isolation-characterization-immune-cells-from-micro-dissected-mouse

Isolation and Characterization of the Immune Cells from Micro-dissected Mouse Choroid Plexuses

The brain is no longer considered as an organ functioning in isolation; accumulating evidence suggests that changes in the peripheral immune system can ...

Institut Pasteur

Bacterial biofilms often form multispecies communities in which complex but ill-understood competition and cooperation interactions occur. In light of the profound physiological modifications associated with this lifestyle, we hypothesized that the biofilm environment might represent an untapped source of natural bioactive molecules interfering with bacterial adhesion or biofilm formation. We produced cell-free solutions extracted from in vitro mature biofilms formed by 122 natural Escherichia coli isolates, and we screened these biofilm extracts for antiadhesion molecules active on a panel of Gram-positive and Gram-negative bacteria. Using this approach, we showed that 20% of the tested biofilm extracts contained molecules that antagonize bacterial growth or adhesion. We characterized a compound, produced by a commensal animal E. coli strain, for which activity is detected only in biofilm extract. Biochemical and genetic analyses showed that this compound corresponds to a new type of released high-molecular-weight polysaccharide whose biofilm-associated production is regulated by the RfaH protein. We demonstrated that the antiadhesion activity of this polysaccharide was restricted to Gram-positive bacteria and that its production reduced susceptibility to invasion and provided rapid exclusion of Staphylococcus aureus from mixed E. coli and S. aureus biofilms. Our results therefore demonstrate that biofilms contain molecules that contribute to the dynamics of mixed bacterial communities and that are not or only poorly detected in unconcentrated planktonic supernatants. Systematic identification of these compounds could lead to strategies that limit pathogen surface colonization and reduce the burden associated with the development of bacterial biofilms on medical devices.

Screening of Escherichia coli species biodiversity reveals new biofilm-associated antiadhesion polysaccharides.

Little is known about Listeria monocytogenes-associated bone and joint infections. Only case reports of this infection have been published.

Listeria monocytogenes-associated joint and bone infections: a study of 43 consecutive cases.

Bacterial infections trigger the expression of type I and II interferon genes but little is known about their effect on type III interferon (IFN-Î») genes, whose products play important roles in epithelial innate immunity against viruses. Here, we studied the expression of IFN-Î» genes in cultured human epithelial cells infected with different pathogenic bacteria and in the mouse placenta infected with Listeria monocytogenes. We first showed that in intestinal LoVo cells, induction of IFN-Î» genes by L. monocytogenes required bacterial entry and increased further during the bacterial intracellular phase of infection. Other Gram-positive bacteria, Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis, also induced IFN-Î» genes when internalized by LoVo cells. In contrast, Gram-negative bacteria Salmonella enterica serovar Typhimurium, Shigella flexneri and Chlamydia trachomatis did not substantially induce IFN-Î». We also found that IFN-Î» genes were up-regulated in A549 lung epithelial cells infected with Mycobacterium tuberculosis and in HepG2 hepatocytes and BeWo trophoblastic cells infected with L. monocytogenes. In a humanized mouse line permissive to fetoplacental listeriosis, IFN-Î»2/Î»3 mRNA levels were enhanced in placentas infected with L. monocytogenes. In addition, the feto-placental tissue was responsive to IFN-Î»2. Together, these results suggest that IFN-Î» may be an important modulator of the immune response to Gram-positive intracellular bacteria in epithelial tissues.

Activation of type III interferon genes by pathogenic bacteria in infected epithelial cells and mouse placenta.

Listeria monocytogenes (Lm) is a ubiquitous bacterium able to survive and thrive within the environment and readily colonizes a wide range of substrates, often as a biofilm. It is also a facultative intracellular pathogen, which actively invades diverse hosts and induces listeriosis. So far, these two complementary facets of Lm biology have been studied independently. Here we demonstrate that the major Lm virulence determinant ActA, a PrfA-regulated gene product enabling actin polymerization and thereby promoting its intracellular motility and cell-to-cell spread, is critical for bacterial aggregation and biofilm formation. We show that ActA mediates Lm aggregation via direct ActA-ActA interactions and that the ActA C-terminal region, which is not involved in actin polymerization, is essential for aggregation in vitro. In mice permissive to orally-acquired listeriosis, ActA-mediated Lm aggregation is not observed in infected tissues but occurs in the gut lumen. Strikingly, ActA-dependent aggregating bacteria exhibit an increased ability to persist within the cecum and colon lumen of mice, and are shed in the feces three order of magnitude more efficiently and for twice as long than bacteria unable to aggregate. In conclusion, this study identifies a novel function for ActA and illustrates that in addition to contributing to its dissemination within the host, ActA plays a key role in Lm persistence within the host and in transmission from the host back to the environment.

ActA promotes Listeria monocytogenes aggregation, intestinal colonization and carriage.

Antivirulence strategies targeting bacterial behavior, such as adhesion and biofilm formation, are expected to exert low selective pressure and have been proposed as alternatives to biocidal antibiotic treatments to avoid the rapid occurrence of bacterial resistance. Here, we tested this hypothesis using group 2 capsule polysaccharide (G2cps), a polysaccharidic molecule previously shown to impair bacterium-surface interactions, and we investigated the nature of bacterial resistance to a nonbiocidal antibiofilm strategy. We screened an Escherichia coli mutant library for an increased ability to form biofilm in the presence of G2cps, and we identified several mutants displaying partial but not total resistance to this antibiofilm polysaccharide. Our genetic analysis showed that partial resistance to G2cps results from multiple unrelated mutations leading to modifications in surface physicochemical properties that counteract the changes in ionic charge and Lewis base properties induced by G2cps. Moreover, some of the identified mutants harboring improved biofilm formation in the presence of G2cps were also partially resistant to other antibiofilm molecules. This study therefore shows that alterations of bacterial surface properties mediate only partial resistance to G2cps. It also experimentally validates the potential value of nonbiocidal antibiofilm strategies, since full resistance to antibiofilm compounds is rare and potentially unlikely to arise in clinical settings. 

Escherichia coli resistance to nonbiocidal antibiofilm polysaccharides is rare and mediated by multiple mutations leading to surface physicochemical modifications.

Listeria monocytogenes (Lm) is ubiquitous and widespread in the environment. It is responsible for one of the most severe human foodborne infection. Lm is a facultative intracellular bacterium that can cross the intestinal barrier, disseminate via the bloodstream and reach the liver, spleen, central nervous system and fetus. The bacterial surface protein ActA is one of the most critical and best characterized virulence factors of Lm. It fulfills many essential functions within host cells, allowing Lm escape from autophagy and recruiting an actin polymerization complex that promotes Lm actin-based motility, cell-to-cell spread and dissemination within host tissues. We have recently shown that ActA also acts extracellularly. It mediates Lm aggregation and biofilm formation in vitro and in vivo, and long-term colonization of the gut lumen. This new property of ActA favors Lm transmission and may participate in the selective pressure on Lm to maintain ActA. 

Listeria monocytogenes ActA: a new function for a 'classic' virulence factor.

At present, little is known regarding Listeria monocytogenes-associated biliary tract infection, a rare form of listeriosis.In this article, we will study 12 culture-proven cases reported to the French National Reference Center for Listeria from 1996 to 2013 and review the 8 previously published cases.Twenty cases were studied: 17 cholecystitis, 2 cholangitis, and 1 biliary cyst infection. Half were men with a median age of 69 years (32-85). Comorbidities were present in 80%, including cirrhosis, rheumatoid arthritis, and diabetes. Five patients received immunosuppressive therapy, including corticosteroids and anti-tumor necrosis factor biotherapies. Half were afebrile. Blood cultures were positive in 60% (3/5). Gallbladder histological lesions were analyzed in 3 patients and evidenced acute, chronic, or necrotic exacerbation of chronic infection. Genoserogroup of the 12 available strains were IVb (n=6), IIb (n=5), and IIa (n=1). Their survival in the bile was not enhanced when compared with isolates from other listeriosis cases. Adverse outcome was reported in 33% (5/15): 3 deaths, 1 recurrence; 75% of the patients with adverse outcome received inadequate antimicrobial therapy (P=0.033).Biliary tract listeriosis is a severe infection associated with high mortality in patients not treated with appropriate therapy. This study provides medical relevance to in vitro and animal studies that had shown Listeria monocytogenes ability to survive in bile and induce overt biliary infections.

Listeria monocytogenes-associated biliary tract infections: a study of 12 consecutive cases and review.

Invasion of nonphagocytic cells, a critical property of Listeria monocytogenes (Lm) that enables it to cross host barriers, is mediated by the interaction of two bacterial surface proteins, InlA and InlB, with their respective receptors E-cadherin and c-Met. Although InlA-E-cadherin interaction is necessary and sufficient for Lm crossing of the intestinal barrier, both InlA and InlB are required for Lm crossing of the placental barrier. The mechanisms underlying these differences are unknown. Phosphoinositide 3-kinase (PI3-K) is involved in both InlA- and InlB-dependent pathways. Indeed, InlA-dependent entry requires PI3-K activity but does not activate it, whereas InlB-c-Met interaction activates PI3-K. We show that Lm intestinal target cells exhibit a constitutive PI3-K activity, rendering InlB dispensable for InlA-dependent Lm intestinal barrier crossing. In contrast, the placental barrier does not exhibit constitutive PI3-K activity, making InlB necessary for InlA-dependent Lm placental invasion. Here, we provide the molecular explanation for the respective contributions of InlA and InlB to Lm host barrier invasion, and reveal the critical role of InlB in rendering cells permissive to InlA-mediated invasion. This study shows that PI3-K activity is critical to host barrier permissiveness to microbes, and that pathogens exploit both similarities and differences of host barriers to disseminate.

PI3-kinase activation is critical for host barrier permissiveness to Listeria monocytogenes.

Secreted exopolysaccharides present important determinants for bacterial biofilm formation, survival, and virulence. Cellulose secretion typically requires the concerted action of a c-di-GMP-responsive inner membrane synthase (BcsA), an accessory membrane-anchored protein (BcsB), and several additional Bcs components. Although the BcsAB catalytic duo has been studied in great detail, its interplay with co-expressed subunits remains enigmatic. Here we show that E. coli Bcs proteins partake in a complex protein interaction network. Electron microscopy reveals a stable, megadalton-sized macromolecular assembly, which encompasses most of the inner membrane and cytosolic Bcs components and features a previously unobserved asymmetric architecture. Heterologous reconstitution and mutational analyses point toward a structure-function model, where accessory proteins regulate secretion by affecting both the assembly and stability of the system. Altogether, these results lay the foundation for more comprehensive models of synthase-dependent exopolysaccharide secretion in biofilms and add a sophisticated secretory nanomachine to the diverse bacterial arsenal for virulence and adaptation.

Insights into the structure and assembly of a bacterial cellulose secretion system.

Neonates are highly susceptible to bacterial meningitis as compared to children and adults. Group B streptococcus (GBS) is a major cause of neonatal meningitis. Neonatal meningitis can result from GBS intestinal colonization and translocation across the intestinal barrier (IB). Here, we show that the immaturity of the neonatal intestinal microbiota leads to low resistance to GBS intestinal colonization and permissiveness of the gut-vascular barrier. Moreover, the age-dependent but microbiota-independent Wnt activity in intestinal and choroid plexus (CP) epithelia results in a lower degree of cell-cell junctions' polarization, which favors bacterial translocation. This study thus reveals that neonatal susceptibility to GBS meningitis results from the age-dependent immaturity of the intestinal microbiota and developmental pathways associated with neonatal tissue growth, which both concur to GBS gut colonization, systemic dissemination, and neuroinvasion. Whereas the activation of developmental pathways is intrinsic to neonates, interventions aimed at maturing the microbiota may help prevent neonatal meningitis.

Article	Year
Screening of Escherichia coli species biodiversity reveals new biofilm-associated antiadhesion polysaccharides. mBio\| PubMed ID: 21558434	2011
Listeria monocytogenes-associated joint and bone infections: a study of 43 consecutive cases. Clinical infectious diseases : an official publication of the Infectious Diseases Society of America\| PubMed ID: 22100574	2012
Activation of type III interferon genes by pathogenic bacteria in infected epithelial cells and mouse placenta. PloS one\| PubMed ID: 22720036	2012
ActA promotes Listeria monocytogenes aggregation, intestinal colonization and carriage. PLoS pathogens\| PubMed ID: 23382675	2013
Escherichia coli resistance to nonbiocidal antibiofilm polysaccharides is rare and mediated by multiple mutations leading to surface physicochemical modifications. Antimicrobial agents and chemotherapy\| PubMed ID: 23733462	2013
Listeria monocytogenes ActA: a new function for a 'classic' virulence factor. Current opinion in microbiology\| PubMed ID: 24581693	2014
Listeria monocytogenes-associated biliary tract infections: a study of 12 consecutive cases and review. Medicine\| PubMed ID: 25319439	2014
PI3-kinase activation is critical for host barrier permissiveness to Listeria monocytogenes. The Journal of experimental medicine\| PubMed ID: 25624443	2015
Insights into the structure and assembly of a bacterial cellulose secretion system. Nature communications\| PubMed ID: 29234007	2017
Neonatal susceptibility to meningitis results from the immaturity of epithelial barriers and gut microbiota. Cell reports\| PubMed ID: 34192531	2021

Laetitia Travier

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