Metabolite cross-feeding enhances virulence in a model polymicrobial infection

Microbes within polymicrobial infections often display synergistic interactions resulting in enhanced pathogenesis; however, the molecular mechanisms governing these interactions are not well understood. Development of model systems that allow detailed mechanistic studies of polymicrobial synergy is a critical step towards a comprehensive understanding of these infections in vivo. In this study, we used a model polymicrobial infection including the opportunistic pathogen Aggregatibacter actinomycetemcomitans and the commensal Streptococcus gordonii to examine the importance of metabolite cross-feeding for establishing co-culture infections. Our results reveal that co-culture with S. gordonii enhances the pathogenesis of A. actinomycetemcomitans in a murine abscess model of infection. Interestingly, the ability of A. actinomycetemcomitans to utilize L-lactate as an energy source is essential for these co-culture benefits. Surprisingly, inactivation of L-lactate catabolism had no impact on mono-culture growth in vitro and in vivo suggesting that A. actinomycetemcomitans L-lactate catabolism is only critical for establishing co-culture infections. These results demonstrate that metabolite cross-feeding is critical for A. actinomycetemcomitans to persist in a polymicrobial infection with S. gordonii supporting the idea that the metabolic properties of commensal bacteria alter the course of pathogenesis in polymicrobial communities.     

Concordance in a World without a Gold Standard: A New Non-Invasive Methodology for Improving Accuracy of Fibrosis Markers

Background

Assessing liver fibrosis is traditionally performed by biopsy, an imperfect gold standard. Non-invasive techniques, liver stiffness measurements (LSM) and biomarkers [FibroTest® (FT)], are widely used in countries where they are available. The aim was to identify factors associated with LSM accuracy using FT as a non-invasive endpoint and vice versa.

Methods

The proof of concept was taken using the manufacturers recommendations for excluding patients at high risk of false negative/positive. The hypothesis was that the concordance between LSM and FT, would be improved by excluding high-risk patients. Thereafter, the impact of potential variability factors was assessed by the same methods. Liver biopsy and independent endpoints were used to validate the results.

Results

Applying manufacturers'' recommendations in 2,004 patients increased the strength of concordance between LSM and FT (P<0.00001). Among the 1,338 patients satisfying recommendations, the methodology identified a significant LSM operator effect (P = 0.001) and the following variability factors (all P<0.01), related to LSM: male gender, older age, and NAFLD as a cause of liver disease. Biopsy confirmed in 391 patients these results.

Conclusion

This study has validated the concept of using the strength of concordance between non-invasive estimates of liver fibrosis for the identification of factors associated with variability and precautions of use.     

Promoter Specificity Elements in Pseudomonas aeruginosa Quorum-Sensing-Controlled Genes

The LasR-dependent and RhlR-dependent quorum-sensing systems are global regulators of gene expression in Pseudomonas aeruginosa. Previous studies have demonstrated that promoter elements of the quorum-sensing-controlled genes lasB and hcnABC are important in density-dependent regulation. We have identified LasR- and RhlR-dependent determinants in promoters of quorum-sensing-controlled genes qsc102, qsc117 (acpP), and qsc131 (phzA to -G) by in silico, deletion, point-mutational, and primer extension analyses. Each of these genes (in addition to lasI and rsaL) is activated by LasR, and qsc117 and qsc131 also respond to RhlR. Point mutations in the promoters of the LasR-specific gene, qsc102, relax specificity so that this promoter can respond to RhlR in addition to LasR. Our findings indicate that quorum-sensing-controlled promoters in P. aeruginosa are either specific for LasR or respond to both LasR and RhlR and that critical bases in the promoter elements determine specificity.     

Molecular Epidemiology of Carbapenem Non-Susceptible Acinetobacter baumannii in France

Carbapenem-resistant Acinetobacter baumannii have emerged globally. The objective of this study was to investigate the epidemiology, clonal diversity and resistance mechanisms of imipenem non-susceptible A. baumannii isolates in France. Between December 2010 and August 2011, 132 notifications were collected, including 37 outbreaks corresponding to 242 cases (2 to 55 per cluster). Multilocus sequence typing, pulsed-field gel electrophoresis (PFGE) and characterisation of carbapenemase-encoding genes were performed on 110 non-repetitive isolates. Gene bla_OXA-23 was the most frequently detected (82%), followed by bla_OXA-24 (11%) and bla_OXA-58 (7%). Eleven sequence types (ST) were distinguished, among which sequence types ST1, ST2 (64%), ST20, ST25, ST85 and ST107. Isolates from epidemiological clusters had the same ST and resistance genes, indicating probable transmission within centres. In contrast, PFGE types of isolates differed among centres, arguing against transmission among centers. This study provides the first epidemiological snapshot of the population of A. baumannii with reduced susceptibility to carbapenems from France, and further underlines the predominance of international clones.     

Transition metal-carbonyl labeling of biotin and avidin for use in solid-phase carbonyl metallo immunoassay (CMIA)

The preparation of several transition metal-carbonyl tracers of biotin and avidin is described. Multiple labeling of avidin was achieved by acylation of some of its amine-bearing residues with N-succinimidyl 4-pentynoate (dicobalt hexacarbonyl). By varying the initial amount of this complex, protein conjugates with the extent of derivatization of up to 13 were obtained. Biotin was labeled with one (eta5-cyclopentadienyl)manganese tricarbonyl moiety by reaction of biotin hydrazide, whereas multiple labeling was reached by successive conjugation of biotin and N-succinimidyl 4-pentynoate (dicobalt hexacarbonyl) or (eta5-cyclopentadienyl)iron dicarbonyl (eta1-N-maleimidato) to poly-L-lysine or fourth generation Starburst dendrimer. All the conjugates displayed a good to excellent bioaffinity toward their respective counterparts, as measured by competitive enzymatic assays.     

Bioactive diterpenoids from Celastraceae species

Celastraceae species have a long tradition of use in folk medicine and agriculture. Intensive research into this family has resulted in the isolation of a large number of secondary metabolites with a wide range of bioactivity, with the most characteristic being the dihydro-β-agarofuran sesquiterpenes and the quinomethide triterpenoids. Despite diterpenoids not often being found in Celastraceae species, the novelty of their structures and their biological activities has stimulated their research. There are several reviews covering studies on Tripterigium wilfordii, a traditional Chinese medicine, and their metabolites, but none focusing on bioactive diterpenoids isolated from other Celastraceae species. This review focuses on diterpenes isolated from Celastraceae species, and their skeleton diversity, species sources, and tested biological activities are discussed. The literature from January 2000 to October 2016 is reviewed, and 64 references are cited. A total of 118 isolated diterpenoids possessing twelve different skeletons are included and classified by the following tested biological activity: cytotoxicity, anti-inflammatory, antitumour-promotion, anti-HIV, immunosuppressant, reversal multi-drug resistance, and antifeedant. This compliance information will be helpful in further research into diterpene Celastraceae isolation, biological activity determination and structure specific modifications.     

Influenza A viruses limit NLRP3‐NEK7‐complex formation and pyroptosis in human macrophages

Pyroptosis is a fulminant form of macrophage cell death, contributing to release of pro‐inflammatory cytokines. In humans, it depends on caspase 1/4‐activation of gasdermin D and is characterized by the release of cytoplasmic content. Pathogens apply strategies to avoid or antagonize this host response. We demonstrate here that a small accessory protein (PB1‐F2) of contemporary H5N1 and H3N2 influenza A viruses (IAV) curtails fulminant cell death of infected human macrophages. Infection of macrophages with a PB1‐F2‐deficient mutant of a contemporary IAV resulted in higher levels of caspase‐1 activation, cleavage of gasdermin D, and release of LDH and IL‐1β. Mechanistically, PB1‐F2 limits transition of NLRP3 from its auto‐repressed and closed confirmation into its active state. Consequently, interaction of a recently identified licensing kinase NEK7 with NLRP3 is diminished, which is required to initiate inflammasome assembly.     

The diversity and phylogeny of the commercially important algal class Eustigmatophyceae,including the new clade Goniochloridales

The Eustigmatophyceae is a class of yellow-green algae allied with the Chrysophyceae and other chlorophyll c possessing stramenopile (heterokont) algae. Some members of the class, especially the marine species of the genus Nannochloropsis, are under intense investigation for their potential for production of biofuels and beneficial fatty acids. The class has generally been thought to comprise a small number of genera and species, and these organisms were considered rare or infrequently encountered. In this study, we examined the phylogeny and diversity of this class by analysis of nuclear 18S rDNA sequence data. Our analysis included sequences from all the named members of the Eustigmatophyceae held in culture collections as well as a number of strains identified in culture collections as Xanthophyceae, new strains with features characteristic of the Eustigmatophyceae, and published data for uncultured DNA clones. The results of these analyses show that the Eustigmatophyceae is far more diverse than generally recognized. Two major lineages are supported in the class, the previously recognized order Eustigmatales and the new clade, Goniochloridales. Additional new lineages were also resolved within each of these major lineages; however, the results of our analyses were considered insufficient for naming these subordinate clades. Several of these lineages comprised only unnamed strains or uncultured DNA clones. Overall, our results indicate that the Eustigmatophyceae is a highly diverse class, with many new species, genera, and families awaiting taxonomic treatment.