Xiankai Liu

Fuerkang Beijing Institute of Biotechnology, Peping, Beijing, China

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Publications (13)46.07 Total impact

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    ABSTRACT: To screen glycoproteins from Campylobacter jejuni NCTC 11168. We introduced a screening strategy based on specific affinity between lectins and glycoproteins. First, the specific affinity between Soybean Agglutinin (SBA) and glycoproteins in C. jejuni was confirmed by western blotting. Then, magnetic beads coating with Soybean Agglutinin (SBA) were used to capture the putative glycoproteins. Putative glycoproteins were competitively eluted by N-acetylgalactosamine and separated by two-dimensional gel electrophoresis. Spots in gels were further analyzed by mass spectrometry. A total of 22 proteins were identified by mass spectrometric analysis, of which 5 have ever been identified to be glycoproteins. Others including Cj0633 have not been proved to be glycosylated so far. The method could be used to screen glycoproteins in bacteria.
    ACTA MICROBIOLOGICA SINICA 05/2013; 53(5):464-9.
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    ABSTRACT: Shigella flexneri, closely related to Escherichia coli, is the most common cause of the endemic form of shigellosis. In this study, a total of 53 homomultimeric protein complexes and 9 heteromultimeric protein complexes of S. flexneri 2a 2457T were separated and identified. Among these, 4 homomultimeric protein complexes are not previously described. Interestingly, one of them may be composed of twelve PhoN1 subunits, which is a periplasmic protein with unknown physiological role encoded by virulence plasmid of S. flexneri. Then, expressions of protein complexes of S. flexneri grown at 37oC and 30oC were compared. The abundance of 3 protein complexes (PyrB/PyrI, GlmS and MglB) related to the synthesis of lipid polysaccharides (LPS), was changed at two different temperatures. Many studies have shown that LPS is essential to the virulence of S. flexneri. Here, we reported the influence of temperature on the expression of LPS. These findings also expand our knowledge about the protein complexes in S. flexneri and its pathogenesis.
    Molecular &amp Cellular Proteomics 02/2013; · 7.25 Impact Factor
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    ABSTRACT: Immunoproteomics was used to screen the immunogenic spore and vegetative proteins of Bacillus anthracis vaccine strain A16R. The spore and vegetative proteins were separated by 2D gel electrophoresis and transferred to polyvinylidene difluoride membranes, and then western blotting was performed with rabbit immune serum against B.anthracis live spores. Immunogenic spots were cut and digested by trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was performed to identify the proteins. As a result, 11 and 45 immunogenic proteins were identified in the spores and vegetative cells, respectively; 26 of which have not been reported previously. To verify their immunogenicity, 12 of the identified proteins were selected to be expressed, and the immune sera from the mice vaccinated by the 12 expressed proteins, except BA0887, had a specific western blot band with the A16R whole cellular lytic proteins. Some of these immunogenic proteins might be used as novel vaccine candidates themselves or for enhancing the protective efficacy of a protective-antigen-based vaccine.
    PLoS ONE 01/2013; 8(3):e57959. · 3.73 Impact Factor
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    ABSTRACT: The large plasmid pXO1 encoding the anthrax toxin is important for the virulence of Bacillus anthracis. It is essential to cure pXO1 from B. anthracis to evaluate its role in the pathogenesis of anthrax infection. Because conventional methods for curing plasmids (e.g., curing agents or growth at elevated temperatures) can induce mutations in the host chromosomal DNA, we developed a specific and reliable method to eliminate pXO1 from B. anthracis using plasmid incompatibility. Three putative replication origins of pXO1 were inserted into a temperature-sensitive plasmid to generate three incompatible plasmids. One of the three plasmids successfully eliminated the large plasmid pXO1 from B. anthracis vaccine strain A16R and wild type strain A16. These findings provided additional information about the replication/partitioning of pXO1 and demonstrated that introducing a small incompatible plasmid can generate plasmid-cured strains of B. anthracis without inducing spontaneous mutations in the host chromosome.
    PLoS ONE 01/2012; 7(1):e29875. · 3.73 Impact Factor
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    ABSTRACT: Lactobacillus plantarum is a widespread probiotic bacteria found in many fermented food products. In this study, the whole-cell proteins and secretory proteins of L. plantarum were separated by two-dimensional electrophoresis method. A total of 434 proteins were identified by tandem mass spectrometry, including a plasmid-encoded hypothetical protein pLP9000_05. The information of first 20 highest abundance proteins was listed for the further genetic manipulation of L. plantarum, such as construction of high-level expressions system. Furthermore, the first interaction map of L. plantarum was established by Blue-Native/SDS-PAGE technique. A heterodimeric complex composed of maltose phosphorylase Map3 and Map2, and two homodimeric complexes composed of Map3 and Map2 respectively, were identified at the same time, indicating the important roles of these proteins. These findings provided valuable information for the further proteomic researches of L. plantarum.
    PLoS ONE 01/2011; 6(10):e25596. · 3.73 Impact Factor
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    ABSTRACT: Plasmid incompatibility, which has no effect on other plasmids or chromosomal genes, can be used to cure a target plasmid. In this report, we successfully cured the plasmid pXO2 from Bacillus anthracis A16 with a newly constructed, incompatible plasmid pKSV7-oriIV and obtained a new pXO2-cured strain, designated A16PI2. This is the first time that a plasmid was cured from the B. anthracis wild-type strain A16 utilizing this principle, which could be considered as an efficacious method to cure large plasmids.
    Current Microbiology 10/2010; 62(3):703-9. · 1.52 Impact Factor
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    ABSTRACT: To upgrade the proteome reference map of Shigella flexneri 2a 2457T, the protein expression profiles of log phase and stationary phase cells grown at 30 and 37 degrees C were thoroughly analyzed using multiple overlapping narrow pH range (between pH 4.0 and 11.0) two-dimensional gel electrophoresis. A total of 723 spots representing 574 protein entries were identified by MALDI-TOF/TOF MS, including the majority of known key virulence factors. 64 hypothetical proteins and six misannotated proteins were also experimentally identified. A comparison between the four proteome maps showed that most of the virulence-related proteins were up-regulated at 37 degrees C, and the differences were more notable in stationary phase cells, suggesting that the expressions of these virulence factors were not only controlled by temperature but also controlled by the nutrients available in the environment. The expression patterns of some virulence-related genes under the four different conditions suggested that they might also be regulated at the post-transcriptional level. A further significant finding was that the expression of the protein ArgT was dramatically up-regulated at 30 degrees C. The results of semiquantitative RT-PCR analysis showed that expression of argT was not regulated at the transcriptional level. Therefore, we carried out a series of experiments to uncover the mechanism regulating ArgT levels and found that the differential expression of ArgT was due to its degradation by a periplasmic protease, HtrA, whose activity, but not its synthesis, was affected by temperature. The cleavage site in ArgT was between position 160 (Val) and position 161 (Ala). These results may provide useful insights for understanding the physiology and pathogenesis of S. flexneri.
    Molecular &amp Cellular Proteomics 02/2010; 9(6):1209-20. · 7.25 Impact Factor
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    ABSTRACT: Shigella flexneri is a gram-negative, facultative pathogen that causes the majority of communicable bacterial dysenteries in developing countries. The virulence factors of S. flexneri have been shown to be produced at 37 degrees C but not at 30 degrees C. To discover potential, novel virulence-related proteins of S. flexneri, we performed differential in-gel electrophoresis (DIGE) analysis to measure changes in the expression profile that are induced by a temperature increase. The ArgT protein was dramatically down-regulated at 37 degrees C. In contrast, the ArgT from the non-pathogenic E. coli did not show this differential expression as in S. flexneri, which suggested that argT might be a potential anti-virulence gene. Competitive invasion assays in HeLa cells and in BALB/c mice with argT mutants were performed, and the results indicated that the over-expression of ArgTY225D would attenuate the virulence of S. flexneri. A comparative proteomic analysis was subsequently performed to investigate the effects of ArgT in S. flexneri at the molecular level. We show that HtrA is differentially expressed among different derivative strains. Gene argT is a novel anti-virulence gene that may interfere with the virulence of S. flexneri via the transport of specific amino acids or by affecting the expression of the virulence factor, HtrA.
    Proteome Science 01/2010; 8:30. · 2.42 Impact Factor
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    ABSTRACT: Shigella flexneri is an important human pathogen that causes dysentery, and remains a significant threat to public health, particularly in developing countries. The virulence of this pathogen is dependent on an acquired virulence plasmid. To investigate the crosstalk between the bacterial chromosome and the exogenous virulence plasmid, a virulence plasmid-cured strain was constructed using plasmid incompatibility. The global patterns of gene expression of this strain compared with the wild-type strain were analyzed using 2-DE combined with MALDI-TOF MS. Most known virulence factors of S. flexneri were identified in the 2-DE gels. Interestingly, the expression of the glycerol 3-phosphate (glp) regulon-encoded proteins was increased when the virulence plasmid was absent. Microarray analysis confirmed that regulation occurred at the transcriptional level. Purification and identification of DNA binding proteins with affinity for the regulatory region of the glp genes revealed that regulation mediated by the virulence plasmid to control the expression of the glp regulon might in turn be mediated by protein GlpR. To our knowledge, this is the first study analyzing the interaction between a pathogen chromosome and a virulence plasmid at the proteomic level.
    Journal of Proteome Research 12/2009; 9(2):843-54. · 5.06 Impact Factor
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    ABSTRACT: Construction of eag deletion mutant of Bacillus anthracis vaccine strain A16R. To study the function of the gene eag of Bacillus anthracis vaccine strain A16R, according to the sequence of Bacillus anthracis Ames strain, we designed primers and constructed a recombinant plasmid by the spectinomycin resistance cassette, upstream homologous fragment and downstream homologous fragment of eag cloned in tandem in pKSV7. We introduced the recombinant into A16R by electroporation and screened the mutant using the principle of homologous recombination. We checked the mutant using the PCR and proteomics. We constructed the recombinant plasmid successfully and got the eag deletion mutant. PCR results showed the gene eag was deleted; SDS PAGE showed evident differences between prime strain and mutant strain. Two-dimensional gel electrophoresis results displayed three EA1 protein points of prime strain were absent in the mutant strain. We constructed eag deletion mutant of Bacillus anthracis vaccine strain A16R. This research will be helpful to study the functions of eag gene and the other important genes of Bacillus anthracis.
    ACTA MICROBIOLOGICA SINICA 02/2009; 49(1):23-31.
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    ABSTRACT: To analyze the function of htpG of S. flexneri 2a 2457T, we constructed an htpG deletion mutant and a recovery mutant. gamma-Red recombination system was used to construct an htpG deletion mutant of S. flexneri 2a 2457T. In addition, a recover mutant was obtained by introducing a low-copy plasmid containing one copy of htpG gene into the deletion mutant. Then, the growth curves of wild-type strain, deletion mutant and recover mutant were measured. Some of biochemical tests were also investigated. Furthermore, the Sereny tests were performed to evaluate the virulence of these strains. No significant difference were observed among three strains. However, the titers of some inflammatory factors evoked by wild-type strain, deletion mutant and recovery mutant in intraperitoneal injected mice were quite different. These results suggest that the HtpG protein of Shigella flexneri 2a strain 2457T might be involved in the immunopathogenesis.
    ACTA MICROBIOLOGICA SINICA 08/2008; 48(7):905-10.
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    ABSTRACT: A comprehensive proteomic study was carried out to identify and characterize proteins expressed by Bifidobacterium longum NCC2705. A total of 708 spots representing 369 protein entries were identified by MALDI-TOF-MS and/or ESI-MS/MS. Isoelectric point values estimated by gel electrophoresis matched closely with their predicted ones, although some discrepancies exist suggesting that post-translational protein modifications might be common in B. longum. The identified proteins represent 21.4% of the predicted 1727 ORFs in the genome and correspond to 30% of the predicted proteome. Moreover 95 hypothetical proteins were experimentally identified. This is the first compilation of a proteomic reference map for the important probiotic organism B. longum NCC2705. The study aimed to define a number of cellular pathways related to important physiological processes at the proteomic level. Proteomic comparison of glucose- and fructose-grown cells revealed that fructose and glucose are catabolized via the same degradation pathway. Interestingly the sugar-binding protein specific to fructose (BL0033) and Frk showed higher levels of expression in cells grown on fructose than on glucose as determined by semiquantitative RT-PCR. BL0033 time course and concentration experiments showed that the induction time and fructose concentration correlates to increased expression of BL0033. At the same time, an ABC (ATP-binding cassette) transporter ATP-binding protein (BL0034) was slightly up-regulated in cells grown on fructose compared with glucose. All of the above results suggest that the uptake of fructose into the cell may be conducted by a specific transport system in which BL0033 might play an important role.
    Molecular &amp Cellular Proteomics 07/2006; 5(6):1105-18. · 7.25 Impact Factor
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    ABSTRACT: Shigella flexneri 2a is an important pathogen causing bacillary dysentery in humans. In order to investigate any potential vaccine candidate proteins present in outer membrane proteins (OMPs) and extracellular proteins of S. flexneri 2a 2457T, we use the proteome mapping and database analyzing techniques. A subproteome map and database of OMPs were established first. One hundred and nine of the total 126 marked spots were cut out and processed to MALDI-TOF-MS and PMF. Eighty-seven spots were identified and they represented 55 OMP entries. Furthermore, immunoproteomics analysis of OMPs and extracellular proteins were performed. Total of 34 immunoreactive spots were identified, in which 22 and 12 were from OMPs and extracellular proteins, respectively. Eight novel antigens were found and some of these antigens may be potential vaccine candidate proteins. These results are useful for future studying of pathogenicity, vaccine, and novel antibacterial drugs. Maps and tables of all identified proteins are available on the Internet at www.proteomics.com.cn.
    PROTEOMICS 01/2006; 5(18):4777-93. · 4.13 Impact Factor

Publication Stats

107 Citations
46.07 Total Impact Points

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Institutions

  • 2006–2013
    • Fuerkang Beijing Institute of Biotechnology
      Peping, Beijing, China
  • 2010
    • Jiangnan University
      Wu-hsi, Jiangsu Sheng, China
  • 2009
    • Huazhong Agricultural University
      • College of Food Science and Technology
      Wuhan, Hubei, China