Mining the Flavoproteome of Brucella ovis, the Brucellosis Causing Agent in Ovis aries.

Flavoproteins are a diverse class of proteins that are mostly enzymes and contain as cofactors flavin mononucleotide (FMN) and/or flavin adenine dinucleotide (FAD), which enable them to participate in a wide range of physiological reactions. We have compiled 78 potential proteins building the flavoproteome of Brucella ovis (B. ovis), the causative agent of ovine brucellosis. The curated list of flavoproteins here reported is based on (i) the analysis of sequence, structure and function of homologous proteins, and their classification according to their structural domains, clans, and expected enzymatic functions; (ii) the constructed phylogenetic trees of enzyme functional classes using 19 Brucella strains and 26 pathogenic and/or biotechnological relevant alphaproteobacteria together with B. ovis; and (iii) the evaluation of the genetic context for each entry. Candidates account for ∼2.7% of the B. ovis proteome, and 75% of them use FAD as cofactor. Only 55% of these flavoproteins belong to the core proteome of Brucella and contribute to B. ovis processes involved in maintenance activities, survival and response to stress, virulence, and/or infectivity. Several of the predicted flavoproteins are highly divergent in Brucella genus from revised proteins and for them it is difficult to envisage a clear function. This might indicate modified catalytic activities or even divergent processes and mechanisms still not identified. We have also detected the lack of some functional flavoenzymes in B. ovis, which might contribute to it being nonzoonotic. Finally, potentiality of B. ovis flavoproteome as the source of antimicrobial targets or biocatalyst is discussed. IMPORTANCE Some microorganisms depend heavily on flavin-dependent activities, but others maintain them at a minimum. Knowledge about flavoprotein content and functions in different microorganisms will help to identify their metabolic requirements, as well as to benefit either industry or health. Currently, most flavoproteins from the sheep pathogen Brucella ovis are only automatically annotated in databases, and only two have been experimentally studied. Indeed, certain homologues with unknown function are not characterized, and they might relate to still not identified mechanisms or processes. Our research has identified 78 members that comprise its flavoproteome, 76 of them flavoenzymes, which mainly relate to bacteria survival, virulence, and/or infectivity. The list of flavoproteins here presented allows us to better understand the peculiarities of Brucella ovis and can be applied as a tool to search for candidates as new biocatalyst or antimicrobial targets.

Proteomic analysis of Brucella melitensis and Brucella ovis for identification of virulence factor using bioinformatics approachs.

The genus Brucella includes several genetically monomorphic species but with different phenotypic and virulence characteristics. In this study, proteins of two Brucella species, B. melitensis type strain 16 M and B. ovis REO198 were compared by proteomics approach, in order to explain the phenotypic and pathophysiological differences among Brucella species and correlate them with virulence factors. Protein extracts from the two Brucella species were separated by SDS-PAGE and 5 areas, which resulted qualitatively and quantitatively different, were analyzed by nLC-MS/MS. A total of 880 proteins (274 proteins of B. melitensis and 606 proteins of B. ovis) were identified; their functional and structural features were analyzed by bioinformatics tools. Four unique peptides belonging to 3 proteins for B. ovis and 10 peptides derived from 7 proteins for B. melitensis were chosen for the high amount of predicted B-cell epitopes exposed to the solvent. Among these proteins, outer-membrane immunogenic protein (N8LTS7) and 25 kDa outer-membrane immunogenic protein (Q45321), respectively of B. ovis and B. melitensis, could be interesting candidates for improving diagnostics tests and vaccines. Moreover, 8 and 13 outer and periplasmic non homologue proteins of B. ovis and B. melitensis were identified to screen the phenotypic differences between the two Brucella strains. These proteins will be used to unravel pathogenesis and ameliorate current diagnostic assays.

A Carbonic Anhydrase Pseudogene Sensitizes Select Brucella Lineages to Low CO2 Tension.

Brucella spp. are intracellular pathogens that cause a disease known as brucellosis. Though the genus is highly monomorphic at the genetic level, species have animal host preferences and some defining physiologic characteristics. Of note is the requirement for CO2 supplementation to cultivate particular species, which confounded early efforts to isolate B. abortus from diseased cattle. Differences in the capacity of Brucella species to assimilate CO2 are determined by mutations in the carbonic anhydrase gene, bcaA Ancestral single-nucleotide insertions in bcaA have resulted in frameshifted pseudogenes in B. abortus and B. ovis lineages, which underlie their inability to grow under the low CO2 tension of a standard atmosphere. Incubation of wild-type B. ovis in air selects for mutations that "rescue" a functional bcaA reading frame, which enables growth under low CO2 and enhances the growth rate under high CO2 Accordingly, we show that heterologous expression of functional Escherichia coli carbonic anhydrases enables B. ovis growth in air. Growth of B. ovis is acutely sensitive to a reduction in CO2 tension, while frame-rescued B. ovis mutants are insensitive to CO2 shifts. B. ovis initiates a gene expression program upon CO2 downshift that resembles the stringent response and results in transcriptional activation of its type IV secretion system. Our study provides evidence that loss-of-function insertion mutations in bcaA sensitize the response of B. ovis and B. abortus to reduced CO2 tension relative to that of other Brucella lineages. CO2-dependent starvation and virulence gene expression programs in these species may influence persistence or transmission in natural hosts.IMPORTANCEBrucella spp. are highly related, but they exhibit differences in animal host preference that must be determined by genome sequence differences. B. ovis and the majority of B. abortus strains require high CO2 tension to be cultivated in vitro and harbor conserved insertional mutations in the carbonic anhydrase gene, bcaA, which underlie this trait. Mutants that grow in a standard atmosphere, first reported nearly a century ago, are easily selected in the laboratory. These mutants harbor varied indel polymorphisms in bcaA that restore its consensus reading frame and rescue its function. Loss of bcaA function has evolved independently in the B. ovis and B. abortus lineages and results in a dramatically increased sensitivity to CO2 limitation.

The CO2-dependence of Brucella ovis and Brucella abortus biovars is caused by defective carbonic anhydrases.

Brucella bacteria cause brucellosis, a major zoonosis whose control requires efficient diagnosis and vaccines. Identification of classical Brucella spp. has traditionally relied on phenotypic characterization, including surface antigens and 5-10% CO2 necessity for growth (CO2-dependence), a trait of Brucella ovis and most Brucella abortus biovars 1-4 strains. Although molecular tests are replacing phenotypic methods, CO2-dependence remains of interest as it conditions isolation and propagation and reflects Brucella metabolism, an area of active research. Here, we investigated the connection of CO2-dependence and carbonic anhydrases (CA), the enzymes catalyzing the hydration of CO2 to the bicarbonate used by anaplerotic and biosynthetic carboxylases. Based on the previous demonstration that B. suis carries two functional CAs (CAI and CAII), we analyzed the CA sequences of CO2-dependent and -independent brucellae and spontaneous mutants. The comparisons strongly suggested that CAII is not functional in CO2-dependent B. abortus and B. ovis, and that a modified CAII sequence explains the CO2-independent phenotype of spontaneous mutants. Then, by mutagenesis and heterologous plasmid complementation and chromosomal insertion we proved that CAI alone is enough to support CO2-independent growth of B. suis in rich media but not of B. abortus in rich media or B. suis in minimal media. Finally, we also found that insertion of a heterologous active CAII into B. ovis reverted the CO2-dependence but did not alter its virulence in the mouse model. These results allow a better understanding of central aspects of Brucella metabolism and, in the case of B. ovis, provide tools for large-scale production of diagnostic antigens and vaccines.

Spray dried microspheres based on chitosan: A promising new carrier for intranasal administration of polymeric antigen BLSOmp31 for prevention of ovine brucellosis.

Previous studies have demonstrated that parenteral immunization with polymeric antigen BLSOmp31 induced a strong immune response and conferred protection against Brucella ovis in rams. This work describes the development of a novel formulation strategy for the delivery of BLSOmp31 in the nasal mucosa. Chitosan microparticles were prepared by spray-drying technology processes and recombinant chimera BLSOmp31 was loaded by passive adsorption onto chitosan microspheres, which were characterized by means of the evaluation of size, zeta potential, morphology, and loading and release rate of BLSOmp31. The mucoadhesive properties of microspheres were evaluated by studying the interaction between microparticles and mucin. The antigen BLSOmp31 integrity was investigated by SDS-PAGE. The yield of production of spray-drying process was 68.95%. Microspheres had a good sphericity, 1-10 µm of particle size and had a positive charge. The loading capacity was found to be 45.19%. The initial fast release of BLSOmp31 from chitosan microparticles was 60%. The BLSOmp31 integrity was not affected by passive adsorption (ionic interaction). The amount of mucin adsorbed on the surface of CMs-BLSOmp31 was lower than on the surface of blank CMs at neutral pH. In vivo studies were carried out in rams. Intranasal immunization induced systemic and local antibodies. In conclusion, the use of BLSOmp31-loaded chitosan spray-drying microspheres offers a promising way for nasal mucosal vaccination in sheep against brucellosis.

The abcEDCBA-Encoded ABC Transporter and the virB Operon-Encoded Type IV Secretion System of Brucella ovis Are Critical for Intracellular Trafficking and Survival in Ovine Monocyte-Derived Macrophages.

Brucella ovis infection is associated with epididymitis, orchitis and infertility in rams. Most of the information available on B. ovis and host cell interaction has been generated using murine macrophages or epithelial cell lines, but the interaction between B. ovis and primary ovine macrophages has not been studied. The aim of this study was to evaluate the role of the B. ovis abcEDCBA-encoded ABC transporter and the virB operon-encoded Type IV Secretion System (T4SS) during intracellular survival of B. ovis in ovine peripheral blood monocyte-derived macrophages. ΔabcBA and ΔvirB2 mutant strains were unable to survive in the intracellular environment when compared to the WT B. ovis at 48 hours post infection (hpi). In addition, these mutant strains cannot exclude the lysosomal marker LAMP1 from its vacuolar membrane, and their vacuoles do not acquire the endoplasmic reticulum marker calreticulin, which takes place in the WT B. ovis containing vacuole. Higher levels of nitric oxide production were observed in macrophages infected with WT B. ovis at 48 hpi when compared to macrophages infected with the ΔabcBA or ΔvirB2 mutant strains. Conversely, higher levels of reactive oxygen species were detected in macrophages infected with the ΔabcBA or ΔvirB2 mutant strains at 48 hpi when compared to macrophages infected with the WT strain. Our results demonstrate that B. ovis is able to persist and multiply in ovine macrophages, while ΔabcBA and ΔvirB2 mutations prevent intracellular multiplication, favor phagolysosome fusion, and impair maturation of the B. ovis vacuole towards an endoplasmic reticulum-derived compartment.

Encapsulated Brucella ovis Lacking a Putative ATP-Binding Cassette Transporter (ΔabcBA) Protects against Wild Type Brucella ovis in Rams.

This study aimed to evaluate protection induced by the vaccine candidate B. ovis ΔabcBA against experimental challenge with wild type B. ovis in rams. Rams were subcutaneously immunized with B. ovis ΔabcBA encapsulated with sterile alginate or with the non encapsulated vaccine strain. Serum, urine, and semen samples were collected during two months after immunization. The rams were then challenged with wild type B. ovis (ATCC25840), and the results were compared to non immunized and experimentally challenged rams. Immunization, particularly with encapsulated B. ovis ΔabcBA, prevented infection, secretion of wild type B. ovis in the semen and urine, shedding of neutrophils in the semen, and the development of clinical changes, gross and microscopic lesions induced by the wild type B. ovis reference strain. Collectively, our data indicates that the B. ovis ΔabcBA strain is an exceptionally good vaccine strain for preventing brucellosis caused by B. ovis infection in rams.

Natural IS711 insertion causing omp31 gene inactivation in Brucella ovis.

The present report describes an atypical Brucella ovis strain (Bo10) isolated from the epididymis and testis of an infected ram. Macroscopic and microscopic lesions characteristic for the infection, including positive Brucella immunostaining, were observed within lesions in the genital organs. Compared to other isolates, strain Bo10 required an additional day (a total of 96 hr) of incubation to form visible colonies, showed a distinct carbon source utilization profile, agglutinated only weakly with rough (R) serum, but showed a high capacity for autoagglutination. Isolate Bo10 failed to produce the 1,071-bp fragment in the outer membrane protein (omp) 31 gene-based part of the "Bruce-ladder" multiplex polymerase chain reaction system but did produce a 1,915-bp amplicon, thus presenting a profile similar to Brucella abortus. Sequence analysis of the 1,915-bp fragment revealed an 842-bp long insertion sequence (IS)711 transposon element inserted into the promoter region of the omp31 gene, immediately upstream from the ribosome binding site (-10 box/Pribnow box). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of a whole-cell lysate showed the absence in Bo10 of the approximately 31-kDa protein fragment associated with omp31. The results demonstrate a natural inactivation of omp31 and, consequently, the absence of the Omp31 protein in this B. ovis isolate. The novel location of IS711 within the genome of a naturally occurring B. ovis strain supports the hypothesis that IS711 could be an active transposon in this Brucella species.

The virB-encoded type IV secretion system is critical for establishment of infection and persistence of Brucella ovis infection in mice.

Brucella spp. are gram-negative intracellular bacterial pathogens that cause chronic infections. Brucella virulence factors include a type IV secretion system (T4SS) and its lipopolysaccharide (LPS), which are essential for persistence. However, the role of the virB-encoded T4SS has not been investigated in naturally rough Brucella species such as Brucella ovis. In this study, male 6-week old BALBc mice were infected with B. ovis, Brucella abortus, and their respective ΔvirB2 mutant strains. During early infection, B. ovis and B. abortus wild type strains were similarly recovered from spleen. Interestingly, in contrast to ΔvirB2 B. abortus that was recovered at similar levels when compared to the wild type strain, the ΔvirB2 B. ovis was markedly attenuated as early as 24h post infection (hpi). The ΔvirB2 B. ovis was unable to survive and multiply in murine peritoneal macrophages and extracellularly within the peritoneal cavity at 12 and 24 hpi with lower splenic colonization than the parental strain at 6, 12 and 24 hpi. In contrast, wild type B. abortus and ΔvirB2 B. abortus had a similar kinetics of infection in this model. As expected, the T4SS was essential for intracellular replication of smooth and rough strains in RAW macrophages at 48 hpi. These results suggest that T4SS is important for survival of B. ovis in murine model, and that a T4SS deficient B. ovis strain is cleared at earlier stages of infection when compared to a similar B. abortus mutant.

Quorum-sensing and BvrR/BvrS regulation, the type IV secretion system, cyclic glucans, and BacA in the virulence of Brucella ovis: similarities to and differences from smooth brucellae.

Brucella ovis is a rough bacterium--lacking O-polysaccharide chains in the lipopolysaccharide--that is virulent in its natural host and whose virulence mechanisms remain almost unexplored. In a search for additional traits that distinguish B. ovis from smooth Brucella, which require O-polysaccharide chains for virulence, we have analyzed the significance in B. ovis of the main virulence factors described for smooth Brucella. Attempts to obtain strains of virulent B. ovis strain PA that are mutated in the BvrR/BvrS two-component regulatory system were unsuccessful, suggesting the requirement of that system for in vitro survival, while the inactivation of bacA--in contrast to the results seen with smooth Brucella--did not affect splenic colonization in mice or behavior in J774.A1 murine macrophages. Defects in the synthesis of cyclic ß-1,2 glucans reduced the uptake of B. ovis PA in macrophages and, although the intracellular multiplication rate was unaffected, led to attenuation in mice. Growth of strains with mutations in the type IV secretion system (encoded by the virB operon) and the quorum-sensing-related regulator VjbR was severely attenuated in the mouse model, and although the mutant strains internalized like the parental strain in J774.A1 murine macrophages, they were impaired for intracellular replication. As described for B. melitensis, VjbR regulates the transcription of the virB operon positively, and the N-dodecanoyl-dl-homoserine lactone (C(12)-HSL) autoinducer abrogates this effect. In contrast, no apparent VjbR-mediated regulation of the fliF flagellar gene was observed in B. ovis, probably due to the two deletions detected upstream of fliF. These results, together with others reported in the text, point to similarities between rough virulent B. ovis and smooth Brucella species as regards virulence but also reveal distinctive traits that could be related to the particular pathogenicity and host tropism characteristics of B. ovis.