Thiostrepton, a resurging drug inhibiting the stringent response to counteract antibiotic-resistance and expression of virulence determinants in Neisseria gonorrhoeae.

Due to the increased resistance to all available antibiotics and the lack of vaccines, Neisseria gonorrhoeae (the gonococcus) poses an urgent threat. Although the mechanisms of virulence and antibiotic resistance have been largely investigated in this bacterium, very few studies have addressed the stringent response (SR) that in pathogenic bacteria controls the expression of genes involved in host-pathogen interaction and tolerance and persistence toward antibiotics. In this study, the results of the transcriptome analysis of a clinical isolate of N. gonorrhoeae, after induction of the SR by serine hydroxamate, provided us with an accurate list of genes that are transcriptionally modulated during the SR. The list includes genes associated with metabolism, cellular machine functions, host-pathogen interaction, genome plasticity, and antibiotic tolerance and persistence. Moreover, we found that the artificial induction of the SR in N. gonorrhoeae by serine hydroxamate is prevented by thiostrepton, a thiopeptide antibiotic that is known to interact with ribosomal protein L11, thereby inhibiting functions of EF-Tu and EF-G, and binding of pppGpp synthase I (RelA) to ribosome upon entry of uncharged tRNA. We found that N. gonorrhoeae is highly sensitive to thiostrepton under in vitro conditions, and that thiostrepton, in contrast to other antibiotics, does not induce tolerance or persistence. Finally, we observed that thiostrepton attenuated the expression of key genes involved in the host-pathogen interaction. These properties make thiostrepton a good drug candidate for dampening bacterial virulence and preventing antibiotic tolerance and persistence. The ongoing challenge is to increase the bioavailability of thiostrepton through the use of chemistry and nanotechnology.

Phenotyping of Fecal Microbiota of Winnie, a Rodent Model of Spontaneous Chronic Colitis, Reveals Specific Metabolic, Genotoxic, and Pro-inflammatory Properties.

Winnie, a mouse carrying a missense mutation in the MUC2 mucin gene, is a valuable model for inflammatory bowel disease (IBD) with signs and symptoms that have multiple similarities with those observed in patients with ulcerative colitis. MUC2 mucin is present in Winnie, but is not firmly compacted in a tight inner layer. Indeed, these mice develop chronic intestinal inflammation due to the primary epithelial defect with signs of mucosal damage, including thickening of muscle and mucosal layers, goblet cell loss, increased intestinal permeability, enhanced susceptibility to luminal inflammation-inducing toxins, and alteration of innervation in the distal colon. In this study, we show that the intestinal environment of the Winnie mouse, genetically determined by MUC2 mutation, selects an intestinal microbial community characterized by specific pro-inflammatory, genotoxic, and metabolic features that could imply a direct involvement in the pathogenesis of chronic intestinal inflammation. We report results obtained by using a variety of in vitro approaches for fecal microbiota functional characterization. These approaches include Caco-2 cell cultures and Caco-2/THP-1 cell co-culture models for evaluation of geno-cytotoxic and pro-inflammatory properties using a panel of 43 marker RNAs assayed by RT-qPCR, and cell-based phenotypic testing for metabolic profiling of the intestinal microbial communities by Biolog EcoPlates. While adding a further step towards understanding the etiopathogenetic mechanisms underlying IBD, the results of this study provide a reliable method for phenotyping gut microbial communities, which can complement their structural characterization by providing novel functional information.

HrpA anchors meningococci to the dynein motor and affects the balance between apoptosis and pyroptosis.

BACKGROUND: In Neisseria meningitidis the HrpA/HrpB two-partner secretion system (TPS) was implicated in diverse functions including meningococcal competition, biofilm formation, adherence to epithelial cells, intracellular survival and vacuolar escape. These diverse functions could be attributed to distinct domains of secreted HrpA. METHODS: A yeast two-hybrid screening, in vitro pull-down assay and immunofluorescence microscopy experiments were used to investigate the interaction between HrpA and the dynein light-chain, Tctex-type 1 (DYNLT1). In silico modeling was used to analyze HrpA structure. Western blot analysis was used to investigate apoptotic and pyroptotic markers. RESULTS: The HrpA carboxy-terminal region acts as a manganese-dependent cell lysin, while the results of a yeast two-hybrid screening demonstrated that the HrpA middle region has the ability to bind the dynein light-chain, Tctex-type 1 (DYNLT1). This interaction was confirmed by in vitro pull-down assay and immunofluorescence microscopy experiments showing co-localization of N. meningitidis with DYNLT1 in infected epithelial cells. In silico modeling revealed that the HrpA-M interface interacting with the DYNLT1 has similarity with capsid proteins of neurotropic viruses that interact with the DYNLT1. Indeed, we found that HrpA plays a key role in infection of and meningococcal trafficking within neuronal cells, and is implicated in the modulation of the balance between apoptosis and pyroptosis. CONCLUSIONS: Our findings revealed that N. meningitidis is able to effectively infect and survive in neuronal cells, and that this ability is dependent on HrpA, which establishes a direct protein-protein interaction with DYNLTI in these cells, suggesting that the HrpA interaction with dynein could be fundamental for N. meningitidis spreading inside the neurons. Moreover, we found that the balance between apoptotic and pyroptotic pathways is heavily affected by HrpA.

Surface architecture of Neisseria meningitidis capsule and outer membrane as revealed by atomic force microscopy.

An extensive morphological analysis of the Neisseria meningitidis cell envelope, including serogroup B capsule and outer membrane, based on atomic force microscopy (AFM) together with mechanical characterization by force spectroscopic measurements, has been carried out. Three meningococcal strains were used: the encapsulated serogroup B strain B1940, and the isogenic mutants B1940 siaD(+C) (lacking capsule), and B1940 cps (lacking both capsule and lipooligosaccharide outer core). AFM experiments with the encapsulated strain B1940 provided unprecedented images of the meningococcal capsule, which seems to be characterized by protrusions ("bumps") with the lateral dimensions of about 30 nm. Measurement of the Young's modulus provided quantitative assessment of the property of the capsule to confer resistance to mechanical stress. Moreover, Raman spectroscopy gave a fingerprint by which it was possible to identify the specific molecular species of the three strains analyzed, and to highlight major differences between them.