Yersinia pestis Biovars and the Proposal of a New Biovar, microtus

Yersinia pestis has been historically divided into three biovars: antiqua, mediaevalis, and orientalis. On the basis of this study, strains from Microtus-related plague foci are proposed to constitute a new biovar, microtus. Based on the ability to ferment glycerol and arabinose and to reduce nitrate, Y. pestis strains can be assigned to one of four biovars: antiqua (glycerol positive, arabinose positive, and nitrate positive), mediaevalis (glycerol positive, arabinose positive, and nitrate negative), orientalis (glycerol negative, arabinose positive, and nitrate positive), and microtus (glycerol positive, arabinose negative, and nitrate negative). A 93-bp in-frame deletion in glpD gene results in the glycerol-negative characteristic of biovar orientalis strains. Two kinds of point mutations in the napA gene may cause the nitrate reduction-negative characteristic in biovars mediaevalis and microtus, respectively. A 122-bp frameshift deletion in the araC gene may lead to the arabinose-negative phenotype of biovar microtus strains. Biovar microtus strains have a unique genomic profile of gene loss and pseudogene distribution, which most likely accounts for the human attenuation of this new biovar. Focused, hypothesis-based investigations on these specific genes will help delineate the determinants that enable this deadly pathogen to be virulent to humans and give insight into the evolution of Y. pestis and plague pathogenesis. Moreover, there may be the implications for development of biovar microtus strains as a potential vaccine.

DNA Microarray Analysis of Genome Dynamics in Yersinia pestis: Insights into Bacterial Genome Microevolution and Niche Adaptation

Genomics research provides an unprecedented opportunity for us to probe into the pathogenicity and evolution of the worlds most deadly pathogenic bacterium, Yersinia pestis, in minute detail. In our present work, extensive microarray analysis in conjunction with PCR validation revealed that there are considerable genome dynamics, due to gene acquisition and loss, in natural populations of Y. pestis. We established a genomotyping system to group homologous isolates of Y. pestis, based on profiling or gene acquisition and loss in their genomes, and then drew an outline of parallel microevolution of the Y. pestis genome. The acquisition of a number of genomic islands and plasmids most likely induced Y. pestis to evolve rapidly from Yersinia pseudotuberculosis to a new, deadly pathogen. Horizontal gene acquisition also plays a key role in the dramatic evolutionary segregation of Y. pestis lineages (biovars and genomovars). In contrast to selective genome expansion by gene acquisition, genome reduction occurs in Y. pestis through the loss of DNA regions. We also theorized about the links between niche adaptation and genome microevolution. The transmission, colonization, and expansion of Y. pestis in the natural foci of endemic plague are parallel and directional and involve gradual adaptation to the complex of interactions between the environment, the hosts, and the pathogen itself. These adaptations are based on the natural selections against the accumulation of genetic changes within genome. Our data strongly support that the modern plague originated from Yunnan Province in China, due to the arising of biovar orientalis from biovar antiqua rather than mediaevalis.

Universal Sample Preparation Method for Characterization of Bacteria by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

ABSTRACT Mass spectrometry has been a very useful method to rapidly identify microorganisms associated with infectious diseases, detect bioterrorism threats, and discriminate among different subtypes of a pathogen. In this study, we developed a universal method for bacterial identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The effects on the mass spectrum of different experimental conditions, including the amount of bacterial cells used and treatment procedures with different solutions, matrix species, and solvents, were examined, and an optimized protocol was developed. Several different bacterial species, including Yersinia pestis, Escherichia coli, Burkholderia cepacia, Bacillus anthracis, and Staphylococcus aureus, which covered the gram-negative and -positive species and spore-producing and non-spore-producing species, were analyzed to evaluate the utility of the protocol. The results showed that five different species and different strains of the same species (9 strains of S. aureus and 10 strains of E. coli) could be discriminated clearly by their peak profiles in a mass range of 1,000 to 20,000 Da. This protocol is simple, rapid, and easy to perform; has excellent reproducibility; and is suitable for the construction of a mass spectrum fingerprinting database, which helps in fast bacterial identification via database searching.

Protein Microarray for Profiling Antibody Responses to Yersinia pestis Live Vaccine

ABSTRACT A protein microarray representing 149 Yersinia pestis proteins was developed to profile antibody responses in EV76-immunized rabbits. Antibodies to 50 proteins were detected. There are 11 proteins besides F1 and V antigens to which the predominant antibody response occurred, and these proteins show promise for further evaluation as candidates for subunit vaccines and/or diagnostic antigens.

Microarray analysis of temperature-induced transcriptome of Yersinia pestis.

Yersinia pestis, the etiologic agent of plague, must acclimatize itself to temperature shifts between the temperature (26 C) for flea blockage and the body temperature (37 C) of warm‐blooded hosts during its life cycle. Here a whole‐genome DNA microarray was used to investigate transcriptional regulation upon the upshift of growth temperature from 26 to 37 C in a chemically defined medium. Four hundred and one genes were regulated differentially under the two temperatures. About 39% of these genes were up‐regulated at 37 C, whereas 61% were down‐regulated. Temperature‐induced changes occurred at the level of transcription of genes encoding proven or predicted virulence factors, regulators, metabolism‐associated proteins, prophages, and hypothetical proteins. Strikingly, many gene clusters displayed a co‐transcription pattern in response to temperature upshift. Our data provided a genome‐wide profile of gene transcription induced by temperature shift and should shed light on the pathogenicity and host‐microbe interaction of this deadly pathogen.

Characterization of the 3a protein of SARS-associated coronavirus in infected vero E6 cells and SARS patients.

n Abstractn n Proteomics was used to identify a protein encoded by ORF 3a in a SARS-associated coronavirus (SARS-CoV). Immuno-blotting revealed that interchain disulfide bonds might be formed between this protein and the spike protein. ELISA indicated that sera from SARS patients have significant positive reactions with synthesized peptides derived from the 3a protein. These results are concordant with that of a spike protein-derived peptide. A tendency exists for co-mutation between the 3a protein and the spike protein of SARS-CoV isolates, suggesting that the function of the 3a protein correlates with the spike protein. Taken together, the 3a protein might be tightly correlated to the spike protein in the SARS-CoV functions. The 3a protein may serve as a new clinical marker or drug target for SARS treatment.n n

Antigenicity Analysis of Different Regions of the Severe Acute Respiratory Syndrome Coronavirus Nucleocapsid Protein

Abstract Background: The widespread threat of severe acute respiratory syndrome (SARS) to human health has made urgent the development of fast and accurate analytical methods for its early diagnosis and a safe and efficient antiviral vaccine for preventive use. For this purpose, we investigated the antigenicity of different regions of the SARS coronavirus (SARS-CoV) nucleocapsid (N) protein. Methods: The cDNA for full-length N protein and its various regions from the SARS-CoV was cloned and expressed in Escherichia coli. After purification, all of the protein fragments were printed on glass slides to fabricate a protein microarray and then probed with the sera from SARS patients to determine the reactivity of these protein fragments. Results: The full-length protein and two other fragments reacted with all 52 sera tested. Four important regions with possible epitopes were identified and named as EP1 (amino acids 51–71), EP2 (134–208), EP3 (249–273), and EP4 (349–422), respectively. EP2 and EP4 possessed linear epitopes, whereas EP1 and EP2 were able to form conformational epitopes that could react with most (>80%) of the tested sera. EP3 and EP4 also formed conformational epitopes, and antibodies against these epitopes existed in all 52 of the sera tested. Conclusion: The N protein is a highly immunogenic protein of the SARS-CoV. Conformational epitopes are important for this protein, and antigenicity of the COOH terminus is higher than that of the NH2 terminus. The N protein is a potential diagnostic antigen and vaccine candidate for SARS-CoV.

Antibody responses to individual proteins of SARS coronavirus and their neutralization activities.

n Abstractn n A novel coronavirus, the severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV), was identified as the causative agent of SARS. The profile of specific antibodies to individual proteins of the virus is critical to the development of vaccine and diagnostic tools. In this study, 13 recombinant proteins associated with four structural proteins (S, E, M and N) and five putative uncharacterized proteins (3a, 3b, 6, 7a and 9b) of the SARS-CoV were prepared and used for screening and monitoring their specific IgG antibodies in SARS patient sera by protein microarray. Antibodies to proteins S, 3a, N and 9b were detected in the sera from convalescent-phase SARS patients, whereas those to proteins E, M, 3b, 6 and 7a were undetected. In the detectable specific antibodies, anti-S and anti-N were dominant and could persist in the sera of SARS patients until week 30. Among the rabbit antisera to recombinant proteins S3, N, 3a and 9b, only anti-S3 serum showed significant neutralizing activity to the SARS-CoV infection in Vero E6 cells. The results suggest (1) that anti-S and anti-N antibodies are diagnostic markers and in particular that S3 is immunogenic and therefore is a good candidate as a subunit vaccine antigen; and (2) that, from a virus structure viewpoint, the presence in some human sera of antibodies reacting with two recombinant polypeptides, 3a and 9b, supports the hypothesis that they are synthesized during the virus cycle.n n

Identification of Signature Genes for Rapid and Specific Characterization of Yersinia pestis

Polymerase chain reaction (PCR) amplification of DNA‐based unique markers, the signature sequences, is ideal for rapid detection and identification of pathogens. We described the discovery of twenty‐eight signature genes of Yersinia pestis by DNA microarray‐based comparative genome hybridization in conjunction with PCR validation. Three pairs of Y. pestis‐specific primers designed from signature genes were demonstrated to have the expected specificity to this target bacterium, without cross‐reaction with the closely related Y. pseudotuberculosis or a large collection of genomic DNAs from other organisms.

Real-Time Polymerase Chain Reaction for Detecting SARS Coronavirus, Beijing, 2003.

During the 2003 severe acute respiratory syndrome (SARS) outbreak, a real-time quantitative polymerase chain reaction, which targets the nucleocapsid gene at the 3′-end of the viral genome, was established to detect and identify the SARS-associated coronavirus. We describe the use of this assay to screen >700 clinical samples.