Wenping Gong

Proteome analysis and serological characterization of surface-exposed proteins of Rickettsia heilongjiangensis.

Background Rickettsia heilongjiangensis, the agent of Far-Eastern spotted fever (FESF), is an obligate intracellular bacterium. The surface-exposed proteins (SEPs) of rickettsiae are involved in rickettsial adherence to and invasion of host cells, intracellular bacterial growth, and/or interaction with immune cells. They are also potential molecular candidates for the development of diagnostic reagents and vaccines against rickettsiosis. Methods R. heilongjiangensis SEPs were identified by biotin-streptavidin affinity purification and 2D electrophoreses coupled with ESI-MS/MS. Recombinant SEPs were probed with various sera to analyze their serological characteristics using a protein microarray and an enzyme-linked immune sorbent assay (ELISA). Results Twenty-five SEPs were identified, most of which were predicted to reside on the surface of R. heilongjiangensis cells. Bioinformatics analysis suggests that these proteins could be involved in bacterial pathogenesis. Eleven of the 25 SEPs were recognized as major seroreactive antigens by sera from R. heilongjiangensis-infected mice and FESF patients. Among the major seroreactive SEPs, microarray assays and/or ELISAs revealed that GroEL, OmpA-2, OmpB-3, PrsA, RplY, RpsB, SurA and YbgF had modest sensitivity and specificity for recognizing R. heilongjiangensis infection and/or spotted fever. Conclusions Many of the SEPs identified herein have potentially important roles in R. heilongjiangensis pathogenicity. Some of them have potential as serodiagnostic antigens or as subunit vaccine antigens against the disease.

Identification of Novel Surface-Exposed Proteins of Rickettsia rickettsii by Affinity Purification and Proteomics

Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever, is the most pathogenic member among Rickettsia spp. Surface-exposed proteins (SEPs) of R. rickettsii may play important roles in its pathogenesis or immunity. In this study, R. rickettsii organisms were surface-labeled with sulfo-NHS-SS-biotin and the labeled proteins were affinity-purified with streptavidin. The isolated proteins were separated by two-dimensional electrophoresis, and 10 proteins were identified among 23 protein spots by electrospray ionization tandem mass spectrometry. Five (OmpA, OmpB, GroEL, GroES, and a DNA-binding protein) of the 10 proteins were previously characterized as surface proteins of R. rickettsii. Another 5 proteins (Adr1, Adr2, OmpW, Porin_4, and TolC) were first recognized as SEPs of R. rickettsii herein. The genes encoding the 5 novel SEPs were expressed in Escherichia coli cells, resulting in 5 recombinant SEPs (rSEPs), which were used to immunize mice. After challenge with viable R. rickettsii cells, the rickettsial load in the spleen, liver, or lung of mice immunized with rAdr2 and in the lungs of mice immunized with other rSEPs excluding rTolC was significantly lower than in mice that were mock-immunized with PBS. The in vitro neutralization test revealed that sera from mice immunized with rAdr1, rAdr2, or rOmpW reduced R. rickettsii adherence to and invasion of vascular endothelial cells. The immuno-electron microscopic assay clearly showed that the novel SEPs were located in the outer and/or inner membrane of R. rickettsii. Altogether, the 5 novel SEPs identified herein might be involved in the interaction of R. rickettsii with vascular endothelial cells, and all of them except TolC were protective antigens.

Recombinant protein YbgF induces protective immunity against Rickettsia heilongjiangensis infection in C3H/HeN mice

BACKGROUND Surface proteins YbgF and PrsA are major seroreactive antigens of Rickettsia heilongjiangensis, the etiological agent of Far-Eastern spotted fever. This study investigated their potential immunogenicity for protective immunity. METHODS Recombinant YbgF and PrsA were used to immunize C3H/HeN mice and rickettsial loads in immunized mouse organs were assessed after R. heilongjiangensis challenge. Anti-sera from immunized mice were applied to neutralize rickettsiae. CD4⁺ and CD8⁺ T cells isolated from R. heilongjiangensis-infected mice were stimulated with YbgF or PrsA, and proliferation and cytokine production assessed. RESULTS The IgG2a/IgG1 ratio of sera was markedly increased in YbgF-immunized mice but was unaltered in PrsA-immunized mice after immunization. The rickettsial load in YbgF-immunized mice was significantly lower than in PrsA-immunized mice after R. heilongjiangensis challenge. Incubation with anti-serum to YbgF, but not PrsA, significantly reduced the number of rickettsiae adhering to and invading endothelial cells. The proliferation level and tumor necrosis factor-α secretion level of CD4⁺ T cells from R. heilongjiangensis-infected mice were significantly higher than in uninfected mice after stimulation with YbgF but not PrsA. CONCLUSION YbgF is a novel protective antigen that induces a Th1-type of protective immune response against R. heilongjiangensis infection.

Exploratory Study on Th1 Epitope-Induced Protective Immunity against Coxiella burnetii Infection

Coxiella burnetii is a Gram-negative bacterium that causes Q fever in humans. In the present study, 131 candidate peptides were selected from the major immunodominant proteins (MIPs) of C. burnetii due to their high-affinity binding capacity for the MHC class II molecule H2 I-Ab based on bioinformatic analyses. Twenty-two of the candidate peptides with distinct MIP epitopes were well recognized by the IFN-γ recall responses of CD4+ T cells from mice immunized with parental proteins in an ELISPOT assay. In addition, 7 of the 22 peptides could efficiently induce CD4+ T cells from mice immunized with C. burnetii to rapidly proliferate and significantly increase IFN-γ production. Significantly higher levels of IL-2, IL-12p70, IFN-γ, and TNF-α were also detected in serum from mice immunized with a pool of the 7 peptides. Immunization with the pool of 7 peptides, but not the individual peptides, conferred a significant protection against C. burnetii infection in mice, suggesting that these Th1 peptides could work together to efficiently activate CD4+ T cells to produce the Th1-type immune response against C. burnetii infection. These observations could contribute to the rational design of molecular vaccines for Q fever.

Rickettsia rickettsii outer membrane protein YbgF induces protective immunity in C3H/HeN mice

Rickettsia rickettsii is the etiological agent of Rocky Mountain spotted fever (RMSF). YbgF and TolC are outer membrane-associated proteins of R. rickettsii that play important roles in its interaction with host cells. We investigated the immunogenicity of YbgF and TolC for protection against RMSF. We immunized C3H/HeN mice with recombinant R. rickettsii YbgF (rYbgF) or TolC (rTolC). Rickettsial burden and impairment in the lungs, spleens, and livers of rYbgF-immunized mice were significantly lower than in rTolC-immunized mice. The ratio of IgG2a to IgG1 in rYbgF-immunized mice continued to increase over the course of our experiments, while that in rTolC-immunized mice was reduced. The proliferation and cytokine secretion of CD4+ and CD8+ T cells isolated from R. rickettsii-infected mice were analyzed following antigen stimulation. The results indicated that proliferation and interferon (IFN)-γ secretion of CD4+ or CD8+ T cells in R. rickettsii-infected mice were significantly greater than in uninfected mice after stimulation with rYbgF. YbgF is a novel protective antigen of R. rickettsii. Protection conferred by YbgF is dependent upon IFN-γ-producing CD4+ and CD8+ T cells and IgG2a, which act in synergy to control R. rickettsii infection.

Surface protein Adr2 of Rickettsia rickettsii induced protective immunity against Rocky Mountain spotted fever in C3H/HeN mice

BACKGROUND Rickettsia rickettsii is the pathogen of Rocky Mountain spotted fever (RMSF), a life-threatening tick-transmitted infection. Adr2 was a surface-exposed adhesion protein of R. rickettsii and its immunoprotection against RMSF was investigated in mice. METHODS Recombinant Adr2 (rAdr2) was used to immunize C3H/HeN mice, and the rickettsial loads in organs of the mice were detected after challenge with R. rickettsii. The levels of specific antibodies of sera from the immunized mice were determined and the sera from immunized mice were applied to neutralize R. rickettsii. Proliferation and cytokine secretion of CD4(+) and CD8(+) T cells isolated from R. rickettsii-infected mice were also assayed after rAdr2 stimulation. RESULTS After R. rickettsii challenge, the rickettsial loads in spleens, livers, and lungs were significantly lower and the impairment degrees of these organs in rAdr2-immunized mice were markedly slighter, compared with those in negative control mice. The ratio of specific IgG2a/IgG1 of rAdr2-immunized mice kept increasing during the immunization. After treatment with rAdr2-immunized sera, the total number of R. rickettsii organisms adhering and invading host cells was significantly lower than that treated with PBS-immunized sera. Interferon-γ secretion by CD4(+) or CD8(+) T cells and tumor necrosis factor-α secretion by CD4(+) T cells from R. rickettsii-infected mice were respectively significantly greater than those from uninfected mice after rAdr2 stimulation. CONCLUSION Adr2 is a protective antigen of R. rickettsii. Protection offered by Adr2 is mainly dependent on antigen-specific cell-mediated immune responses, including efficient activity of CD4(+) and CD8(+) T cells to produce great amount of TNF-α and/or IFN-γ as well as rapid increase of specific IgG2a, which synergistically activate and opsonize host cells to killing intracellular rickettsiae.

Enhanced protection against Rickettsia rickettsii infection in C3H/HeN mice by immunization with a combination of a recombinant adhesin rAdr2 and a protein fragment rOmpB-4 derived from outer membrane protein B

BACKGROUND Two surface proteins of Rickettsia rickettsii, outer membrane protein B (OmpB) and adhesion 2 (Adr2), have been recognized as protective antigens. Herein, the immunization with both OmpB and Adr2 was performed in mice so as to explore whether their combination could induce an enhanced immunoprotection against R. rickettsii infection. METHODS C3H/HeN mice were immunized with recombinant protein rAdr2 or/and rOmp-4, a fragment derived from OmpB, and then mice were challenged with R. rickettsii. After which rickettsial loads in mice were measured by quantitative PCR. The specific antibodies in mouse sera were determined by ELISA and antigen-specific cytokines secretion by mouse T cells were analyzed in vitro. RESULTS After challenge with R. rickettsii, the mice immunized with rAdr2 or/and rOmpB-4 had significant lower rickettsial load in livers, spleens, or lungs compared to PBS mock-immunized mice. Particularly, the load in lungs of mice immunized with both rAdr2 and rOmpB-4 was significantly lower than that with either of them. High levels of specific antibodies were detected in sera from mice immunized with rAdr2 or/and rOmpB-4, but the ratios of specific IgG2a to IgG1 induced by their combination were significantly higher than that by either rAdr2 or rOmpB-4. Following stimulation with rAdr2 or/and rOmpB-4, the INF-γ secreted by CD4(+) T cells from infected mice was significantly higher than that by cognate cells from uninfected mice. And the TNF-α secreted by CD4(+) or CD8(+) T cells from infected mice was markedly greater than that by cognate cells from uninfected mice after stimulation by their combination but not either of them. CONCLUSION The combination of rAdr2 and rOmpB-4 conferred an enhanced protection against R. rickettsii infection in mice, which was mainly dependent on a stronger Th1-oriented immunoresponse with greater INF-γ and TNF-α secretion by antigen-specific T cells and specific IgG2a elicited by the combination.

Serological characterization of surface-exposed proteins of Coxiella burnetii.

The obligate intracellular Gram-negative bacterium Coxiella burnetii causes Q fever, a worldwide zoonosis. Here we labelled Cox. burnetii with biotin and used biotin-streptavidin affinity chromatography to isolate surface-exposed proteins (SEPs). Using two-dimensional electrophoresis combined with mass spectrometry, we identified 37 proteins through bioinformatics analysis. Thirty SEPs expressed in Escherichia coli (recombinant SEPs, rSEPs) were used to generate microarrays, which were probed with sera from mice experimentally infected with Cox. burnetii or sera from Q fever patients. Thirteen rSEPs were recognized as seroreactive, and the majority reacted with at least 50 % of the sera from mice infected with Cox. burnetii but not with sera from mice infected with Rickettsia rickettsii, R. heilongjiangensis, or R. typhi. Further, 13 proteins that reacted with sera from patients with Q fever did not react with sera from patients with brucellosis or mycoplasma pneumonia. Our results suggest that these seroreactive SEPs have potential as serodiagnostic antigens or as subunit vaccine antigens against Q fever.

Genomic and comparative genomic analyses of Rickettsia heilongjiangensis provide insight into its evolution and pathogenesis

Rickettsia heilongjiangensis, the causative agent of far eastern spotted fever, is an obligate intracellular gram-negative bacterium that belongs to the spotted fever group rickettsiae. To understand the evolution and pathogenesis of R. heilongjiangensis, we analyzed its genome and compared it with other rickettsial genomes available in GenBank. The R. heilongjiangensis chromosome contains 1333 genes, including 1297 protein coding genes and 36 RNA coding genes. The genome also contains 121 pseudogenes, 54 insertion sequences, and 39 tandem repeats. Sixteen genes encoding the major components of the type IV secretion systems were identified in the R. heilongjiangensis genome. In total, 37 β-barrel outer membrane proteins were predicted in the genome, eight of which have been previously confirmed to be outer membrane proteins. In addition, 266 potential virulence factor genes, seven partially deleted antibiotic resistance genes, and a genomic island were identified in the genome. The codon usage in the genome is compatible with its low GC content, and the amino acid usage shows apparent bias. A comparative genomic analysis showed that R. heilongjiangensis and R. japonica share one unique fragment that may be a target sequence for a diagnostic assay. The orthologs of 37 genes of R. heilongjiangensis were found in pathogenic R. rickettsii str. Sheila Smith but not in non-pathogenic R. rickettsii str. Iowa, which may explain why R. heilongjiangensis is pathogenic. Pan-genome analysis showed that R. heilongjiangensis and 42 other rickettsiae strains share 693 core genes with a pan-genome size of 4837 genes. The pan-genome-based phylogeny showed that R. heilongjiangensis was closely related to R. japonica.

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 obligate intracellular bacteria, Rickettsia rickettsii and Coxiella burnetii, are the potential agents of bio-warfare/bio-terrorism. Here C3H/HeN mice were immunized with a recombinant protein fragment rOmp-4 derived from outer membrane protein B, a major protective antigen of R. rickettsii, combined with chloroform-methanol residue (CMR) extracted from phase I C. burnetii organisms, a safer Q fever vaccine. These immunized mice had significantly higher levels of IgG1 and IgG2a to rOmpB-4 and interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α), two crucial cytokines in resisting intracellular bacterial infection, as well as significantly lower rickettsial loads and slighter pathological lesions in organs after challenge with R. rickettsii, compared with mice immunized with rOmpB-4 or CMR alone. Additionally, after challenge with C. burnetii, the coxiella loads in the organs of these mice were significantly lower than those of mice immunized with rOmpB-4 alone. Our results prove that CMR could markedly potentiate enhance the rOmpB-4-specific immunoprotection by promoting specific and non-specific immunoresponses and the immunization with the protective antigen of R. rickettsii combined with CMR of C. burnetii could confer effective protection against infection of R. rickettsii or C. burnetii.