Las islas de patogenicidad de Saphylococcus aureus son capaces de controlar factores de virulencia cromosómicos

Trabajo presentado en la X Reunión de Microbiología Molecular, celebrada en Segovia del 9 al 11 de junio de 2014.Staphylococcus aureus es una importante bacteria patógena, debido a la enorme variedad de infecciones que es capaz de producir. La enorme versatilidad de S. aureus se debe a su habilidad para persistir y multiplicarse en diferentes ambientes junto con su capacidad para producir una gran variedad de factores de virulencia, algunos de las cuales están codificadas en elementos genéticos móviles (EGMs), como bacteriófagos e islas de patogenicidad (SaPI) (1). Sobre el 90% de las cepas de S. aureus aisladas de infecciones humanas están pigmentadas. Staphyloxanthin (STX) es un carotenoide considerado como factor de virulencia, ya que contribuye a la evasión del sistema inmune. Por otro lado, la N-acetiltransferasa (GNAT) está implicada en resistencia a antibióticos. En este trabajo describimos como las SaPIs son capaces de regular la producción de factores de virulencia como son STX y GNAT.Peer Reviewe

The Brucella abortus two-component system response regulator BvrR binds to three DNA regulatory boxes in the upstream region of omp25

Brucella abortus is a facultative extracellular-intracellular bacterial zoonotic pathogen worldwide. It is also a major cause of abortion in bovines, generating economic losses. The two-component regulatory system BvrR/BvrS modulates the expression of genes required to transition from extracellular to intracellular lifestyles. However, few regulatory regions of BvrR direct target genes have been studied. In this study, we characterized the regulatory region of omp25, a gene encoding an outer membrane protein that is positively regulated by TCS BvrR/BvrS. By omp25-lacZ reporter fusions and β-galactosidase activity assays, we found that the region between-262 and + 127 is necessary for transcriptional activity, particularly a 111-bp long fragment located from-262 to −152. In addition, we demonstrated the binding of P-BvrR to three sites within the −140 to +1 region. Two of these sites were delimited between −18 to +1 and − 99 to −76 by DNase I footprinting and called DNA regulatory boxes 1 and 2, respectively. The third binding site (box 3) was delimited from −140 to −122 by combining EMSA and fluorescence anisotropy results. A molecular docking analysis with HDOCK predicted BvrR-DNA interactions between 11, 13, and 12 amino acid residue-nucleotide pairs in boxes 1, 2, and 3, respectively. A manual sequence alignment of the three regulatory boxes revealed the presence of inverted and non-inverted repeats of five to eight nucleotides, partially matching DNA binding motifs previously described for BvrR. We propose that P-BvrR binds directly to up to three regulatory boxes and probably interacts with other transcription factors to regulate omp25 expression. This gene regulation model could apply to other BvrR target genes and to orthologs of the TCS BvrR/BvrS and Omp25 in phylogenetically closed Rhizobiales

Brucella ceti infection in dolphins from the Western Mediterranean sea

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The Differential Interaction of Brucella and Ochrobactrum with Innate Immunity Reveals Traits Related to the Evolution of Stealthy Pathogens

International audienceBACKGROUND: During evolution, innate immunity has been tuned to recognize pathogen-associated molecular patterns. However, some alpha-Proteobacteria are stealthy intracellular pathogens not readily detected by this system. Brucella members follow this strategy and are highly virulent, but other Brucellaceae like Ochrobactrum are rhizosphere inhabitants and only opportunistic pathogens. To gain insight into the emergence of the stealthy strategy, we compared these two phylogenetically close but biologically divergent bacteria. METHODOLOGY/PRINCIPAL FINDINGS: In contrast to Brucella abortus, Ochrobactrum anthropi did not replicate within professional and non-professional phagocytes and, whereas neutrophils had a limited action on B. abortus, they were essential to control O. anthropi infections. O. anthropi triggered proinflammatory responses markedly lower than Salmonella enterica but higher than B. abortus. In macrophages and dendritic cells, the corresponding lipopolysaccharides reproduced these grades of activation, and binding of O. anthropi lipopolysaccharide to the TLR4 co-receptor MD-2 and NF-kappaB induction laid between those of B. abortus and enteric bacteria lipopolysaccharides. These differences correlate with reported variations in lipopolysaccharide core sugars, sensitivity to bactericidal peptides and outer membrane permeability. CONCLUSIONS/SIGNIFICANCE: The results suggest that Brucellaceae ancestors carried molecules not readily recognized by innate immunity, so that non-drastic variations led to the emergence of stealthy intracellular parasites. They also suggest that some critical envelope properties, like selective permeability, are profoundly altered upon modification of pathogen-associated molecular patterns, and that this represents a further adaptation to the host. It is proposed that this adaptive trend is relevant in other intracellular alpha-Proteobacteria like Bartonella, Rickettsia, Anaplasma, Ehrlichia and Wolbachia

Neurobrucellosis in Stranded Dolphins, Costa Rica

Ten striped dolphins, Stenella coeruleoalba, stranded along the Costa Rican Pacific coast, had meningoencephalitis and antibodies against Brucella spp. Brucella ceti was isolated from cerebrospinal fluid of 6 dolphins and 1 fetus. S. coeruleoalba constitutes a highly susceptible host and a potential reservoir for B. ceti transmission

Genetic and Phenotypic Characterization of the Etiological Agent of Canine Orchiepididymitis Smooth Brucella sp. BCCN84.3

Members of the genus Brucella cluster in two phylogenetic groups: classical and non-classical species. The former group is composed of Brucella species that cause disease in mammals, including humans. A Brucella species, labeled as Brucella sp. BCCN84.3, was isolated from the testes of a Saint Bernard dog suffering orchiepididymitis, in Costa Rica. Following standard microbiological methods, the bacterium was first defined as "Brucella melitensis biovar 2." Further molecular typing, identified the strain as an atypical "Brucella suis." Distinctive Brucella sp. BCCN84.3 markers, absent in other Brucella species and strains, were revealed by fatty acid methyl ester analysis, high resolution melting PCR and omp25 and omp2a/omp2b gene diversity. Analysis of multiple loci variable number of tandem repeats and whole genome sequencing demonstrated that this isolate was different from the currently described Brucella species. The smooth Brucella sp. BCCN84.3 clusters together with the classical Brucella Glade and displays all the genes required for virulence. Brucella sp. BCCN84.3 is a species nova taxonomical entity displaying pathogenicity; therefore, relevant for differential diagnoses in the context of brucellosis. Considering the debate on the Brucella species concept, there is a need to describe the extant taxonomical entities of these pathogens in order to understand the dispersion and evolution

Virulent Brucella nosferati infecting Desmodus rotundus has emerging potential due to the broad foraging range of its bat host for humans and wild and domestic animals

Desmodus rotundus, vampire bats, transmit dangerous infections, and brucellosis is a hazardous zoonotic disease, two adversities that coexist in the subtropical and tropical areas of the American continent. Here, we report a 47.89% Brucella infection prevalence in a colony of vampire bats inhabiting the tropical rainforest of Costa Rica. The bacterium induced placentitis and fetal death in bats. Wide-range phenotypic and genotypic characterization placed the Brucella organisms as a new pathogenic species named Brucella nosferati sp. nov., isolated from bat tissues, including the salivary glands, suggesting feeding behavior might favor transmission to their prey. Overall analyses placed B. nosferati as the etiological agent of a reported canine brucellosis case, demonstrating its potential for infecting other hosts. To assess the putative prey hosts, we analyzed the intestinal contents of 14 infected and 23 non-infected bats by proteomics. A total of 54,508 peptides sorted into 7,203 unique peptides corresponding to 1,521 proteins were identified. Twenty-three wildlife and domestic taxa, including humans, were foraged by B. nosferati-infected D. rotundus, suggesting contact of this bacterium with a broad range of hosts. Our approach is appropriate for detecting, in a single study, the prey preferences of vampire bats in a diverse area, demonstrating its suitability for control strategies where vampire bats thrive. IMPORTANCE: The discovery that a high proportion of vampire bats in a tropical area is infected with pathogenic Brucella nosferati and that bats forage on humans and many wild and domestic animals is relevant from the perspective of emerging disease prevention. Indeed, bats harboring B. nosferati in their salivary glands may transmit this pathogenic bacterium to other hosts. This potential is not trivial since, besides the demonstrated pathogenicity, this bacterium possesses all the required virulent arsenal of dangerous Brucella organisms, including those that are zoonotic for humans. Our work has settled the basis for future surveillance actions in brucellosis control programs where these infected bats thrive. Moreover, our strategy to identify the foraging range of bats may be adapted for exploring the feeding habits of diverse animals, including arthropod vectors of infectious diseases, and therefore of interest to a broader audience besides experts on Brucella and bats. The discovery that a high proportion of vampire bats in a tropical area is infected with pathogenic Brucella nosferati and that bats forage on humans and many wild and domestic animals is relevant from the perspective of emerging disease prevention. Indeed, bats harboring B. nosferati in their salivary glands may transmit this pathogenic bacterium to other hosts. This potential is not trivial since, besides the demonstrated pathogenicity, this bacterium possesses all the required virulent arsenal of dangerous Brucella organisms, including those that are zoonotic for humans. Our work has settled the basis for future surveillance actions in brucellosis control programs where these infected bats thrive. Moreover, our strategy to identify the foraging range of bats may be adapted for exploring the feeding habits of diverse animals, including arthropod vectors of infectious diseases, and therefore of interest to a broader audience besides experts on Brucella and bats

Analysis of TcdB Proteins within the Hypervirulent Clade 2 Reveals an Impact of RhoA Glucosylation on Clostridium difficile Proinflammatory Activities

Clostridium difficile strains within the hypervirulent clade 2 are responsible for nosocomial outbreaks worldwide. The increased pathogenic potential of these strains has been attributed to several factors but is still poorly understood. During a C. difficile outbreak, a strain from this clade was found to induce a variant cytopathic effect (CPE), different from the canonical arborizing CPE. This strain (NAP1V) belongs to the NAP1 genotype but to a ribotype different from the epidemic NAP1/RT027 strain. NAP1V and NAP1 share some properties, including the overproduction of toxins, the binary toxin, and mutations in tcdC. NAP1V is not resistant to fluoroquinolones, however. A comparative analysis of TcdB proteins from NAP1/RT027 and NAP1V strains indicated that both target Rac, Cdc42, Rap, and R-Ras but only the former glucosylates RhoA. Thus, TcdB from hypervirulent clade 2 strains possesses an extended substrate profile, and RhoA is crucial for the type of CPE induced. Sequence comparison and structural modeling revealed that TcdBNAP1 and TcdBNAP1V share the receptor-binding and autoprocessing activities but vary in the glucosyltransferase domain, consistent with the different substrate profile. Whereas the two toxins displayed identical cytotoxic potencies, TcdBNAP1 induced a stronger proinflammatory response than TcdBNAP1V as determined in ex vivo experiments and animal models. Since immune activation at the level of intestinal mucosa is a hallmark of C. difficile-induced infections, we propose that the panel of substrates targeted by TcdB is a determining factor in the pathogenesis of this pathogen and in the differential virulence potential seen among C. difficile strains.Wellcome Trust, United States a Trevor D Lawley bajo el número 098051Consejo Nacional de Rectores/[803-B1-654]/CONARE/Costa RicaConsejo Nacional de Rectores/[803-B4-652]/CONARE/Costa RicaUniversidad de Costa Rica/[803-B5-107]/UCR/Costa RicaUniversidad de Costa Rica/[803-B5-108]/UCR/Costa RicaConsejo Nacional para Investigaciones Científicas y Tecnológicas/[FV-0004-13. HHS]/CONICIT/Costa RicaNational Institutes of Health/[R01AI095755]/NIH/Estados UnidosUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET

Brucella Genetic Variability in Wildlife Marine Mammals Populations Relates to Host Preference and Ocean Distribution

Intracellular bacterial pathogens probably arose when their ancestor adapted from a free-living environment to an intracellular one, leading to clonal bacteria with smaller genomes and less sources of genetic plasticity. Still, this plasticity is needed to respond to the challenges posed by the host. Members of the Brucella genus are facultative-extracellular intracellular bacteria responsible for causing brucellosis in a variety of mammals. The various species keep different host preferences, virulence, and zoonotic potential despite having 97–99% similarity at genome level. Here, we describe elements of genetic variation in Brucella ceti isolated from wildlife dolphins inhabiting the Pacific Ocean and the Mediterranean Sea. Comparison with isolates obtained from marine mammals from the Atlantic Ocean and the broader Brucella genus showed distinctive traits according to oceanic distribution and preferred host. Marine mammal isolates display genetic variability, represented by an important number of IS711 elements as well as specific IS711 and SNPs genomic distribution clustering patterns. Extensive pseudogenization was found among isolates from marine mammals as compared with terrestrial ones, causing degradation in pathways related to energy, transport of metabolites, and regulation/ transcription. Brucella ceti isolates infecting particularly dolphin hosts, showed further degradation of metabolite transport pathways as well as pathways related to cell wall/membrane/envelope biogenesis and motility. Thus, gene loss through pseudogenization is a source of genetic variation in Brucella, whichinturn, relates to adaptation to different hosts.This is relevant to understand the natural history of bacterial diseases, their zoonotic potential, and the impact of human interventions such as domestication.Comisión Nacional para la Gestión de la Biodiversidad/[R-028-203-OT]/CONAGEBIO/Costa RicaMinisterio de Ciencia, Tecnología y Telecomunicaciones/[FV-004-13]/MICITT/Costa RicaWellcome Trust/[098051]/WT/LondresWellcome Trust/[106690/Z/14/Z]/WT/LondresUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET

Brucella abortus Uses a Stealthy Strategy to Avoid Activation of the Innate Immune System during the Onset of Infection

To unravel the strategy by which Brucella abortus establishes chronic infections, we explored its early interaction with innate immunity. Methodology/Principal Findings Brucella did not induce proinflammatory responses as demonstrated by the absence of leukocyte recruitment, humoral or cellular blood changes in mice. Brucella hampered neutrophil (PMN) function and PMN depletion did not influence the course of infection. Brucella barely induced proinflammatory cytokines and consumed complement, and was strongly resistant to bactericidal peptides, PMN extracts and serum. Brucella LPS (BrLPS), NH-polysaccharides, cyclic glucans, outer membrane fragments or disrupted bacterial cells displayed low biological activity in mice and cells. The lack of proinflammatory responses was not due to conspicuous inhibitory mechanisms mediated by the invading Brucella or its products. When activated 24 h post-infection macrophages did not kill Brucella, indicating that the replication niche was not fusiogenic with lysosomes. Brucella intracellular replication did not interrupt the cell cycle or caused cytotoxicity in WT, TLR4 and TLR2 knockout cells. TNF-α-induction was TLR4- and TLR2-dependent for live but not for killed B. abortus. However, intracellular replication in TLR4, TLR2 and TLR4/2 knockout cells was not altered and the infection course and anti-Brucella immunity development upon BrLPS injection was unaffected in TLR4 mutant mice. Conclusion/Significance We propose that Brucella has developed a stealth strategy through PAMPs reduction, modification and hiding, ensuring by this manner low stimulatory activity and toxicity for cells. This strategy allows Brucella to reach its replication niche before activation of antimicrobial mechanisms by adaptive immunity. This model is consistent with clinical profiles observed in humans and natural hosts at the onset of infection and could be valid for those intracellular pathogens phylogenetically related to Brucella that also cause long lasting infections