Identification of Brucella abortus genes in elk (Cervus elaphus) using in vivo-induced antigen technology (IVIAT) reveals novel markers of infection

Lowry, Jake E.; Goodridge, Lawrence; Vernati, Giulia; Fluegel, A.M.; Edwards, William H.; Andrews, Gerard P.

doi:10.1016/j.vetmic.2009.10.010

Cited by 17 publications

(14 citation statements)

References 28 publications

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“…Hia was not identified as an IVI gene in our initial study [20], and consistent with this finding, was subsequently was found to be constitutively expressed based on our mRNA analysis (data not shown). Our selection of this gene product was based on similar homology to Y. pestis protein and previous reports in the literature of involvement as a virulence factor [32].…”

Section: Discussionsupporting

confidence: 80%

“…In contrast to AfuA and Mdh, D15 expression was not evident early in S19 colonization, thus D15 may be relevant at a later stage of infection in the mouse. Curiously, in our previous study [20], we failed to detect antibody to Mdh and D15 in S19-immunized elk, indicating that both proteins may be regulated differently in cervids.…”

Section: Discussionmentioning

confidence: 68%

“…We previously applied the gene discovery methodology, known as in vivo-induced antigen technology (IVIAT), to identify B. abortus virulence genes up-regulated during infection in elk ( Cervis elaphus ), and as a result have identified ten loci with gene products potentially important to survival of the pathogen in this host [20]. Furthermore, the conserved nature of most of these gene products has led us to extend our hypothesis - that they also may be requisite virulence effectors in other Brucella susceptible hosts.…”

Section: Introductionmentioning

confidence: 99%

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Vaccination with Brucella abortus Recombinant In Vivo-Induced Antigens Reduces Bacterial Load and Promotes Clearance in a Mouse Model for Infection

et al. 2011

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Current vaccines used for the prevention of brucellosis are ineffective in inducing protective immunity in animals that are chronically infected with Brucella abortus, such as elk. Using a gene discovery approach, in vivo-induced antigen technology (IVIAT) on B. abortus, we previously identified ten loci that encode products up-regulated during infection in elk and consequently may play a role in virulence. In our present study, five of the loci (D15, 0187, VirJ, Mdh, AfuA) were selected for further characterization and compared with three additional antigens with virulence potential (Hia, PrpA, MltA). All eight genes were PCR-amplified from B. abortus and cloned into E. coli. The recombinant products were then expressed, purified, adjuvanted, and delivered subcutaneously to BALB/c mice. After primary immunization and two boosts, mice were challenged i.p. with 5×104 CFU of B. abortus strain 19. Spleens from challenged animals were harvested and bacterial loads determined by colony count at various time points. While vaccination with four of the eight individual proteins appeared to have some effect on clearance kinetics, mice vaccinated with recombinant Mdh displayed the most significant reduction in bacterial colonization. Furthermore, mice immunized with Mdh maintained higher levels of IFN-γ in spleens compared to other treatment groups. Collectively, our in vivo data gathered from the S19 murine colonization model suggest that vaccination with at least three of the IVIAT antigens conferred an enhanced ability of the host to respond to infection, reinforcing the utility of this methodology for the identification of potential vaccine candidates against brucellosis. Mechanisms for immunity to one protein, Mdh, require further in vitro exploration and evaluation against wild-type B. abortus challenge in mice, as well as other hosts. Additional studies are being undertaken to clarify the role of Mdh and other IVI antigens in B. abortus virulence and induction of protective immunity.

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Section: Discussionsupporting

confidence: 80%

Section: Discussionmentioning

confidence: 68%

Section: Introductionmentioning

confidence: 99%

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Vaccination with Brucella abortus Recombinant In Vivo-Induced Antigens Reduces Bacterial Load and Promotes Clearance in a Mouse Model for Infection

et al. 2011

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“…abortus MDH was an important antigenic protein, which plays important roles during the infection [10]. Furthermore, vaccination with bacterial-expressed B .…”

Section: Discussionmentioning

confidence: 99%

“…Recent studies reported that B . abortus MDH was an important antigenic protein during the infection [10] and vaccination with bacterial-expressed B . abortus MDH enhanced the protection to Brucella infection [11].…”

Section: Introductionmentioning

confidence: 99%

Enzymatic Activity Analysis and Catalytic Essential Residues Identification ofBrucella abortusMalate Dehydrogenase

Han

Tong

Tian

et al. 2014

The Scientific World Journal

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Malate dehydrogenase (MDH) plays important metabolic roles in bacteria. In this study, the recombinant MDH protein (His-MDH) of Brucella abortus was purified and its ability to catalyze the conversion of oxaloacetate (OAA) to L-malate (hereon referred to as MDH activity) was analyzed. Michaelis Constant (Km) and Maximum Reaction Velocity (Vmax) of the reaction were determined to be 6.45 × 10−3 M and 0.87 mM L−1 min−1, respectively. In vitro studies showed that His-MDH exhibited maximal MDH activity in pH 6.0 reaction buffer at 40°C. The enzymatic activity was 100%, 60%, and 40% inhibited by Cu2+, Zn2+, and Pb2+, respectively. In addition, six amino acids in the MDH were mutated to investigate their roles in the enzymatic activity. The results showed that the substitutions of amino acids Arg 89, Asp 149, Arg 152, His 176, or Thr 231 almost abolished the activity of His-MDH. The present study will help to understand MDH's roles in B. abortus metabolism.

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Characterization and reactivity of broiler chicken sera to selected recombinant Campylobacter jejuni chemotactic proteins

et al. 2014

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Campylobacter jejuni, a Gram-negative rod bacterium, is the leading causative agent of human acute bacterial gastroenteritis worldwide. Consumption and handling of raw or undercooked poultry are regarded as a major source for human infection. Because bacterial chemotaxis guides microorganisms to colonization and invasion in the host cells, proteins involved in chemotactic processes can be novel targets for vaccine development. In this communication, we report amplification, cloning and expression of the C. jejuni chemotactic proteins in an Escherichia coli expression system. A total of 15 chemotactic protein genes were successfully expressed. These recombinant proteins were confirmed by nucleotide sequencing, SDS-PAGE analysis and immunoblot analysis of six-His and hemagglutinin tags. Twelve recombinant chemotactic proteins were further tested whether they were antigenic using sera from broiler chickens older than 4 weeks. The immunoblot results show that each chicken serum reacted to a variety of the recombinant proteins, but all sera reacted to the Cjj0473 gene product (annotated as a methyl-accepting chemotaxis protein), suggesting that anti-Campylobacter antibodies may be prevalent in the poultry population. These antibody screening results provide a rationale for further evaluation of the Cjj0473 protein as a potential vaccine for broilers to improve human food safety.

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Identification of Brucella abortus genes in elk (Cervus elaphus) using in vivo-induced antigen technology (IVIAT) reveals novel markers of infection

Cited by 17 publications

References 28 publications

Vaccination with Brucella abortus Recombinant In Vivo-Induced Antigens Reduces Bacterial Load and Promotes Clearance in a Mouse Model for Infection

Vaccination with Brucella abortus Recombinant In Vivo-Induced Antigens Reduces Bacterial Load and Promotes Clearance in a Mouse Model for Infection

Enzymatic Activity Analysis and Catalytic Essential Residues Identification ofBrucella abortusMalate Dehydrogenase

Characterization and reactivity of broiler chicken sera to selected recombinant Campylobacter jejuni chemotactic proteins

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