Cloning, Nucleotide Sequence, and Expression of the Brucella melitensis sucB Gene Coding for an Immunogenic Dihydrolipoamide Succinyltransferase Homologous Protein

Abstract

The Brucella melitensis sucB gene encoding the dihydrolipoamide succinyltransferase (E2o) enzyme (previously identified as an immunogenic protein in infected sheep) was cloned and sequenced. The amino acid sequence predicted from the cloned gene revealed 88.8 and 51.2% identity to the dihydrolipoamide succinyltransferase SucB protein from Brucella abortus and Escherichia coli, respectively. Sera from naturally infected sheep showed antibody reactivity against the recombinant SucB protein.Brucella spp. are gram-negative, facultative intracellular bacterial pathogens that cause brucellosis, an infectious disease affecting animals and humans. Brucella melitensis is the most important species involved in ovine and caprine brucellosis, which is characterized by abortion, low production, and infertility in infected animals. B. melitensis is also the most pathogenic species for humans.One of the principal aims in brucellosis research is the identification of Brucella antigens eliciting humoral and/or cellmediated responses, which might be of interest for the development of diagnostic tests or subcellular vaccines that avoid the drawbacks of those currently used. B. melitensis Rev 1, a live attenuated vaccine strain that is currently used in sheep and goats, has been successful in disease eradication and control programs in some countries (1). However, there have been significant problems associated with its use. The most important among them are the residual virulence of Rev1 for humans and the development of agglutinating antibodies in animals vaccinated as adults which are indistinguishable from those elicited by natural infection (8). The construction of new brucellosis vaccines and associated diagnostic tests lacking these indesirable properties would be of great interest to veterinary medicine.A number of immunogenic proteins have been previously identified by immunoblotting, such as the BP26 protein, and are currently being considered for the development of new diagnostic tests for ovine brucellosis (3,6,7,10). Recently, two-dimensional electrophoresis, immunoblotting, and N-terminal microsequencing have considerably facilitated the identification of immunogenic proteins in ovine brucellosis (15,16). Among the proteins identified by these methods, one with an apparent molecular mass of 45 kDa was recognized by sera from Brucella-infected sheep, and its N-terminal sequence showed homology to a dihydrolipoamide succinyltransferase (SucB) described in many bacteria (2,5,9,13,19). A monoclonal antibody (MAb) was raised against this protein (16) to allow easy screening of genomic libraries to clone the corresponding gene. The present report describes the cloning and the nucleotide sequence of the gene termed sucB encoding dihydrolipoamide succinyltransferase (E2o), an enzyme of the ␣-ketoglutarate dehydrogenase complex, and its expression in Escherichia coli.Specificity of the anti-SucB MAb. The MAb raised against Brucella SucB did not cross-react with E. coli and other bacteria closely genetically related to Brucella...

Cloning and Expression of Bartonella henselae sucB Gene Encoding an Immunogenic Dihydrolipoamide Succinyltransferase Homologous Protein

“…In the case of C. burnetii, the antibody responses to SucB protein have been shown to be detected from Q fever patients (16). Furthermore, the SucB proteins of Brucella species, such as B. ovis and B. melitensis have also been found to react with the sera from naturally infected ram and sheep, respectively (24,27). In the present study, the authors showed that the recombinant B. henselae SucB is an immunogenic protein reacting with serum from the immunized mice.…”

Molecular and immunological characterisation of recombinant Brucella abortus glyceraldehyde-3-phosphate-dehydrogenase, a T- and B-cell reactive protein that induces partial protection when co-administered with an interleukin-12-expressing plasmid in a DNA vaccine formulation

“…Several Brucella genes have been cloned, and the proteins that they encode have been characterised [9][10][11][12][13]. However, little is understood regarding the importance of these bacterial proteins in the immune response.…”

Francisella tularensisInfection-Derived Monoclonal Antibodies Provide Detection, Protection, and Therapy

“…Therefore, it is not surprising that the MAbs recognize these antigens from both strains. These two proteins were among the earliest antigens to be identified and have been studied as potential vaccine immunogens (19,20,50,51,62). Both proteins were expressed on the surface of S. enterica serovar Typhimurium (20,51), and in both instances, partial protection resulted, much of which was attributed to the cross-reactivity between the surface antigens of S. enterica serovar Typhimurium and those of F. tularensis.…”

“…(23,33), Brucella spp. (55,62), and Coxiella burnetii (38), and SucC is considered a diagnostic target as well as a potential vaccine candidate in Leptospira spp. (48).…”