Background & aims Several animal studies have emphasized the role of gut microbiota in non-alcoholic fatty liver disease (NAFLD). However, data about gut dysbiosis in human NAFLD remains scarce in the literature, especially studies including the whole spectrum of NAFLD lesions. We aimed to evaluate the association between gut dysbiosis and severe NAFLD lesions, i.e. non-alcoholic steatohepatitis (NASH) and fibrosis, in a well-characterized population of adult NAFLD. Methods 57 patients with biopsy-proven NAFLD were enrolled. The taxonomic composition of gut microbiota was determined using 16S ribosomal RNA gene sequencing of stool samples. Results 30 patients had F0/1 fibrosis stage at liver biopsy (10 with NASH), and 27 patients had significant F≥2 fibrosis (25 with NASH). Bacteroides abundance was significantly increased in NASH and F≥2 patients, whereas Prevotella abundance was decreased. Ruminococcus abundance was significantly higher in F≥2 patients. By multivariate analysis, Bacteroides abundance was independently associated with NASH and Ruminococcus with F≥2 fibrosis. Stratification according to the abundance of these 2 bacteria generated 3 patient subgroups with increasing severity of NAFLD lesions. Based on imputed metagenomic profiles, KEGG pathways significantly related to NASH and fibrosis F≥2 were mostly related to carbohydrate, lipid, and amino acid metabolism. Conclusion NAFLD severity associates with gut dysbiosis and a shift in metabolic function of the gut microbiota. We identified Bacteroides as independently associated with NASH and Ruminococcus with significant fibrosis. Thus, gut microbiota analysis adds information to classical predictors of NAFLD severity and suggests novel metabolic targets for pre/probiotics therapies.

The production of several virulence factors by Pseudomonas aeruginosa is controlled according to cell density through two quorum-sensing systems, las and rhl. The las system is comprised of the transcriptional activator protein LasR and of LasI, which directs the synthesis of the autoinducer PAI-1. Similarly, the rhl system consists of the transcriptional activator protein RhlR and of RhlI, which directs synthesis of the autoinducer PAI-2 (formerly referred to as factor 2). To study the interrelation between the two P. aeruginosa quorumsensing systems, we fused a lacZ reporter gene to lasR, rhlR, and rhlA and monitored expression of these three genes under various conditions. Our data indicate that lasR and rhlR are expressed in a growth-dependent manner, with activation of each gene occurring during the last half of log-phase growth. We also show that the las quorum-sensing system controls the rhl quorum-sensing system in two ways. First, we found that LasR and PAI-1 activated rhlR transcription. Second, we showed that PAI-1 blocked PAI-2 from binding to RhlR, thereby inhibiting the expression of rhlA. Our data thus indicate that the las system exerts two levels of control on RhlR, transcriptional and posttranslational.At least two complete quorum-sensing systems, las and rhl, are present in the opportunistic human pathogen Pseudomonas aeruginosa. These systems are known to control the expression of a number of virulence genes in response to bacterial cell density (5), but their specific effect on each other has not been studied. The las and rhl systems each contain homologs of the LuxR and LuxI proteins of the prototypic lux quorum-sensing system from Vibrio fischeri (see reference 5 for a review). The las system consists of the transcriptional activator protein LasR and of LasI, which directs the synthesis of the autoinducer PAI-1 [N-(3-oxododecanoyl)-L-homoserine lactone] (6,20,22). This system has been shown to activate the expression of lasI, lasB, lasA, apr, and toxA (7,20,27,30). Similarly, the rhl system consists of the transcriptional activator protein RhlR and RhlI, which directs the synthesis of the autoinducer PAI-2 (N-butyryl-L-homoserine lactone; formerly known as factor 2) (16,17,23). This system controls the expression of rhlI and rhlAB, which codes for a rhamnosyltransferase required for rhamnolipid (heat-stabile hemolysin) production (11,(15)(16)(17). It has also been reported that rhl quorum sensing activates the expression of rpoS, a stationary-phase sigma factor that controls numerous genes (11).The general model for quorum sensing (5) begins with the autoinducer, which is a diffusible molecule, being produced at a basal level at low cell densities. The autoinducer concentration then increases with cell density until a threshold concentration is reached. At this concentration, the autoinducer binds to its specific target protein (i.e., LasR or RhlR), and the autoinducer-protein complex activates genes that it controls.Experiments on the interchangeability of the las and rhl system component...

Curli are functional extracellular amyloid fibers produced by uropathogenic Escherichia coli (UPEC) and other Enterobacteriaceae. Ring-fused 2-pyridones, such as FN075 and BibC6, inhibited curli biogenesis in UPEC and prevented the in vitro polymerization of the major curli subunit protein CsgA. The curlicides FN075 and BibC6 share a common chemical lineage with other ring-fused 2-pyridones termed pilicides. Pilicides inhibit the assembly of type 1 pili, which are required for pathogenesis during urinary tract infection. Notably, the curlicides retained pilicide activities and inhibited both curli-dependent and type 1-dependent biofilms. Furthermore, pretreatment of UPEC with FN075 significantly attenuated virulence in a mouse model of urinary tract infection. Curli and type 1 pili exhibited exclusive and independent roles in promoting UPEC biofilms, and curli provided a fitness advantage in vivo. Thus, the ability of FN075 to block the biogenesis of both curli and type 1 pili endows unique anti-biofilm and anti-virulence activities on these compounds.Bacterial biofilms are complex microbial communities that exhibit reduced sensitivity to conventional antibiotics, host defenses and external stresses 1,2 . Multiple determinants contribute to biofilm development and maintenance, and their requirements in biofilm formation may vary depending on environmental conditions. In addition, factors important in

To establish disease, an infecting organism must overcome a vast array of host defenses. During cystitis, uropathogenic Escherichia coli (UPEC) subvert innate defenses by invading superficial umbrella cells and rapidly increasing in numbers to form intracellular bacterial communities (IBCs). In the late stages of the IBC pathway, filamentous and bacillary UPEC detach from the biofilm-like IBC, fluxing out of this safe haven to colonize the surrounding epithelium and initiate subsequent generations of IBCs, and eventually they establish a quiescent intracellular reservoir. Filamentous UPEC are not observed during acute infection in mice lacking functional Toll-like receptor 4 (TLR4), suggesting that the filamentous phenotype arises in response to host innate immunity. We investigated SulA, a cell division inhibitor associated with the SOS response, to gain insight into the role of filamentous UPEC in pathogenesis. A transcriptional reporter from PsulA revealed spatial and temporal differences in expression within IBCs, and it was active in the majority of filamentous UPEC. Although UTI89 and UTI89 ⌬sulA both formed first-generation IBCs equally well, UTI89 ⌬sulA was sharply attenuated in formation of second-generation IBCs and establishment of the quiescent intracellular reservoir. The virulence of UTI89 ⌬sulA was restored in TLR4-deficient mice, suggesting that filamentation facilitates the transition to additional rounds of IBC formation by subverting innate immune responses. These findings demonstrate that transient SulA-mediated inhibition of cell division is essential for UPEC virulence in the murine model of cystitis.bacterial filamentation ͉ SOS response ͉ pathogenesis

As an interface with the environment, the skin is a complex ecosystem colonized by many microorganisms that coexist in an established balance. The cutaneous microbiome inhibits colonization with pathogens, such as Staphylococcus aureus, and is a crucial component for function of the epidermal barrier. Moreover, crosstalk between commensals and the immune system is now recognized because microorganisms can modulate both innate and adaptive immune responses. Host-commensal interactions also have an effect on the developing immune system in infants and, subsequently, the occurrence of diseases, such as asthma and atopic dermatitis (AD). Later in life, the cutaneous microbiome contributes to the development and course of skin disease. Accordingly, in patients with AD, a decrease in microbiome diversity correlates with disease severity and increased colonization with pathogenic bacteria, such as S aureus. Early clinical studies suggest that topical application of commensal organisms (eg, Staphylococcus hominis or Roseomonas mucosa) reduces AD severity, which supports an important role for commensals in decreasing S aureus colonization in patients with AD. Advancing knowledge of the cutaneous microbiome and its function in modulating the course of skin disorders, such as AD, might result in novel therapeutic strategies.

SummaryUropathogenic Escherichia coli, the predominant causative agent of urinary tract infections, use type 1 pili to bind and invade bladder epithelial cells. Upon entry, the bacteria rapidly replicate and enter a complex developmental pathway ultimately forming intracellular bacterial communities (IBCs), a niche with biofilm-like properties protected from innate defences and antibiotics. Paradoxically, bacteria within IBCs produce type 1 pili, an organelle thought only to be an extracellular colonization factor. Thus, we investigated the function of type 1 pili in IBC development. The cystitis isolate, UTI89, was genetically manipulated for conditional fim expression under control of the tet promoter. In this strain, UTI89-tetR/P tet fim, piliation is constitutively inhibited by the tetracycline repressor, TetR. Repression is relieved by anhydrotetracycline (AHT) treatment. UTI89-tetR/ Ptet fim and the isogenic control strain, UTI89-tetR, grown in the presence of AHT, colonized the bladder and invaded the superficial umbrella cells at similar levels at early times in a murine model of infection. However, after invasion UTI89-tetR/Ptet fim became non-piliated and was unable to form typical IBCs comprised of tightly packed, coccoid-shaped bacteria in contrast to the control strain, UTI89-tetR. Thus, this work changes the extracellular colonization functional paradigm of pili by demonstrating their intracellular role in biofilm formation.

A chemical synthesis platform with broad applications and flexibility was rationally designed to inhibit biogenesis of adhesive pili assembled by the chaperone-usher pathway in Gram-negative pathogens. The activity of a family of bicyclic 2-pyridones, termed pilicides, was evaluated in two different pilus biogenesis systems in uropathogenic Escherichia coli. Hemagglutination mediated by either type 1 or P pili, adherence to bladder cells, and biofilm formation mediated by type 1 pili were all reduced by Ϸ90% in laboratory and clinical E. coli strains. The structure of the pilicide bound to the P pilus chaperone PapD revealed that the pilicide bound to the surface of the chaperone known to interact with the usher, the outer-membrane assembly platform where pili are assembled. Point mutations in the pilicide-binding site dramatically reduced pilus formation but did not block the ability of PapD to bind subunits and mediate their folding. Surface plasmon resonance experiments confirmed that the pilicide interfered with the binding of chaperone-subunit complexes to the usher. These pilicides thus target key virulence factors in pathogenic bacteria and represent a promising proof of concept for developing drugs that function by targeting virulence factors.antimicrobials ͉ chaperone-usher pathway ͉ pilicide ͉ urinary tract infection

In Pseudomonas aeruginosa, the transcriptional activator LasR and the Pseudomonas autoinducer PAI, are necessary for efficient transcriptional activation of the lasB gene, encoding elastase (L. Passador, J. M. Cook, M. J. Gambello, L. Rust, and B. H. Iglewski, Science 260:1127-1130, 1993). The transcriptional start points of lasI in Escherichia coli and P. aeruginosa were determined by S1 nuclease mapping. In the presence of both LasR and PAI, the start site, T1, is located at position ؊25 relative to the ATG translational start codon. A minor transcriptional start, T2, is found at position ؊13 when lasI is transcribed in the absence of either LasR or PAI in P. aeruginosa and E. coli, respectively. To begin to closely examine the regulation of lasI, whose product is involved in the synthesis of PAI, a lasI-lacZ fusion on a lambda phage was constructed to form monolysogens of E. coli MG4. Lysogens supplied only with either lasI or lasR via multicopy plasmids demonstrated no significant increase in ␤-galactosidase expression compared with control levels. Lysogens in which both lasR and lasI were supplied in multicopy exhibited a 62-fold increase in expression, and a lysogen in which lasR was supplied in trans and which was grown in the presence of exogenous PAI exhibited a 60-fold increase. Thus, LasR and PAI are necessary for the full expression of lasI in E. coli. The interchangeability of the P. aeruginosa and Vibrio fischeri homologs LasR and LuxR and their respective autoinducers, PAI and VAI, as activators of lasI-lacZ was examined. Only the combination of LasR and PAI significantly increased the expression of lasI. The comparison of lasI-lacZ and lasB-lacZ expression in lysogens grown in the presence of lasR and PAI revealed that half-maximal expression of lasI required 0.1 nM PAI, in contrast to the 1.0 nM PAI necessary for lasB half-maximal expression. These results suggest an autoinduction regulatory hierarchy in which LasR and low PAI concentrations primarily activate lasI expression in a regulatory loop. With the accumulation of PAI, secondary activation of virulence product genes such as lasB occurs.Multiple homologous regulatory systems which entail the interaction of a diffusible effector molecule, termed an autoinducer, with a transcriptional activating protein to induce expression of different target genes have been described for gram-negative bacteria. The transcriptional activators and autoinducers in these processes are closely related, and the protein activators display significant peptide sequence homology, particularly in their putative DNA-binding domains. Vibrio fischeri (6,7,20), Agrobacterium tumefaciens (24, 33), Erwinia carotovora (2, 17), and Pseudomonas aeruginosa (22) are among the prokaryotic organisms which utilize this type of regulation, to control bioluminescence (V. fischeri), conjugation (A. tumefaciens), and virulence factor (E. carotovora and P. aeruginosa) regulation.P. aeruginosa, a gram-negative opportunistic human pathogen, uses the transcriptional activator protein, La...