Brucella melitensis is a facultative intracellular bacterial pathogen that causes brucellosis, a zoonotic disease primarily infecting sheep and goats, characterized by undulant fever, arthritic pain and other neurological disorders in humans. A comprehensive proteomic study of strain 16M was conducted to identify and characterize the proteins expressed in laboratory-grown culture. Using overlapping narrow range immobilized pH gradient strips for two-dimensional gel electrophoresis, 883 protein spots were detected between pH 3.5 and 11. The average isoelectric point and molecular weight values of the detected spots were 5.22 and 46.5 kDa, respectively. Of the 883 observed protein spots, 440 have been identified by matrix-assisted laser desorption/ionization-mass spectrometry. These proteins represent 187 discrete open reading frames (ORFs) or 6% of the predicted 3197 ORFs contained in the genome. The corresponding ORFs of the identified proteins are distributed evenly between each of the two circular B. melitensis chromosomes, indicating that both replicons are functionally active. The presented proteome map lists those protein spots identified to date in this study. This map may serve as a baseline reference for future proteomic studies aimed at the definition of biochemical pathways associated with stress responses, host specificity, pathogenicity and virulence. It will also assist in characterization of global proteomic effects in gene-knockout mutants. Ultimately, it may aid in our overall understanding of the cell biology of B. melitensis, an important bacterial pathogen.

The genus Brucella consists of bacterial pathogens that cause brucellosis, a major zoonotic disease characterized by undulant fever and neurological disorders in humans. Among the different Brucella species, Brucella melitensis is considered the most virulent. Despite successful use in animals, the vaccine strains remain infectious for humans. To understand the mechanism of virulence in B. melitensis, the proteome of vaccine strain Rev 1 was analyzed by two-dimensional gel electrophoresis and compared to that of virulent strain 16M. The two strains were grown under identical laboratory conditions. Computer-assisted analysis of the two B. melitensis proteomes revealed proteins expressed in either 16M or Rev 1, as well as up-or down-regulation of proteins specific for each of these strains. These proteins were identified by peptide mass fingerprinting. It was found that certain metabolic pathways may be deregulated in Rev 1. Expression of an immunogenic 31-kDa outer membrane protein, proteins utilized for iron acquisition, and those that play a role in sugar binding, lipid degradation, and amino acid binding was altered in Rev 1.Brucellosis is a major infectious disease afflicting humans and a wide range of domesticated animals worldwide. The disease is caused by several Brucella species, which are aerobic, nonmotile, gram-negative, facultative intracellular coccobacilli. The genus Brucella belongs to the ␣-2 subgroup of the class Proteobacteria. It is subdivided, on the basis of its pathogenicity and host preference, into six nomen species: Brucella abortus, B. canis, B. melitensis, B. neotomae, B. ovis, and B. suis (12). In addition, a new strain affecting marine mammals was recently isolated and tentatively named B. maris (32). On the basis of DNA hybridization data, it was suggested that all of these organisms be placed into a single species, B. melitensis (39). Among the various nomen species, B. abortus, B. canis, B. suis, B. maris, and B. melitensis have been reported to infect humans (21,35,13). B. melitensis is a pathogen of goats and sheep and is considered the most virulent species for humans. Human infection can result from either occupational contact or ingestion of contaminated food.Vaccination and eradication of infected hosts have been key factors in the control of brucellosis. Rev 1, an attenuated strain of virulent B. melitensis, was developed in 1957 (19). It is considered the most effective vaccine for the control of brucellosis in small ruminants and was used in comprehensive vaccination programs in many countries, including Saudi Arabia, Kuwait, Mongolia, Spain, and Turkey (8).Our laboratory has been involved in a comprehensive analysis of the B. melitensis 16M proteome, and initial results have been published recently (40). Previous proteomics studies using B. melitensis cells grown under different conditions have been reported (36, 37), and initial work on the B. abortus proteome has been described (26, 29). A comparative study was conducted with B. abortus vaccine strains S19 and RB51 and...

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Brucella species are pathogenic agents that cause brucellosis, a debilitating zoonotic disease that affects a large variety of domesticated animals and humans. Brucella melitensis and Brucella abortus are considered major health threats because of their highly infectious nature and worldwide occurrence. The availability of the annotated genomes for these two species has allowed a comparative proteomics study of laboratory grown B. melitensis 16M and B. abortus 2308 by two-dimensional (2-D) gel electrophoresis and peptide mass fingerprinting. Computer-assisted analysis of the different 2-D gel images of strains 16M and 2308 revealed significant quantitative and qualitative differences in their protein expression patterns. Proteins involved in membrane transport, particularly the high affinity amino acids binding proteins, and those involved in Sec-dependent secretion systems related to type IV and type V secretion systems, were differentially expressed. Differential expression of these proteins may be responsible for conferring specific host preference in the two strains 2308 and 16M.

Detection of microbial nucleic acids in body fluids has become the preferred method for rapid diagnosis of many infectious diseases. However, culture-based diagnostics that are time-consuming remain the gold standard approach in certain cases, such as sepsis. New culture-free methods are urgently needed. Here, we describe Single MOLecule Tethering or SMOLT, an amplification-free and purification-free molecular assay that can detect microorganisms in body fluids with high sensitivity without the need of culturing. The signal of SMOLT is generated by the displacement of micron-size beads tethered by DNA probes that are between 1 and 7 microns long. The molecular extension of thousands of DNA probes is determined with sub-micron precision using a robust and rapid optical approach. We demonstrate that SMOLT can detect nucleic acids directly in blood, urine and sputum at sub-femtomolar concentrations, and microorganisms in blood at 1 CFU mL−1 (colony forming unit per milliliter) threefold faster, with higher multiplexing capacity and with a more straight-forward protocol than amplified methodologies. SMOLT’s clinical utility is further demonstrated by developing a multiplex assay for simultaneous detection of sepsis-causing Candida species directly in whole blood.

Brucella melitensis is a facultative, intracellular, gram-negative cocco-bacillus that causes Malta fever in humans and brucellosis in animals. There are at least six species in the genus, and the disease is classified as zoonotic because several species infect humans. Using 2-D gel electrophoresis and mass spectrometry, we have initiated (i) a comprehensive mapping and identification of all the expressed proteins of B. melitensis virulent strain 16M, and (ii) a comparative study of its proteome with the attentuated vaccinal strain Rev 1. Comprehensive proteome maps of all six Brucella species will be generated in order to obtain vital information for vaccine development, identification of pathogenicity islands, and establishment of host specificity and evolutionary relatedness.

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