Xin Pang

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.

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.

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

Abstract 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.

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.

Bacterial mRNA purification by magnetic capture-hybridization method.

A magnetic capture‐hybridization method was developed for purification of bacterial messenger RNA (mRNA) from total RNA by removing 5S, 16S and 23S ribosomal RNAs (rRNA). The quality of mRNA was evaluated by A260nm/A280nm value, denatured gel electrophoresis and RT‐PCR. The results showed that highly purified and intact mRNA was obtained by this method. The magnetic capture‐hybridization is a rapid and simple method for bacterial mRNA purification and has promising potential for improving studies using bacterial mRNA.