Chloroform-Methanol Residue of Coxiella burnetii Markedly Potentiated the Specific Immunoprotection Elicited by a Recombinant Protein Fragment rOmpB-4 Derived from Outer Membrane Protein B of Rickettsia rickettsii in C3H/HeN Mice

The outer-membrane protein OmpW of Vibrio cholerae was studied with respect to its structure, functional properties and regulation of expression. On SDS-PAGE, the membrane-associated form of OmpW protein (solubilized by either 0·1 % or 2 % SDS at 25 °C) migrated as a monomer of 19 kDa that changed to 21 kDa on boiling. The protein was hyperexpressed in Escherichia coli in the histidine-tagged form and the purified His6-OmpW (heated or unheated) migrated as a 23 kDa protein on SDS-PAGE. Circular dichroism and Fourier-transform infrared spectroscopic analyses of the recombinant protein showed the presence of β-structures (∼40 %) with minor amounts (8–15 %) of α-helix. These results were consistent with those obtained by computational analysis of the sequence data of the protein using the secondary structure prediction program Jnet. The recombinant protein did not exhibit any porin-like property in a liposome-swelling assay. An antiserum to the purified protein induced a moderate level (66·6 % and 33·3 % at 1 : 50 and 1 : 100 dilutions, respectively) of passive protection against live vibrio challenge in a suckling mouse model. OmpW-deficient mutants of V. cholerae strains were generated by insertion mutagenesis. In a competitive assay in mice, the intestinal colonization activities of these mutants were found to be either only marginally diminished (for O1 strains) or 10-fold less (for an O139 strain) as compared to those of the corresponding wild-type strains. The OmpW protein was expressed in vivo as well as in vitro in liquid culture medium devoid of glucose. Interestingly, the glucose-dependent regulation of OmpW expression was less prominent in a ToxR− mutant of V. cholerae. Further, the expression of OmpW protein was found to be dependent on in vitro cultural conditions such as temperature, salinity, and availability of nutrients or oxygen. These results suggest that the modulation of OmpW expression by environmental factors may be linked to the adaptive response of the organism under stress conditions.

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Protective immunity against Rickettsia heilongjiangensis in a C3H/HeN mouse model mediated by outer membrane protein B-pulsed dendritic cells

Science China Life Sciences ◽

10.1007/s11427-014-4720-4 ◽

2014 ◽

Vol 58 (3) ◽

pp. 287-296 ◽

Cited By ~ 3

Author(s):

YanFen Meng ◽

XiaoLu Xiong ◽

Yong Qi ◽

ChangSong Duan ◽

WenPing Gong ◽

...

Keyword(s):

Dendritic Cells ◽

Membrane Protein ◽

Mouse Model ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Protective Immunity ◽

Rickettsia Heilongjiangensis ◽

Protein B

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Serological Reactivity and Biochemical Characterization of Methylated and Unmethylated Forms of a Recombinant Protein Fragment Derived from Outer Membrane Protein B of Rickettsia typhi

Clinical and Vaccine Immunology ◽

10.1128/cvi.00281-07 ◽

2008 ◽

Vol 15 (4) ◽

pp. 684-690 ◽

Cited By ~ 15

Author(s):

Chien-Chung Chao ◽

Zhiwen Zhang ◽

Hui Wang ◽

Abdulnaser Alkhalil ◽

Wei-Mei Ching

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Mass Spectrometry Analysis ◽

Enzyme Linked Immunosorbent Assay ◽

Composition Analysis ◽

Rickettsia Typhi ◽

Serological Reactivity ◽

Lysine Residues ◽

Protein B

ABSTRACT Rickettsia typhi, an obligate intracellular bacterium that causes murine typhus, possesses a heavily methylated outer membrane protein B (OmpB) antigen. This immunodominant antigen is responsible for serological reactions and is capable of eliciting protective immune responses with a guinea pig model. Western blot analysis of partially digested OmpB with patient sera revealed that most of the reactive fragments are larger than 20 kDa. One of these fragments, which is located at the N terminus (amino acids 33 to 273), fragment A (At), has been expressed in Escherichia coli. The expressed protein (rAt) was purified by chromatography and properly refolded by sequential dialysis. The refolded rAt protein was recognized by at least 87% of the typhus group patient sera as determined by enzyme-linked immunosorbent assay (ELISA). However, the titers were lower than those obtained with OmpB of R. typhi. Since native OmpB is hypermethylated at lysine residues, we chemically methylated the lysine residues in rAt. The methylation was confirmed by amino acid composition analysis, and the methylation pattern of the methylated rAt (mrAt) protein was similar to that of native At from OmpB, as revealed by liquid chromatography-mass spectrometry analysis. Both rAt and mrAt were evaluated in an ELISA for their serological reactivity with patient sera. Among patient sera tested, 83% exhibited higher titers with mrAt than with rAt. These results suggest that rAt, with or without methylation, can potentially replace rickettsia-derived OmpB or whole-cell antigen for the diagnosis of R. typhi infection.

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