christophe.lambert@fundp.ac.be; http://www.fundp.ac.be/~lambertc

BackgroundAlterations in the composition of the lung microbiome associated with adverse clinical outcomes, known as dysbiosis, have been implicated with disease severity and exacerbations in COPD.ObjectiveTo characterise longitudinal changes in the lung microbiome in the AERIS study (Acute Exacerbation and Respiratory InfectionS in COPD) and their relationship with associated COPD outcomes.MethodsWe surveyed 584 sputum samples from 101 patients with COPD to analyse the lung microbiome at both stable and exacerbation time points over 1 year using high-throughput sequencing of the 16S ribosomal RNA gene. We incorporated additional lung microbiology, blood markers and in-depth clinical assessments to classify COPD phenotypes.ResultsThe stability of the lung microbiome over time was more likely to be decreased in exacerbations and within individuals with higher exacerbation frequencies. Analysis of exacerbation phenotypes using a Markov chain model revealed that bacterial and eosinophilic exacerbations were more likely to be repeated in subsequent exacerbations within a subject, whereas viral exacerbations were not more likely to be repeated. We also confirmed the association of bacterial genera, including Haemophilus and Moraxella, with disease severity, exacerbation events and bronchiectasis.ConclusionsSubtypes of COPD have distinct bacterial compositions and stabilities over time. Some exacerbation subtypes have non-random probabilities of repeating those subtypes in the future. This study provides insights pertaining to the identification of bacterial targets in the lung and biomarkers to classify COPD subtypes and to determine appropriate treatments for the patient.Trial registration numberResults, NCT01360398.

In order to identify transcriptional regulators involved in virulence gene control in Brucella melitensis, we generated a collection of 88 mutants in the AraC, ArsR, Crp, DeoR, GntR, IclR, LysR, MerR, RpiR, and TetR families of regulators. This collection was named LiMuR (library of mutants for regulators). We developed a method to test several mutants simultaneously in one animal in order to identify those unable to survive. This method, called the plasmid-tagged mutagenesis method, was used to test the residual virulence of mutants after 1 week in a mouse model of infection. Ten attenuated mutants, of which six and three belong to the GntR and LysR families, respectively, were identified and individually confirmed to replicate at lower rates in mice. Among these 10 mutants, only gntR10 and arsR6 are attenuated in cellular models. The LiMuR also allows simple screenings to identify regulators of a particular gene or operon. As a first example, we analyzed the expression of the virB operon in the LiMuR mutants. We carried out Western blottings of whole-cell extracts to analyze the production of VirB proteins using polyclonal antisera against VirB proteins. Four mutants produced small amounts of VirB proteins, and one mutant overexpressed VirB proteins compared to the wild-type strain. In these five mutants, reporter analysis using the virB promoter fused to lacZ showed that three mutants control virB at the transcriptional level. The LiMuR is a resource that will provide straightforward identification of regulators involved in the control of genes of interest.Bacteria have selected mechanisms to express only the appropriate subset of genes conferring a growth or survival advantage in a given situation (10). The expression of many bacterial genes is regulated at the initiation of transcription by regulators which, in response to specific environmental and/or cellular signals, bind at the promoter of target genes to activate or repress them (23).The availability of complete bacterial genome sequences has opened up doors to the development of new strategies to study the biology of microorganisms (59). For instance, the complete Brucella melitensis sequence (15) allowed us to conduct a systematic study of several families of regulators in this organism. Bacteria of the genus Brucella are facultative intracellular gram-negative coccobacilli that are pathogenic for domestic animals and occasionally for humans (57). Some transcriptional regulators of Brucella that have a role in the control of virulence have already been identified. Indeed, the two-component system BvrR/BvrS controls cell invasion, intracellular survival, and expression of two outer membrane proteins (25, 58). Some transcriptional regulators required for Brucella's virulence were also identified in several screens in cellular models and in BALB/c mice (19,31,35,36). Random screening for attenuated mutants may not be viewed as a comprehensive analysis that would allow the identification of all regulators involved in the virulence of the model tested. We...

For this study, we screened 1,152 signature-tagged mutagenesis mutants of Brucella melitensis 16M in a mouse model of infection and found 36 of them to be attenuated in vivo. Molecular characterization of transposon insertion sites showed that for four mutants, the affected genes were only present in Rhizobiaceae. Another mutant contained a disruption in a gene homologous to mosA, which is involved in rhizopine biosynthesis in some strains of Rhizobium, suggesting that this sugar may be involved in Brucella pathogenicity. A mutant was disrupted in a gene homologous to fliF, a gene potentially coding for the MS ring, a basal component of the flagellar system. Surprisingly, a mutant was affected in the rpoA gene, coding for the essential ␣-subunit of the RNA polymerase. This disruption leaves a partially functional protein, impaired for the activation of virB transcription, as demonstrated by the absence of induction of the virB promoter in the Tn5::rpoA background. The results presented here highlight the fact that the ability of Brucella to induce pathogenesis shares similarities with the molecular mechanisms used by both Rhizobium and Agrobacterium to colonize their hosts.Brucella spp. are gram-negative, facultative, intracellular bacteria that cause abortion and sterility in domestic mammals and a chronic undulant fever in humans (6, 52). On the basis of ribosomal 16S sequence comparison, Brucella spp. are members of the alpha subdivision of the class Proteobacteria. Within the alpha subgroup, brucellae are specifically related to rickettsiae, agrobacteria, and rhizobiae, organisms that also have the ability or requirement to live in close association with eukaryotic cells (37). The complete genomes of Brucella melitensis and Brucella suis have been sequenced recently (11,43); genomic analysis showed only pinpoint differences between the two species and suggested that Brucella may have evolved from a soil-plant-associated bacterium related to organisms like Rhizobium and Agrobacterium (43).Brucella is able to survive in professional and nonprofessional phagocytes by subverting the intracellular trafficking of eukaryotic cells (3,44,45). Studies in epithelial cells have shown that the ability of Brucella to escape from the classical cellular trafficking pathway, which normally leads to the lysosome, needs at least the VirB system (homologous to a type IV secretion machinery) and the BvrR/BvrS two-component system (8, 10, 53). It has also been shown that Brucella recruits actin and activates small GTPases during its internalization in HeLa cells (20).While genome analysis revealed some genes that could be related to virulence (e.g., adhesins, hemolysins, and invasins), it showed that Brucella lacks classical virulence-related sequences and genes, such as pathogenicity islands, type III secretion systems, toxins, pilus biogenesis genes, etc. (36). Therefore, to draw a complete map of the molecular basis of Brucella pathogenesis, unbiased approaches are still needed. Moreover, these approaches will help in the fun...

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