Brucellosis is a worldwide disease of humans and livestock and is caused by closely related classical Brucella species, which are adapted to intracellular life within cells of a variety of mammals: Brucella melitensis (sheep and goats), B. suis (hogs), B. abortus (cattle), B. ovis (sheep), B. canis (dogs), B. neotomae (wood rats), and B. maris (marine mammals). Different Brucella strains have also been isolated from a great variety of wildlife species, and wildlife should always be carefully monitored in order to prevent the re-emergence of brucellosis. Transmission to humans occurs through the consumption of infected, unpasteurized animal-milk products, through direct contact with infected animal parts, and through the inhalation of infected aerosolized particles. Brucellosis is an occupational disease in shepherds, abattoir workers, veterinarians, dairy-industry professionals, and personnel in microbiologic laboratories. Brucellosis remains a major worldwide zoonosis and is of particular concern in South America, given that Brazil and Argentina are the first and fourth world beef and cattle exporters, respectively, accounting for 33 percent of the total world exports in year 2004. The annual economic losses caused by the disease have been estimated at U.S $100,000,000 for both countries. According to official reports, seven to nine percent of farms in this area are infected, with an individual rate of four to five percent infected cattle. Human brucellosis is an important disease that persists in South America, where the infection in animals has not been brought under control; the number of cases reported in remote regions is likely underestimated. Eradication of brucellosis depends largely on socioeconomic and political circumstances. Progress in understanding the molecular pathogenesis of the disease, vaccine engineering, and postgenomic approaches aimed at the discovery of new pathways used by this pathogen to modify the intracellular environment may lead to new preventive interventions.
Brucella can be considered a furtive pathogen. No classical virulence factors, such as exotoxins, cytolysins, capsules, fimbria, flagella, plasmids, lysogenic phages, resistant forms, antigenic variation, endotoxic lipopolysaccharide (LPS), or apoptotic inducers have been described in Brucella organisms. Instead, the true virulence elements of Brucella are those molecular determinants that allow it to invade, resist intracellular killing, and reach a replicating niche in professional and nonprofessional phagocytes. Long-term residence of Brucella in the phagosomal compartment of host macrophages is essential to its ability to produce disease in both natural and experimental host.
Brucella uses anaerobic or microaerobic respiration in the replicative compartment. Riboflavin metabolism and respiration at low oxygen tension are likely to be highly related in pathogens that thrive in microaerobic conditions. We recently reported that Brucella bears an atypical riboflavin metabolic pathway. We are studying the role of riboflavin metabolism on bacterial virulence. We also aim to identify and characterize the sensor and/or regulator molecules involved in sensing oxygen and redox levels of Brucella spp. We conduct these studies using a multidisciplinary approach involving genetic, microbiologic, biochemical, biophysical and structural analyses.
Brucella has an atypical riboflavin-biosynthetic pathway. Riboflavin is the precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), essential cofactors for a multitude of mainstream metabolic enzymes that mediate hydride, oxygen, and electron transfer reactions. Only plants, fungi, and microorganisms can synthesize riboflavin, whereas higher animals, including man, must obtain it through diet. The last two steps in the biosynthesis of riboflavin are catalyzed by 6,7-dimethyl-8-ribityllumazine synthase (LS) and riboflavin synthase. The enzyme LS catalyzes the penultimate step in the biosynthesis of riboflavin One important characteristic of this enzyme is the structural quaternary divergence found in different species. The protein exists as pentameric and icosahedral forms, built from similar structural monomeric units. We recently characterized the quaternary structure of B. abortus lumazine synthase (BLS). BLS folds as a highly stable dimer of pentamers, representing a third category of quaternary assembly for lumazine synthase. The new quaternary arrangement described for us raised the question about the biological implications of decameric LS. Recent genomic and bioinformatic analyses suggested that Brucella spp. have two similar genes that code for this catalytic function, namely ribH1 and ribH2. The gene ribH2 codes for the decameric protein that we previously called BLS. We demonstrated by enzymatic and crystallographic studies that RibH2 shows only residual activity as LS and likely evolved for a different, yet undescribed function. We also recently characterized the enzymatic activity and three-dimensional structure of RibH1. Belonging to the pentameric LS family, RibH1 is an enzyme with standard lumazine synthase activity; its gene is included in the riboflavin synthesis operon, located in chromosome I. In contrast, RibH2 is an isolated gene located in chromosome II and regulated by an RFN-box, a highly conserved RNA-regulatory element found frequently in untranslated regions of prokaryotic mRNAs. RibH2 has been extensively studied as a serological marker for active brucellosis, indicating that this protein is expressed at high levels during the host-bacterium interaction. Thus, positive regulation of RibH2 expression by a FMN riboswitch appears to occur during Brucella multiplication inside host macrophages. Besides, recent experiments with RibH2 null mutants of B. abortus show that this protein is a virulence factor, which confers resistance to oxidative stress inside macrophages. We aim to characterize the role of this atypical riboflavin-biosynthetic pathway on the pathogenicity of Brucella spp.
PAS (PER-ARNT-SIM) domains are signaling domains that are widely distributed in proteins from members of the Archaea and Bacteria and from fungi, plants, insects, and vertebrates. They function as input modules in proteins that sense oxygen, redox potential, light, and several other stimuli, the specificity in sensing arising, in part, from different cofactors that may be associated with the PAS fold. In prokaryotes, PAS domains are found almost exclusively in the input domain of sensors of two-component signal transduction systems. Many of the newly identified PAS-containing proteins have been shown to specifically sense oxygen or redox potential. They include the Aer protein, an E. coli signal transducer that responds to changes in the concentration of oxygen, redox carriers, and carbon sources, and FixL from Rhizobium meliloti, which has been shown to be an oxygen-sensing protein. Mycobacterium tuberculosis also has a PAS-like domain two-component system required for protective response to oxidative stress and virulence; the system is known to be involved in oxygen and redox sensing. All these sensors exhibit a sensor PAS module followed by a histidine kinase transmitter domain. A bioinformatics analysis that we recently undertook found that Brucella spp. genomes code for several two-component signal transduction proteins with LOV and PAS domains. We aim to study the function of the Brucella two-component signal transduction proteins that contain PAS and LOV domains by means of genetic, structural, biophysics, and biochemical studies, in order to determine its role in oxygen and redox sensing and in the overall virulence of the bacteria. In collaboration with Roberto Bogomolni´s laboratory (Department of Chemistry and Biochemistry, University of California, Santa Cruz, California), we were able to demonstrate recently that a Light-Activated LOV-Histidine Kinase acts as a virulence factor of Brucella.
Last updated July 2010
