Bacteria-cell Interactions Research Articles

Integrins as a bridge between bacteria and cells: key targets for therapeutic wound healing.

Integrins are heterodimers composed of α and β subunits that are bonded through non-covalent interactions. Integrins mediate the dynamic connection between extracellular adhesion molecules and the intracellular actin cytoskeleton. Integrins are present in various tissues and organs where these heterodimers participate in diverse physiological and pathological responses at the molecular level in living organisms. Wound healing is a crucial process in the recovery from traumatic diseases and comprises three overlapping phases: inflammation, proliferation and remodeling. Integrins are regulated during the entire wound healing process to enhance processes such as inflammation, angiogenesis and re-epithelialization. Prolonged inflammation may result in failure of wound healing, leading to conditions such as chronic wounds. Bacterial colonization of a wound is one of the primary causes of chronic wounds. Integrins facilitate the infectious effects of bacteria on the host organism, leading to chronic inflammation, bacterial colonization, and ultimately, the failure of wound healing. The present study investigated the role of integrins as bridges for bacteria-cell interactions during wound healing, evaluated the role of integrins as nodes for bacterial inhibition during chronic wound formation, and discussed the challenges and prospects of using integrins as therapeutic targets in wound healing.

Effect of Tryptophan Metabolites on Cell Damage Revealed by Bacteria-Cell Interactions in Hydrogel Microspheres.

This work presented an alternative approach for studying bacteria-cell interactions in three-dimensional (3D) hydrogel microspheres formed by the cross-linking reaction of alginate and calcium-ethylenediaminetetraacetic acid (EDTA-Ca) produced in a microfluidic chip. During the co-culture process of hepatocytes (HepG2) and Escherichia coli (E. coli) 25922, we concluded that the content change of tryptophan metabolites detected via ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was related to the cell damage level and the change of interleukin (IL-22) detected by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) was related to the ways of co-cultivation. Compared to the two-dimensional (2D) adherent cell culture process in a Petri dish (2D), the co-culture process of HepG2 and E. coli 25922 in hydrogel microspheres indicated more information about metabolism such as the appearance of indole-3-propionic acid (IPA) and possibly IL-22. The method provides a new perspective to investigate the bacteria-cell interaction and it could be a promising tool in the study of gut microbiota and human health.

The anti-adhesive effect of glycoclusters on Pseudomonas aeruginosa bacteria adhesion to epithelial cells studied by AFM single cell force spectroscopy.

The human opportunistic pathogen Pseudomonas aeruginosa (PA) is responsible for chronic infections of the respiratory epithelium in cystic fibrosis patients. PA takes advantage of an arsenal of virulence factors to infect and colonize human lungs. Among them, the lectin LecA favours epithelium invasion by interacting with host cell globotriaosylceramide (Gb3). A new therapeutic approach is based on the development of synthetic multivalent molecules (glycoclusters) targeting LecA with a higher affinity than its natural ligand. Atomic force microscopy-single cell force spectroscopy has been used to study the effect of glycoclusters on the bacteria-cell interaction. Glycoclusters have been shown to affect the detachment work and detachment force of the bacteria-cell interaction. The specificity and the efficiency of the glycocluster in targeting the lectin and destabilizing the PA-epithelial cell adhesion are demonstrated and discussed.

Spotlight on…Pascale Cossart.

#### Biographical summary I was educated as a chemist at the University of Lille, France. After a Master of Science at Georgetown University, Washington, DC, I arrived at the Pasteur Institute in 1971, where I am still…I only interrupted this long period for 3 months in 1973 for the birth of my first daughter, 1 year to teach biochemistry in Laos in 1974 and during which my second daughter was born; my third daughter was born while I was a post doc in 1979. I am now the head of the Bacteria-Cell Interactions Unit in the Department of Cell Biology and Infection. I have just published a book entitled ‘La nouvelle Microbiology’ edited by Odile Jacob, Francois Jacob's daughter. I have also been elected Secretaire perpetuel at the Academie des Sciences since January 2016. 1. My current research is addressing the molecular and cellular mechanisms underlying how an intracellular pathogen is behaving during life in different environments, in particular during the switch from saprophytism to virulence. We take as a model the bacterium Listeria monocytogenes . Our research has an impact in infection biology but also in cell biology and in fundamental biology, in particular RNA biology. 2. I cannot say that I had decided to have a career in microbiology. I was educated as a pure chemist in Lille. I obtained a Master degree in chemistry and started to be interested in biochemistry. I then moved to the States and at Georgetown University obtained a Master degree in chemistry/biochemistry. I then came back to France and was advised to go to the Pasteur Institute, which seemed as one of the best research places in France. At Pasteur, a succession of …

Lactoferrin prevents invasion and inflammatory response following E. coli strain LF82 infection in experimental model of Crohn's disease

Crohn's disease is a multifactorial disease in which an aberrant immune response to commensal intestinal microbiota leads to chronic inflammation. The small intestine of patients with Crohn's disease is colonized by a group of adherent-invasive Escherichia coli strongly able to adhere and invade intestinal epithelial cells lactoferrin is an iron-binding glycoprotein known to have anti-bacterial and anti-inflammatory activities. We explore the ability of bovine lactoferrin to modulate the interactions between the adherent-invasive E. coli strain LF82 and intestinal epithelial cells as well as the inflammatory response. Bacterial adhesion and invasion assays were used to assess the antimicrobial activity of lactoferrin. Electron microscopy was used to characterize bacteria-cell interactions. The mRNA expression of pro-inflammatory cytokines was measured both in cultured cells and in biopsies taken from intestine of patients affected by Crohn's disease. Lactoferrin inhibited bacterial invasion through minimally affecting adhesion. This divergence was due to a mannose-dependent lactoferrin binding to the bacterial type 1 pili and consequent bacterial aggregation on the intestinal epithelial cell surface. Expression of pro-inflammatory cytokines, such as TNF-alpha, IL-8, and IL-6, was markedly inhibited by lactoferrin both in cultured and Crohn-derived intestinal cells. Bovine lactoferrin might function via an antibacterial and/or anti-inflammatory mechanism in the treatment of Crohn's disease.

Thyroid Hormone Enhances Nitric Oxide-Mediated Bacterial Clearance and Promotes Survival after Meningococcal Infection

Euthyroid sick syndrome characterized by reduced levels of thyroid hormones (THs) is observed in patients with meningococcal shock. It has been found that the level of THs reflects disease severity and is predictive for mortality. The present study was conducted to investigate the impact of THs on host defense during meningococcal infection. We found that supplementation of thyroxine to mice infected with Neisseria meningitidis enhanced bacterial clearance, attenuated the inflammatory responses and promoted survival. In vitro studies with macrophages revealed that THs enhanced bacteria-cell interaction and intracellular killing of meningococci by stimulating inducible nitric oxide synthase (iNos)-mediated NO production. TH treatment did not activate expression of TH receptors in macrophages. Instead, the observed TH-directed actions were mediated through nongenomic pathways involving the protein kinases PI3K and ERK1/2 and initiated at the membrane receptor integrin αvβ3. Inhibition of nongenomic TH signaling prevented iNos induction, NO production and subsequent intracellular bacterial killing by macrophages. These data demonstrate a beneficial role of THs in macrophage-mediated N. meningitidis clearance. TH replacement might be a novel option to control meningococcal septicemia.

Kefiran protects Caco-2 cells from cytopathic effects induced by Bacillus cereus infection

The aim of this work was to evaluate the ability of kefiran to antagonize cytopathic effects triggered by Bacillus cereus strain B10502 on cultured human enterocytes (Caco-2 cells). Cell damage was evaluated by F-actin labelling, scanning electron microscopy and determination of ratios of necrotic and detached cells. To assess the interaction between kefiran and bacteria or eukaryotic cells, flow cytometric analysis was conducted with FITC-labelled kefiran. Kefiran significantly protected infected cells from cytopathic effects induced by B. cereus such as cell necrosis, F-actin disorganisation and microvilli effacement, although presence of kefiran did not modify the adhesion of microorganisms to cultured human enterocytes. Results could be ascribed to the ability of kefiran to interact with both bacteria and eukaryotic cells thus antagonizing interactions necessary for maximal biological effects. Our findings encourage further research on the use of bacterial exopolysaccharides to antagonize virulence factors associated to direct bacteria-cell interactions.

A Sphingomonas bacterium interacting with epithelial cells

Bacteria of the genus Sphingomonas are environmental organisms that have recently been implicated in a variety of community-acquired and nosocomial infections. During studies on bacteria–cell interactions, we incurred a microorganism contaminating our HeLa cell culture, possibly from water utilized for reagent preparation; this bacterium appeared to tightly adhere to cell monolayers and to survive, with only limited growth rate, which did not seem to alter cells as far as shape, growth rate or survival were concerned. The contaminating organism was isolated and partially characterized by morphological, genetic, and biochemical assays. Mechanisms of cell interaction and entry into epithelial cells were investigated by electron microscopy, immunofluorescence, and biochemical inhibitors. Morphological and biochemical features indicated that the microorganism belonged to the genus Sphingomonas. Electron microscopy showed that contact between the Sphingomonas bacterium and epithelial cells leads to a dramatic alteration of the cell surface, with formation of numerous microvillar extensions plus membrane ruffling. Confocal microscopy and the use of inhibitors showed that actin microfilaments were involved during attachment and entry into HeLa cells. Macropinosome formation and an inhibitory effect by amiloride indicate that internalization occurs in part via a macropinocytosis mechanism. Moreover, cholesterol distribution at the site of bacterial binding suggests that Sphingomonas bacteria could use the lipid rafts as initial binding sites.

Cholera toxin and extracellular Ca2+ induce adherence of non-piliated Neisseria: evidence for an important role of G-proteins and Rho in the bacteria-cell interaction

In this study, we characterize the interaction between non-piliated (P-) Neisseria gonorrhoeae and human epithelial cells. P- mutants lacking the pilus subunit protein PilE attach at low levels to cells. Although the binding may not lead to heavy inflammatory responses, the interaction between P- Neisseria and host cells most probably play a role in colonization and asymptomatic carriage of the pathogen. Here we show that the adherence of P N. gonorrhoeae is blocked by GDP-beta-S [guanosine 5'-O(thio)diphosphate], a non-hydrolyzable GTP analogue, and by C3 exotoxin, an inhibitor of the small G-protein Rho. G-protein activators such as cholera toxin, that activates Gs, and fluoroaluminate, a general G-protein activator, induced bacterial adherence. Furthermore, increase of the extracellular free [Ca2+] dramatically enhanced adherence of non-piliated Neisseria. The pharynx and the urogenital tract are natural entry sites of the pathogenic Neisseria species, and at both sites the epithelial cells can be exposed to wide variations in Ca2+ concentration. Taken together, these data show the importance of extracellular Ca2+ in the pathogenic Neisseria-host interaction, and reveal a novel function of cholera toxin, namely induction of bacterial adherence.

Intimate adhesion of Neisseria meningitidis to human epithelial cells is under the control of the crgA gene, a novel LysR-type transcriptional regulator.

PilC1, a pilus-associated protein in Neisseria menin- gitidis, is a key element in initial meningococcal adhesion to target cells. A promoter element (CREN, contact regulatory element of Neisseria) is responsible for the transient induction of this gene upon cell contact. crgA (contact-regulated gene A) encodes a transcriptional regulator whose expression is also induced upon cell contact from a promoter region similar to the CREN of pilC1. CrgA shows significant sequence homologies to LysR-type transcriptional regulators. Its inactivation in meningococci provokes a dramatic reduction in bacterial adhesion to epithelial cells. Moreover, this mutant is unable to undergo intimate adhesion to epithelial cells or to provoke effacing of microvilli on infected cells. Purified CrgA is able to bind to pilC1 and crgA promoters, and CrgA seems to repress the expression of pilC1 and crgA. Our results support a dynamic model of bacteria-cell interaction involving a network of regulators acting in cascade. CrgA could be an intermediate regulator in such a network.

13] Electron microscopical methods in adhesion

Publisher Summary This chapter focuses on electron microscopical methods used in adhesion. Electron microscopy, mainly of negatively stained preparations, have been used to identify and characterize the proteinaceous flamentous bacterial surface structures, termed as “fimbriae’ or “pili,” which functions as adhesins. Electron microscopical studies of bacteria-cell interactions have mainly employ scanning electron microscopy (SEM) and transmission electron microscopy (TEM) of thin sections, together with associated immunological applications of other methods. An objective of all microscopy is to attain good contrast and resolution of a specimen. In negative staining, this is achieved by surrounding/embedding the specimen in a thin film of heavy metal stain such that unstained structures show up against the heavy metal stain in negative contrast. Immunocytochemical techniques are powerful tools for the localization of specific antigens. The development of colloidal gold probes revolutionized the electron microscopical application of such techniques. Bacterial adhesion is a cell surface interaction that makes it ideal for examination by SEM. SEM is mainly used to examine mucosal surfaces from both natural and experimental infections of animals and humans and to examine tissue and tissue culture cells experimentally infected in vitro .

Quantitative analysis of lymphocyte-Salmonella interaction and effect of lymphocyte irradiation by Helium-Neon laser

A model system has been developed to quantitatively investigate bacteria-cell interaction using a rough mutant of Salmonella and human peripheral blood lymphocytes. The effect of lymphocyte irradiation by low-power continuous wave Helium-Neon laser has been investigated since laser therapy in wound and decoubitous ulcer healing could involve the lymphoyd cell function. Helium-Neon laser irradiation is shown here to enhance the adherence of Salmonella to lymphocytes. In particular, changes in newly defined binding parameters show that laser irradiation increases the frequency of binding-lymphocytes, the affinity of Salmonella for lymphocytes and the number of lymphocyte receptor sites as well.

In vitro interaction of Mycobacterium avium with intestinal epithelial cells.

Human intestinal epithelial cell monolayers were inoculated with cultures of Mycobacterium avium serotype 2, 8, or 10 that were viable, autoclaved, Formalin killed, exposed to UV light, or suspended in anti-M. avium serotype 2 serum. The effects of four reagents known to block phagocytosis or endocytosis (cytochalasin B, dibutyryl cyclic adenosine monophosphate, iodoacetate, and 2,4-dinitrophenol) on the bacteria-cell interaction were also studied. The maximum uptake of pathogenic M. avium by human intestinal epithelial cells occurred after 2 to 3 h of incubation. Serotype 2 was taken up in greater quantity than serotype 8 or 10. Saprophytic mycobacteria did not attach to or penetrate the host cells. The data showed that viable mycobacteria are ingested by host cells, whereas dead organisms are not. Components of the bacterial cells are partially, but not solely, responsible for the phagocytosis of M. avium serotype 2 by human intestinal epithelial cells. Furthermore, uptake of M. avium by human intestinal epithelial cells was suppressed by reagents which inhibit uptake by known phagocytic cells, suggesting that the mechanism of uptake is an endocytic process induced by virulent mycobacteria.

Surface Properties Of Staphylococcus Saprophyticus and Staphylococcus Epidermidis as Studied by Adherence Tests and Two‐Polymer, Aqueous Phase Systems

S. saprophyticus is an established pathogen in man, devoided of characteristics associated with pathogenicity in Staphylococcus aureus. The ability of this species to attach to cells from regions, viz. the urinary tract, where it acts as an invador and to cells from areas where it is known as a commensal, was compared to its behaviour in this respect with another staphylococcal species, viz. S. epidermidis. S. saprophyticus showed a preferenital adherence to human exfoliated urogenital cells, when compared with its ability to attach to skin and buccal cells from man and also when compared with procine cells from these regions. The profound ability to adhere to human exfoliated urogenital epithelial cells by far exceeded that of S. epidermidis, while no such species difference was found when testing porcine cells (S. saprophyticus is unknown as a urogenital tract pathogen in pigs). When studied in a two-polymer, aqueous phase system, S. saprophyticus and S. epidermidis were found to have a negative surface charge at pH 7.2, but the former carried a considerably higher surface charge density. Both staphylococcal species exhibited a poor hydrophobic interaction liability. These physico-chemical surface characteristics are briefly discussed with regard to the differential bacteria-cell interactions of these species.