Increased Brucella abortus asRNA_0067 expression under intraphagocytic stressors is associated with enhanced virB2 transcription.

Intracellular pathogens like Brucella face challenges during the intraphagocytic adaptation phase, where the modulation of gene expression plays an essential role in taking advantage of stressors to persist inside the host cell. This study aims to explore the expression of antisense virB2 RNA strand and related genes under intracellular simulation media. Sense and antisense virB2 RNA strands increased expression when nutrient deprivation and acidification were higher, being starvation more determinative. Meanwhile, bspB, one of the T4SS effector genes, exhibited the highest expression during the exposition to pH 4.5 and nutrient abundance. Based on RNA-seq analysis and RACE data, we constructed a regional map depicting the 5' and 3' ends of virB2 and the cis-encoded asRNA_0067. Without affecting the CDS or a possible autonomous RBS, we generate the deletion mutant ΔasRNA_0067, significantly reducing virB2 mRNA expression and survival rate. These results suggest that the antisense asRNA_0067 expression is promoted under exposure to the intraphagocytic adaptation phase stressors, and its deletion is associated with a lower transcription of the virB2 gene. Our findings illuminate the significance of these RNA strands in modulating the survival strategy of Brucella within the host and emphasize the role of nutrient deprivation in gene expression.

An altered fecal microbiota profile in patients with non-alcoholic fatty liver disease (NAFLD) associated with obesity.

INTRODUCTION: increasing evidence suggests a role of intestinal dysbiosis in obesity and non-alcoholic fatty liver disease (NAFLD). The advances in recent years with regard to the role of the gut microbiota raise the potential utility of new therapeutic approaches based on the modification of the microbiome. OBJECTIVE: the aim of this study was to compare the bacterial communities in obese patients with or without NAFLD to those of healthy controls. PATIENTS AND METHODS: the fecal microbiota composition of 20 healthy adults, 36 obese patients with NAFLD and 17 obese patients without NAFLD was determined by 16S ribosomal RNA sequencing using the Illumina MiSeq system. RESULTS: the results highlighted significant differences in the phylum Firmicutes between patients with and without NAFLD, which was a determining factor of the disease and supported its possible role as a marker of NAFLD. At the genus level, the relative abundance of Blautia, Alkaliphilus, Flavobacterium and Akkermansia was reduced in obese patients, both with or without NAFLD, compared to healthy controls. Furthermore, the number of sequences from the genus Streptococcus was significantly higher in patients with NAFLD in comparison with individuals without the disease, constituting another possible marker. Comparison of bacterial communities at the genus level by a principal coordinate analysis indicated that the bacterial communities of patients with NAFLD were dispersed and did not form a group. CONCLUSION: in conclusion, these results indicate the role of intestinal dysbiosis in the development of NAFLD associated with obesity. There was a differential microbiota profile between obese patients, with and without NAFLD. Thus, supporting gut microbiota modulation as a therapeutic alternative for the prevention and treatment of NAFLD.

Survival of Brucella abortus aqpX mutant in fresh and ripened cheeses.

The objective of this work was to evaluate the survival of a Brucella abortus aqpX mutant during the elaboration and conservation of fresh and ripened cheeses at 4 °C and 24 °C. The pH values and water activity were monitored for each type of cheese. The fresh cheese was elaborated with raw milk inoculated with 6×108 colony-forming units (CFU)/mL each of parental and mutant strain. Ripening cheeses were elaborated with both raw and pasteurized milk and inoculated with 12×108 CFU/mL each of parental and mutant strains. In fresh cheese, survival was observed during elaboration and conservation for 7 days at 4 °C in mutant and parental strains. The number of survivors of the mutant strain was 10 times lower compared with the parental strain at pH 5 and a(w) of 0.930. In the cheese elaborated with raw milk and ripened at 24 °C, both strains survived until day 17 at pH 4.0 and a(w) of 0.89. However, when the cheese was elaborated with pasteurized milk, the parental strain survived until day 31 of ripening, and the mutant strain survived 24 days at pH 4 and a(w) of 0.886. The survival of the mutant strain showed a diminution of one logarithm during elaboration and ripening of cheese as compared with the parental strain. When the cheese was elaborated with raw milk and ripened at 4 °C, survival of the parental strain was 24 days, whereas the mutant strain survived only 17 days (pH 5 and a(w) 0.90). Regarding the cheese elaborated with pasteurized milk and maturated at 4 °C, both strains survived 31 days (pH 5 and a(w) 0.90), with the same survival diminution during elaboration and ripening. Our results show that in both types of cheese, the mutated aqpX strain survived 10 times less than the parental strain, which shows that the aqpX gene can be related to the survival of Brucella abortus in this type of cheese.

A new family of enzymes catalyzing the first committed step of the methylerythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in bacteria.

Isoprenoids are a large family of compounds with essential functions in all domains of life. Most eubacteria synthesize their isoprenoids using the methylerythritol 4-phosphate (MEP) pathway, whereas a minority uses the unrelated mevalonate pathway and only a few have both. Interestingly, Brucella abortus and some other bacteria that only use the MEP pathway lack deoxyxylulose 5-phosphate (DXP) reductoisomerase (DXR), the enzyme catalyzing the NADPH-dependent production of MEP from DXP in the first committed step of the pathway. Fosmidomycin, a specific competitive inhibitor of DXR, inhibited growth of B. abortus cells expressing the Escherichia coli GlpT transporter (required for fosmidomycin uptake), confirming that a DXR-like (DRL) activity exists in these bacteria. The B. abortus DRL protein was found to belong to a family of uncharacterized proteins similar to homoserine dehydrogenase. Subsequent experiments confirmed that DRL and DXR catalyze the same biochemical reaction. DRL homologues shown to complement a DXR-deficient E. coli strain grouped within the same phylogenetic clade. The scattered taxonomic distribution of sequences from the DRL clade and the occurrence of several paralogues in some bacterial strains might be the result of lateral gene transfer and lineage-specific gene duplications and/or losses, similar to that described for typical mevalonate and MEP pathway genes. These results reveal the existence of a novel class of oxidoreductases catalyzing the conversion of DXP into MEP in prokaryotic cells, underscoring the biochemical and genetic plasticity achieved by bacteria to synthesize essential compounds such as isoprenoids.

Imputation of SNPs associated with presbycusis through linkage disequilibrium analysis in the ILDR1 gene.

The single-nucleotide polymorphisms (SNP) ILDR1 rs2332035 has shown a high statistical association with presbycusis (hearing loss with age or age-related hearing impairment (ARHI)), according to genetic association studies in European populations. However, linked markers have not been surveyed. Here linkage disequilibrium (LD) of markers in ILDR1, in relation to rs2332035, is explored in the 2504 individuals from the 1000Genomes database. Of the 920 SNPs retrieved, 10 showed strong LD (r2= 0.8) in Europeans and Latin Americans, which are proposed here as candidate markers for both control-case association and cause-effect studies in both populations.

Analysis of laccase-like enzymes secreted by fungi isolated from a cave in northern Spain.

Laccases belong to a family of multicopper enzymes able to oxidize a broad spectrum of organic compounds. Despite the well-known property of laccases to carry out bleaching and degradation of industrial dyes and polyphenolic compounds, their industrial use is often limited by the high cost, low efficiency, or instability of these enzymes. To look for new microorganisms which produce laccases that are potentially suitable for industrial applications, we have isolated several fungal strains from a cave in northern Spain. Their phenotypic analysis on agar plates supplemented with ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) disclosed two laccase-positive strains. Further genotyping revealed that they belonged to the Gliomastix murorum and Conidiobolus thromboides species. The secretion of G. murorum and C. thromboides laccase-like enzymes was then confirmed by zymography. Further identification of these polypeptides by mass-spectroscopy revealed the nature of the laccases and made it possible to predict their functional domains and other features. In addition, plate assays revealed that the laccases secreted by both G. murorum and C. thromboides were capable of degrading industrial dyes (Congo Red, Indigo, and Eriochrome Black T). Homology modeling and substrate docking predicted the putative structure of the currently uncrystallized G. murorum enzyme as well as its amino acid residues potentially involved in interactions with these dyes. In summary, new biochemical and structural insights into decolorization mediated by G. murorum laccase as well as identification of laccase-like oxidase in C. thromboides point to a promising future for these enzymes in biotechnology.

Brucella abortus ure2 region contains an acid-activated urea transporter and a nickel transport system.

BACKGROUND: Urease is a virulence factor that plays a role in the resistance of Brucella to low pH conditions, both in vivo and in vitro. Brucella contains two separate urease gene clusters, ure1 and ure2. Although only ure1 codes for an active urease, ure2 is also transcribed, but its contribution to Brucella biology is unknown. RESULTS: Re-examination of the ure2 locus showed that the operon includes five genes downstream of ureABCEFGDT that are orthologs to a nikKMLQO cluster encoding an ECF-type transport system for nickel. ureT and nikO mutants were constructed and analyzed for urease activity and acid resistance. A non-polar ureT mutant was unaffected in urease activity at neutral pH but showed a significantly decreased activity at acidic pH. It also showed a decreased survival rate to pH 2 at low concentration of urea when compared to the wild type. The nikO mutant had decreased urease activity and acid resistance at all urea concentrations tested, and this phenotype could be reverted by the addition of nickel to the growth medium. CONCLUSIONS: Based on these results, we concluded that the operon ure2 codes for an acid-activated urea transporter and a nickel transporter necessary for the maximal activity of the urease whose structural subunits are encoded exclusively by the genes in the ure1 operon.

Polymorphisms in Brucella Carbonic Anhydrase II Mediate CO2 Dependence and Fitness in vivo.

Some Brucella isolates are known to require an increased concentration of CO2 for growth, especially in the case of primary cultures obtained directly from infected animals. Moreover, the different Brucella species and biovars show a characteristic pattern of CO2 requirement, and this trait has been included among the routine typing tests used for species and biovar differentiation. By comparing the differences in gene content among different CO2-dependent and CO2-independent Brucella strains, we have confirmed that carbonic anhydrase (CA) II is the enzyme responsible for this phenotype in all the Brucella strains tested. Brucella species contain two CAs of the ß family, CA I and CA II; genetic polymorphisms exist for both of them in different isolates, but only those putatively affecting the activity of CA II correlate with the CO2 requirement of the corresponding isolate. Analysis of these polymorphisms does not allow the determination of CA I functionality, while the polymorphisms in CA II consist of small deletions that cause a frameshift that changes the C-terminus of the protein, probably affecting its dimerization status, essential for the activity. CO2-independent mutants arise easily in vitro, although with a low frequency ranging from 10-6 to 10-10 depending on the strain. These mutants carry compensatory mutations that produce a full-length CA II. At the same time, no change was observed in the sequence coding for CA I. A competitive index assay designed to evaluate the fitness of a CO2-dependent strain compared to its corresponding CO2-independent strain revealed that while there is no significant difference when the bacteria are grown in culture plates, growth in vivo in a mouse model of infection provides a significant advantage to the CO2-dependent strain. This could explain why some Brucella isolates are CO2 dependent in primary isolation. The polymorphism described here also allows the in silico determination of the CO2 requirement status of any Brucella strain.

Demonstration of IS711 transposition in Brucella ovis and Brucella pinnipedialis.

BACKGROUND: The Brucella genome contains an insertion sequence (IS) element called IS711 or IS6501, which is specific to the genus. The copy number of IS711 varies in the genome of the different Brucella species, ranging from 7 in B. abortus, B. melitensis and B. suis to more than 30 in B. ovis and in Brucella strains isolated from marine mammals. At present, there is no experimental evidence of transposition of IS711, but the occurrence of this element with a high copy number in some species, and the isolation of Brucella strains with "ectopic" copies of IS711 suggested that this IS could still transpose. RESULTS: In this study we obtained evidence of transposition of IS711 from the B. ovis and B. pinnipedialis chromosomes by using the "transposon trap" plasmid pGBG1. This plasmid expresses resistance to tetracycline only if the repressor gene that it contains is inactivated. The strains B. melitensis 16 M, B. abortus RB51, B. ovis BOC22 (field strain) and B. pinnipedialis B2/94, all containing the plasmid pGBG1, were grown in culture media with tetracycline until the appearance of tetracycline resistant mutants (TcR). TcR mutants due to IS711 transposition were only detected in B. ovis and B. pinnipedialis strains. CONCLUSION: Four different copies of IS711 were found to transpose to the same target sequence in the plasmid pGBG1. This demonstrated that IS711 are active in vivo, specially in Brucella species with a high number of IS711 copies as B. ovis and B. pinnipedialis.

A virus-encoded type I interferon decoy receptor enables evasion of host immunity through cell-surface binding.

Soluble cytokine decoy receptors are potent immune modulatory reagents with therapeutic applications. Some virus-encoded secreted cytokine receptors interact with glycosaminoglycans expressed at the cell surface, but the biological significance of this activity in vivo is poorly understood. Here, we show the type I interferon binding protein (IFNα/ßBP) encoded by vaccinia and ectromelia viruses requires of this cell binding activity to confer full virulence to these viruses and to retain immunomodulatory activity. Expression of a variant form of the IFNα/ßBP that inhibits IFN activity, but does not interact with cell surface glycosaminoglycans, results in highly attenuated viruses with a virulence similar to that of the IFNα/ßBP deletion mutant viruses. Transcriptomics analysis and infection of IFN receptor-deficient mice confirmed that the control of IFN activity is the main function of the IFNα/ßBP in vivo. We propose that retention of secreted cytokine receptors at the cell surface may largely enhance their immunomodulatory activity.

RNA-Seq Based Transcriptome Analysis of the Type I Interferon Host Response upon Vaccinia Virus Infection of Mouse Cells.

Vaccinia virus (VACV) encodes the soluble type I interferon (IFN) binding protein B18 that is secreted from infected cells and also attaches to the cell surface, as an immunomodulatory strategy to inhibit the host IFN response. By using next generation sequencing technologies, we performed a detailed RNA-seq study to dissect at the transcriptional level the modulation of the IFN based host response by VACV and B18. Transcriptome profiling of L929 cells after incubation with purified recombinant B18 protein showed that attachment of B18 to the cell surface does not trigger cell signalling leading to transcriptional activation. Consistent with its ability to bind type I IFN, B18 completely inhibited the IFN-mediated modulation of host gene expression. Addition of UV-inactivated virus particles to cell cultures altered the expression of a set of 53 cellular genes, including genes involved in innate immunity. Differential gene expression analyses of cells infected with replication competent VACV identified the activation of a broad range of host genes involved in multiple cellular pathways. Interestingly, we did not detect an IFN-mediated response among the transcriptional changes induced by VACV, even after the addition of IFN to cells infected with a mutant VACV lacking B18. This is consistent with additional viral mechanisms acting at different levels to block IFN responses during VACV infection.

Functional characterization of SDF-1 proximal promoter.

Stromal-cell derived factor 1 (SDF1) is a CXC chemokine that binds and signals through the CXCR4 receptor, playing an essential role in embryonic B lymphopoiesis, myelopoiesis and organogenesis. The CXCR4/SDF1 pathway is associated with several pathologies. CXCR4 serves as a fusion cofactor for lymphotropic strains of human immunodeficiency virus type 1 and SDF1 inhibits viral entry. Moreover, recent works suggest an important role for SDF1 in metastasis progression and autoimmune diseases such as rheumatoid arthritis. To understand the molecular mechanisms that regulate SDF1 expression, we have cloned and functionally analysed its 5' flanking regulatory region. An SDF1-promoter luciferase construct showed high levels of reporter gene activity in transient transfection experiments. DNase I footprinting analysis revealed that the proximal promoter was occupied by six putative Sp1-binding motifs. Binding of Sp1 to the promoter was confirmed by electrophoretic mobility shift assay, and its importance in SDF1 gene expression verified by in vitro mutagenesis. Particularly, mutation of an Sp1 motif located between -57 and -39 upstream of the main transcription start-site resulted in a marked reduction in promoter activity. It has been shown that the SDF1 expression could be induced by mitogenic stimuli, X-ray radiation or treatment with IL1beta, depending on cell environment. We have analysed the effect of these stimuli on SDF1 promoter transactivation in three different cell lines. Phorbol myristated acetate plus ionomycin increased promoter activity in U373 and LC5 but repressed it in MS5 cells. On the contrary, gamma irradiation promoted SDF1 transcription in MS5 cells but not in the other cell lines. Interferon-gamma acted as a transcriptional repressor in U373 and LC5 but not in MS5 cells. Finally, IL1beta functions as mild activator only in U373 cells. The present study demonstrates that these stimuli mediate SDF1 production through promoter activation in a cell-specific manner.

Metagenomic analysis of lacustrine viral diversity along a latitudinal transect of the Antarctic Peninsula.

Environmental viruses constitute the most abundant biological entities on earth, and harbor an enormous genetic diversity. While their strong influence on the ecosystem is widely acknowledged, current knowledge about their diversity and distribution remains limited. Here we present the metagenomic study of viral communities from freshwater bodies located along a transect of the Antarctic Peninsula. These ecosystems were chosen on the basis of environmental and biogeographical variation. The results obtained indicate that the virus assemblages were diverse, and that the larger fraction represented viruses with no close relatives in the databases. Comparisons to existing metaviromes showed that the communities studied were dissimilar to other freshwater viromes including those from the Arctic. Finally, we observed no indication of there being a reduction in either viral richness or diversity estimates with increasing latitude along the studied transect, further adding to the controversy regarding the possible existence of latitudinal gradients of diversity in the microbial world.

First-in-class inhibitor of the T cell receptor for the treatment of autoimmune diseases.

Modulating T cell activation is critical for treating autoimmune diseases but requires avoiding concomitant opportunistic infections. Antigen binding to the T cell receptor (TCR) triggers the recruitment of the cytosolic adaptor protein Nck to a proline-rich sequence in the cytoplasmic tail of the TCR's CD3ε subunit. Through virtual screening and using combinatorial chemistry, we have generated an orally available, low-molecular weight inhibitor of the TCR-Nck interaction that selectively inhibits TCR-triggered T cell activation with an IC50 (median inhibitory concentration) ~1 nM. By modulating TCR signaling, the inhibitor prevented the development of psoriasis and asthma and, furthermore, exerted a long-lasting therapeutic effect in a model of autoimmune encephalomyelitis. However, it did not prevent the generation of a protective memory response against a mouse pathogen, suggesting that the compound might not exert its effects through immunosuppression. These results suggest that inhibiting an immediate TCR signal has promise for treating a broad spectrum of human T cell-mediated autoimmune and inflammatory diseases.

Development of a new PCR assay to identify Brucella abortus biovars 5, 6 and 9 and the new subgroup 3b of biovar 3.

One hundred twenty-nine Brucella field strains isolated from cattle in Cantabria, Spain, from March 1999 to February 2003, were analysed by using the AMOS-ERY PCR assay and by Southern blot hybridisation with a probe from insertion sequence IS711. Most of the field isolates produced only the ery band in the AMOS-ERY assay and showed a hybridisation pattern identical to that exhibited by reference strains of biovars 5, 6 and 9 of Brucella abortus, but different from strain Tulya, belonging to biovar 3 of B. abortus. However, typing of these strains by standard methods demonstrated that they belonged to biovar 3 of B. abortus. These results indicated that B. abortus biovar 3 was not genetically homogeneous and at least could be divided in two. In one class, that we called biovar 3a, would be the Tulya strain, while the local field strains would belong to biovar 3b. Cloning and nucleotide sequencing of a DNA fragment containing an IS711 copy exclusive of the B. abortus field strains from biovar 3b and reference strains from biovars 5, 6 and 9, revealed the existence of a 5.4 kb deletion close to an IS711 copy. Based on these data, we designed a new primer, which together with the IS711 AMOS primer produced a PCR fragment of 1.7 kb only from the isolates of biovars 3b, 5, 6 and 9 of B. abortus. No amplification products were produced with these primers from strains of the rest of species and biovars of Brucella and from bacteria phylogenetically close to Brucella analysed in this work. Addition of this primer to the AMOS-ERY PCR primer cocktail allows the positive distinction of B. abortus biovars 3b, 5, 6 and 9 from the rest of Brucella species and biovars.

Neoplasia maligna ginecológica: incidencia en el nuevo milenio: experiencia del servicio oncológico hospitalario (IVSS) / Gynecological malignant neoplasm: incidence in the new millennium: experience of the hospital oncology service (IVSS)

Conocer los indicadores de salud, como una forma de evaluar calidad del servicio que una institución presta a la población. La incidencia, prevalencia y tasas de mortalidad, son tres elementos básicos a conocer, esto permite planificar priorizar las necesidades de una determinada población, mejorando la optimización de recursos y conocer en que eslabón de la historia natural de la enfermedad se puede actuar. Queremos determinar la incidencia registrada en nuestro servicio, desde el 2000 hasta el 2015, de cada una de las patologías malignas atendidas. Un total de 1 824 historias de un universo de 4 911; las restantes no pudieron ser revisadas, por su desincorporación del archivo activo. Apreciamos que la patología con mayor incidencia fue el cáncer de cuello uterino, con un pequeño orcentaje (10 %) iagnosticado en estadio I. Seguidamente encontramos al cáncer de endometrio representando un 12 % de los casos. Dentro de la patología de ovario, el carcinoma epitelial representó el 75 %. El carcinoma de trompa de Falopio solo el 0,3 % de todas las patologías malignas del área inecológica, similar a lo eportado en la literatura mundial. Igualmente el cáncer de vulva, vagina y sarcoma uterino, representaron un escaso porcentaje de incidencia. Este trabajo constituye una fase inicial de investigaciones futuras, en las cuales se deben calcular tasas de upervivencia y período libre de enfermedad, además de incentivar la actualización anual, para evitar sub-registro por la pérdida de datos.(AU)

To know health indicators, is a way to assess the quality of service an institution provides to the population. The incidence, the prevalence and the mortality rates are three basic known elements, which allow you to plan and prioritize the needs of a given population, the improving resource optimization and know that link the natural history of the disease can act. With our research we want to determine the impact registered in our department from the year 2000 to the year 2015, each of the malignant athologies treated. A total of 1 824 stories of a universe of 4 911 were reviewed; the other could not be reviewed by the divestiture of the active file. However, with the data analyzed appreciate that the disease was highest incidence was the cervical cancer, with a small percentage (8 %) diagnosed with stage I, and then found the endometrial cancer representing 12 % of cases. Within pathology ovarian epithelial carcinoma he represented the most frequent with 75 %. The Fallopian tube carcinoma represented only 0.3 % of all malignant gynecological pathologies area, similar to that reported in the literature. Likewise cancer of the vulva, the vagina and the uterine sarcoma, accounted for a small percentage of incidences. This paper is an initial phase of future investigations, which must be calculated survival rates and the disease-free period, in addition to encouraging the annual update, to avoid underreporting by data loss.(AU)

Evaluation of the effects of erythritol on gene expression in Brucella abortus.

Bacteria of the genus Brucella have the unusual capability to catabolize erythritol and this property has been associated with their virulence mainly because of the presence of erythritol in bovine foetal tissues and because the attenuated S19 vaccine strain is the only Brucella strain unable to oxydize erythritol. In this work we have analyzed the transcriptional changes produced in Brucella by erythritol by means of two high throughput approaches: RNA hybridization against a microarray containing most of Brucella ORF's constructed from the Brucella ORFeome and next generation sequencing of Brucella mRNA in an Illumina GAIIx platform. The results obtained showed the overexpression of a group of genes, many of them in a single cluster around the ery operon, able to co-ordinately mediate the transport and degradation of erythritol into three carbon atoms intermediates that will be then converted into fructose-6P (F6P) by gluconeogenesis. Other induced genes participating in the nonoxidative branch of the pentose phosphate shunt and the TCA may collaborate with the ery genes to conform an efficient degradation of sugars by this route. On the other hand, several routes of amino acid and nucleotide biosynthesis are up-regulated whilst amino acid transport and catabolism genes are down-regulated. These results corroborate previous descriptions indicating that in the presence of erythritol, this sugar was used preferentially over other compounds and provides a neat explanation of the the reported stimulation of growth induced by erythritol.

Differences in two-component signal transduction proteins among the genus Brucella: implications for host preference and pathogenesis.

Two-component systems (TCSs) are the predominant bacterial signal transduction mechanisms. Species of the genus Brucella are genetically highly related and differ mainly in mammalian host adaptation and pathogenesis. In this study, TCS proteins encoded in the available genome sequences of Brucella species have been identified using bioinformatic methods. All the Brucella species share an identical set of TCS proteins, and the number of TCS proteins in the closely related opportunistic human pathogen Ochrobactrum anthropi was higher than in Brucella species as expected from its lifestyle. O. anthropi lacks orthologs of the Brucella TCSs NodVW, TceSR and TcfSR, suggesting that these TCS proteins could be necessary for the adaptation of Brucella as an intracellular pathogen. This genomic analysis revealed the presence of a differential distribution of TCS pseudogenes among Brucella species. Moreover, there were also differences in TCS pseudogenes between strains belonging to the same Brucella species, and in particular between B. suis biovars 1 and 2.

Transcriptome analysis of the Brucella abortus BvrR/BvrS two-component regulatory system.

BACKGROUND: The two-component BvrR/BvrS system is essential for Brucella abortus virulence. It was shown previously that its dysfunction alters the expression of some major outer membrane proteins and the pattern of lipid A acylation. To determine the genes regulated by BvrR/BvrS, we performed a whole-genome microarray analysis using B. abortus RNA obtained from wild type and bvrR mutant cells grown in the same conditions. METHODOLOGY/PRINCIPAL FINDINGS: A total of 127 differentially expressed genes were found: 83 were over expressed and 44 were less expressed in the bvrR mutant. Two operons, the phosphotransferase system and the maltose transport system, were down-regulated. Several genes involved in cell envelope or outer membrane biogenesis were differentially expressed: genes for outer membrane proteins (omp25a, omp25d), lipoproteins, LPS and fatty acid biosynthesis, stress response proteins, chaperones, flagellar genes, and twelve genes encoding ABC transport systems. Ten genes related with carbon metabolism (pckA and fumB among others) were up-regulated in the bvrR mutant, and denitrification genes (nirK, norC and nosZ) were also regulated. Notably, seven transcriptional regulators were affected, including VjbR, ExoR and OmpR that were less expressed in the bvrR mutant. Finally, the expression of eleven genes which have been previously related with Brucella virulence was also altered. CONCLUSIONS/SIGNIFICANCE: All these data corroborate the impact of BvrR/BvrS on cell envelope modulation, confirm that this system controls the carbon and nitrogen metabolism, and suggest a cross-talk among some regulators to adjust the Brucella physiology to the shift expected to occur during the transit from the extracellular to the intracellular niche.