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2.
Infect Immun ; 92(4): e0006224, 2024 Apr 09.
Article in English | MEDLINE | ID: mdl-38514466

ABSTRACT

Streptococcus agalactiae also named Group B Streptococcus (GBS) is the most significant pathogen causing invasive infections, such as bacteremia and meningitis, in neonates. Worldwide epidemiological studies have shown that a particular clonal complex (CC) of capsular serotype III, the CC17, is strongly associated with meningitis in neonates and is therefore, designated as the hypervirulent clone. Macrophages are a permissive niche for intracellular bacteria of all GBS clones. In this study, we deciphered the specific interaction of GBS CC17 strains with macrophages. Our study revealed that CC17 strains are phagocytosed at a higher rate than GBS non-CC17 strains by human monocytes and macrophages both in cellular models and in primary cells. CC17-enhanced phagocytosis is due to an initial enhanced-attachment step to macrophages mediated by the CC17-specific surface protein HvgA and the PI-2b pilus (Spb1). We showed that two different inhibitors of scavenger receptors (fucoidan and poly(I)) specifically inhibited CC17 adhesion and phagocytosis while not affecting those of non-CC17 strains. Once phagocytosed, both CC17 and non-CC17 strains remained in a LAMP-1 positive vacuole that ultimately fuses with lysosomes where they can survive at similar rates. Finally, both strains displayed a basal egress which occurs independently from actin and microtubule networks. Our findings provide new insights into the interplay between the hypervirulent GBS CC17 and major players of the host's innate immune response. This enhanced adhesion, leading to increased phagocytosis, could reflect a peculiar capacity of the CC17 lineage to subvert the host immune defenses, establish a niche for persistence or disseminate.


Subject(s)
Meningitis , Streptococcal Infections , Infant, Newborn , Humans , Streptococcus agalactiae , Streptococcal Infections/microbiology , Macrophages , Clone Cells
3.
J Clin Invest ; 131(5)2021 03 01.
Article in English | MEDLINE | ID: mdl-33465054

ABSTRACT

Group B Streptococcus (GBS) is the major cause of human neonatal infections. A single clone, designated CC17-GBS, accounts for more than 80% of meningitis cases, the most severe form of the infection. However, the events allowing blood-borne GBS to penetrate the brain remain largely elusive. In this study, we identified the host transmembrane receptors α5ß1 and αvß3 integrins as the ligands of Srr2, a major CC17-GBS-specific adhesin. Two motifs located in the binding region of Srr2 were responsible for the interaction between CC17-GBS and these integrins. We demonstrated in a blood-brain-barrier cellular model that both integrins contributed to the adhesion and internalization of CC17-GBS. Strikingly, both integrins were overexpressed during the postnatal period in the brain vessels of the blood-brain barrier and blood-cerebrospinal fluid barrier and contributed to juvenile susceptibility to CC17 meningitis. Finally, blocking these integrins decreased the ability of CC17-GBS to cross into the CNS of juvenile mice in an in vivo model of meningitis. Our study demonstrated that CC17-GBS exploits integrins in order to cross the brain vessels, leading to meningitis. Importantly, it provides host molecular insights into neonate's susceptibility to CC17-GBS meningitis, thereby opening new perspectives for therapeutic and prevention strategies of GBS-elicited meningitis.


Subject(s)
Adhesins, Bacterial/metabolism , Blood-Brain Barrier/metabolism , Integrin alphaVbeta3/metabolism , Meningitis, Bacterial/metabolism , Receptors, Vitronectin/metabolism , Streptococcal Infections/metabolism , Streptococcus agalactiae/metabolism , Adhesins, Bacterial/genetics , Animals , Animals, Newborn , Bacterial Adhesion/genetics , Blood-Brain Barrier/microbiology , Cell Line , Humans , Integrin alphaVbeta3/genetics , Meningitis, Bacterial/genetics , Rats , Receptors, Vitronectin/genetics , Streptococcal Infections/genetics , Streptococcus agalactiae/genetics
4.
Elife ; 82019 11 11.
Article in English | MEDLINE | ID: mdl-31710290

ABSTRACT

Group B Streptococcus (GBS) is the leading cause of invasive bacterial neonatal infections. Late-onset diseases (LOD) occur between 7 and 89 days of life and are largely due to the CC17 GBS hypervirulent clone. We studied the impact of estradiol (E2) and progesterone (P4), which impregnate the fetus during pregnancy, on GBS neonatal infection in cellular and mouse models of hormonal exposure corresponding to concentrations found at birth (E2-P4 C0) and over 7 days old (E2-P4 C7). Using representative GBS isolates, we show that E2-P4 C7 concentrations specifically favor CC17 GBS meningitis following mice oral infection. CC17 GBS crosses the intestinal barrier through M cells. This process mediated by the CC17-specific surface protein Srr2 is enhanced by E2-P4 C7 concentrations which promote M cell differentiation and CC17 GBS invasiveness. Our findings provide an explanation for CC17 GBS responsibility in LOD in link with neonatal gastrointestinal tract maturation and hormonal imprint.


Subject(s)
Bacterial Translocation , Estradiol/metabolism , Host-Pathogen Interactions , Neonatal Sepsis/physiopathology , Progesterone/metabolism , Streptococcal Infections/microbiology , Streptococcus agalactiae/physiology , Animals , Animals, Newborn , Cells, Cultured , Disease Models, Animal , Humans , Mice , Models, Theoretical
5.
Microbes Infect ; 21(2): 99-103, 2019 03.
Article in English | MEDLINE | ID: mdl-30419351

ABSTRACT

The core PI-2b pilus present in "hypervirulent" ST-17 Streptococcus agalactiae strains consists of three pilin subunits (Spb1, Ap1 and Ap2) assembled by sortase SrtC1 and cell-wall anchored by Srt2. Spb1 was shown to be the major pilin and Ap2 the anchor pilin. Ap1 is a putative adhesin. Two additional genes, orf and lep, are part of this operon. The contribution of Lep and Ap1 to the biogenesis of the PI-2b pilus was investigated. Concerning the role of PI-2b, we found that higher PI-2b expression resulted in higher adherence to human brain endothelial cells and higher phagocytosis by human THP1 macrophages.


Subject(s)
Adhesins, Bacterial/metabolism , Bacterial Adhesion , Fimbriae, Bacterial/metabolism , Operon/genetics , Streptococcal Infections/microbiology , Streptococcus agalactiae/genetics , Adhesins, Bacterial/genetics , Cell Wall/metabolism , Endothelial Cells/microbiology , Humans , Macrophages/microbiology , Phagocytosis , Streptococcus agalactiae/pathogenicity , Streptococcus agalactiae/physiology
6.
PLoS Pathog ; 13(2): e1006224, 2017 02.
Article in English | MEDLINE | ID: mdl-28222186

ABSTRACT

During infection by invasive bacteria, epithelial cells contribute to innate immunity via the local secretion of inflammatory cytokines. These are directly produced by infected cells or by uninfected bystanders via connexin-dependent cell-cell communication. However, the cellular pathways underlying this process remain largely unknown. Here we perform a genome-wide RNA interference screen and identify TIFA and TRAF6 as central players of Shigella flexneri and Salmonella typhimurium-induced interleukin-8 expression. We show that threonine 9 and the forkhead-associated domain of TIFA are necessary for the oligomerization of TIFA in both infected and bystander cells. Subsequently, this process triggers TRAF6 oligomerization and NF-κB activation. We demonstrate that TIFA/TRAF6-dependent cytokine expression is induced by the bacterial metabolite heptose-1,7-bisphosphate (HBP). In addition, we identify alpha-kinase 1 (ALPK1) as the critical kinase responsible for TIFA oligomerization and IL-8 expression in response to infection with S. flexneri and S. typhimurium but also to Neisseria meningitidis. Altogether, these results clearly show that ALPK1 is a master regulator of innate immunity against both invasive and extracellular gram-negative bacteria.


Subject(s)
Adaptor Proteins, Signal Transducing/immunology , Gram-Negative Bacterial Infections/immunology , Immunity, Innate/immunology , TNF Receptor-Associated Factor 6/immunology , Chemokines/biosynthesis , Enzyme-Linked Immunosorbent Assay , Epithelial Cells/immunology , Fluorescent Antibody Technique , Gram-Negative Bacteria/immunology , HEK293 Cells , HeLa Cells , Heptoses/immunology , Humans , Image Processing, Computer-Assisted , Immunoblotting , Immunoprecipitation , Neisseria meningitidis/immunology , Salmonella typhimurium/immunology , Shigella flexneri/immunology
7.
Front Immunol ; 7: 84, 2016.
Article in English | MEDLINE | ID: mdl-27014264

ABSTRACT

Type III secretion systems (T3SSs) are specialized secretion apparatus involved in the virulence of many Gram-negative pathogens, enabling the injection of bacterial type III effectors into host cells. The T3SS-dependent injection of effectors requires the insertion into host cell membranes of a pore-forming "translocon," whose effects on cell responses remain ill-defined. As opposed to pore-forming toxins that damage host cell plasma membranes and induce cell survival mechanisms, T3SS-dependent pore formation is transient, being regulated by cell membrane repair mechanisms or bacterial effectors. Here, we review host cell responses to pore formation induced by T3SSs associated with the loss of plasma membrane integrity and regulation of innate immunity. We will particularly focus on recent advances in mechanisms controlling pore formation and the activity of the T3SS linked to type III effectors or bacterial proteases. The implications of the regulation of the T3SS translocon activity during the infectious process will be discussed.

8.
Pathog Dis ; 74(2)2016 Mar.
Article in English | MEDLINE | ID: mdl-26702632

ABSTRACT

Group A Streptococcus (GAS) infections present high morbidity and mortality rates and consequently remain a significant health problem. The emm3 isolates induce more severe pathologies than all others. In this study, we tested, on a collection of invasive and non-invasive emm3 clinical isolates, whether in that genotype the invasive status of the strains affects the innate immune response. We show that phagocytosis is dependent on the invasiveness of the isolates. Interestingly, all emm3 isolates compromise macrophage integrity, already noticeable 1 h after infection. Inflammatory modulators (IL-6, TNF-α and IFN-ß) are nevertheless detected during at least 6 h post-infection. This is a likely consequence of the macrophages not being all infected. The efficient and rapid induction of macrophage death could explain the virulence of the emm3 strains.


Subject(s)
Macrophages/immunology , Macrophages/microbiology , Streptococcal Infections/immunology , Streptococcal Infections/microbiology , Streptococcus pyogenes/physiology , Animals , Caspase 3/metabolism , Cell Death/immunology , Cytokines/metabolism , Disease Models, Animal , Female , Genetic Variation , Genotype , Inflammation Mediators/metabolism , Macrophages/metabolism , Mice , Phagocytosis/immunology , Streptococcal Infections/metabolism , Streptococcus pyogenes/classification , Streptococcus pyogenes/isolation & purification , Streptococcus pyogenes/pathogenicity , Virulence
9.
PLoS Pathog ; 11(7): e1005013, 2015 Jul.
Article in English | MEDLINE | ID: mdl-26132339

ABSTRACT

Type III secretion systems (T3SSs) are specialized macromolecular machines critical for bacterial virulence, and allowing the injection of bacterial effectors into host cells. The T3SS-dependent injection process requires the prior insertion of a protein complex, the translocon, into host cell membranes consisting of two-T3SS hydrophobic proteins, associated with pore-forming activity. In all described T3SS to date, a hydrophilic protein connects one hydrophobic component to the T3SS needle, presumably insuring the continuum between the hollow needle and the translocon. In the case of Enteropathogenic Escherichia coli (EPEC), the hydrophilic component EspA polymerizes into a filament connecting the T3SS needle to the translocon composed of the EspB and EspD hydrophobic proteins. Here, we identify EspA and EspD as targets of EspC, a serine protease autotransporter of Enterobacteriaceae (SPATE). We found that in vitro, EspC preferentially targets EspA associated with EspD, but was less efficient at proteolyzing EspA alone. Consistently, we found that EspC did not regulate EspA filaments at the surface of primed bacteria that was devoid of EspD, but controlled the levels of EspD and EspA secreted in vitro or upon cell contact. While still proficient for T3SS-mediated injection of bacterial effectors and cytoskeletal reorganization, an espC mutant showed increased levels of cell-associated EspA and EspD, as well as increased pore formation activity associated with cytotoxicity. EspP from enterohaemorrhagic E. coli (EHEC) also targeted translocator components and its activity was interchangeable with that of EspC, suggesting a common and important function of these SPATEs. These findings reveal a novel regulatory mechanism of T3SS-mediated pore formation and cytotoxicity control during EPEC/EHEC infection.


Subject(s)
Enteropathogenic Escherichia coli/pathogenicity , Escherichia coli Infections/metabolism , Escherichia coli Proteins/metabolism , Pore Forming Cytotoxic Proteins/metabolism , Type III Secretion Systems/metabolism , Bacterial Proteins/metabolism , Electrophoresis, Polyacrylamide Gel , Enteropathogenic Escherichia coli/metabolism , HeLa Cells , Humans , Immunohistochemistry , Microscopy, Confocal , Mutagenesis, Site-Directed
10.
J Mol Cell Biol ; 1(2): 64-5, 2009 Dec.
Article in English | MEDLINE | ID: mdl-19720630

ABSTRACT

OspE, a Shigella type III effector binds to integrin-like kinase and enhances cell adhesion to better disseminate and colonize the intestinal epithelium. Because of the existence of OspE orthologues in other enteropathogens such as enteropathogenic Escherichia coli or Salmonella sp., maintenance of cell adhesion appears as a widespread strategy for bacteria that interact with the intestinal epithelium.


Subject(s)
Host-Pathogen Interactions , Shigella/physiology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Focal Adhesions/microbiology , Gene Expression Regulation, Bacterial , Humans , Shigella/genetics , Shigella/pathogenicity
11.
Infect Immun ; 77(1): 517-31, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19015254

ABSTRACT

We used transfected epithelial CHO-B2 cells as a model to identify the mechanism mediating internalization of Afa/Dr diffusely adhering Escherichia coli. We provide evidence that neither the alpha5 or beta1 integrin subunits nor alpha5beta1 integrin functioned as a receptor mediating the adhesion and/or internalization of Dr or Afa-III fimbria-positive bacteria. We also demonstrated that (i) whether or not the AfaD or DraD invasin subunits were present, there was no difference in the cell association and entry of bacteria and that (ii) DraE or AfaE-III adhesin subunits are necessary and sufficient to promote the receptor-mediated bacterial internalization into epithelial cells expressing human decay-accelerating factor (DAF), CEACAM1, CEA, or CEACAM6. Internalization of Dr fimbria-positive E. coli within CHO-DAF, CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cells occurs through a microfilament-independent, microtubule-dependent, and lipid raft-dependent mechanism. Wild-type Dr fimbria-positive bacteria survived better within cells expressing DAF than bacteria internalized within CHO-CEACAM1, CHO-CEA, or CHO-CEACAM6 cells. In DAF-positive cells, internalized Dr fimbria-positive bacteria were located in vacuoles that contained more than one bacterium, displaying some of the features of late endosomes, including the presence of Lamp-1 and Lamp-2, and some of the features of CD63 proteins, but not of cathepsin D, and were acidic. No interaction between Dr fimbria-positive-bacterium-containing vacuoles and the autophagic pathway was observed.


Subject(s)
Bacterial Adhesion , Cytoplasm/microbiology , Escherichia coli/physiology , Receptors, Cell Surface/physiology , Adhesins, Escherichia coli/metabolism , Animals , Antigens, CD/metabolism , CD55 Antigens/metabolism , CHO Cells , Cell Adhesion Molecules/metabolism , Cricetinae , Cricetulus , Epithelial Cells/microbiology , GPI-Linked Proteins , HeLa Cells , Humans , Microbial Viability , Receptors, Cell Surface/metabolism , Vacuoles/microbiology
12.
Microb Pathog ; 45(5-6): 415-22, 2008.
Article in English | MEDLINE | ID: mdl-18977288

ABSTRACT

Until recently, intermediate filaments (IF) were thought to be only involved in resistance to physical stress and mechanical integrity of cells and tissues. Recent data indicate that IF play a much more important role in cellular physiology including organelle structure and positioning within the cell. Here, we show that Salmonella enterica serovar Typhimurium (S. typhimurium) induces in epithelial cells and macrophages the formation of an aggresome-like structure with a dramatic remodelling of cytoplasmic IF (vimentin and cytokeratin) networks and the adaptor proteins 14-3-3 which are recruited around intracellular S. typhimurium microcolonies. These rearrangements are not necessary for bacterial replication. Depletion of vimentin and cytokeratin by siRNA indicates that IF remodelling is required to maintain Salmonella microcolonies in the juxtanuclear area.


Subject(s)
Cell Nucleus/microbiology , Intermediate Filaments/metabolism , Salmonella Infections/metabolism , Salmonella typhimurium/physiology , Vacuoles/microbiology , Animals , Cell Line , Cell Nucleus/metabolism , Humans , Intermediate Filament Proteins/genetics , Intermediate Filament Proteins/metabolism , Intermediate Filaments/chemistry , Intermediate Filaments/microbiology , Mice , Protein Biosynthesis , Salmonella Infections/microbiology , Vacuoles/metabolism
13.
Cell Microbiol ; 9(1): 204-21, 2007 Jan.
Article in English | MEDLINE | ID: mdl-16903846

ABSTRACT

Afa/Dr diffusely adhering Escherichia coli (DAEC) strains are responsible for urinary tract and intestinal infections. Both in intestine and kidney, the epithelial cells forming epithelium are sealed by junctional domains. We provide evidence that the Secreted autotransporter toxin, Sat, belonging to the subfamily of serine protease autotransporters of Enterobacteriaceae (SPATEs), acts as a virulence factor in Afa/Dr DAEC by promoting lesions in the tight junctions (TJs) of polarized epithelial Caco-2/TC7 cells. Southern blot analysis reveals that the prototype strains of the subclass-1 and subclass-2 typical Afa/Dr DAEC strains, hybridize with a sat probe. Using the wild-type IH11128 strain, the recombinant E. coli AAEC185 strain that expresses Sat, the recombinant E. coli that expresses both Dr adhesin and Sat, we report that Sat in monolayers of cultured enterocyte-like Caco-2/TC7 cells, induces rearrangements of the TJs-associated proteins ZO-1, ZO-3 and occludin, and increases the formation of domes as the result of an increase in the paracellular permeability without affecting the transepithelial electrical resistance of the cell monolayers. Moreover, we observe that Sat-induced disassembly of TJs-associated proteins is dependent on the serine protease motif. Finally, an analysis of the prevalence of the sat gene in three collections of Afa/Dr DAEC strains collected from the stools of children with and without diarrhoea, and from the urine of patients with urinary tract infection (UTI) shows that: (i) the sat gene is highly prevalent in UTI-associated Afa/Dr DAEC strains (88% positive), (ii) the sat gene is generally absent from Afa/Dr DAEC strains collected from the stools of children without diarrhoea (16% positive); whereas (iii) it is present in about half of the strains collected from the stools of children with diarrhoea (46% positive).


Subject(s)
Adhesins, Escherichia coli/metabolism , Bacterial Toxins/metabolism , Epithelial Cells/microbiology , Escherichia coli Infections/microbiology , Escherichia coli Proteins/metabolism , Escherichia coli/pathogenicity , Tight Junctions/ultrastructure , Actins/metabolism , Amino Acid Motifs , Bacterial Adhesion , Bacterial Toxins/chemistry , Bacterial Toxins/genetics , Caco-2 Cells , Child , Diarrhea/microbiology , Epithelial Cells/cytology , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Feces/microbiology , Humans , Mutagenesis, Site-Directed , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Tight Junctions/metabolism , Urinary Tract Infections/microbiology , Virulence Factors/metabolism
14.
Mol Microbiol ; 62(2): 356-66, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16965519

ABSTRACT

Afa/Dr family of adhesins are produced by pathogenic Escherichia coli strains that are especially prevalent in chronic diarrhoeal and recurrent urinary tract infections. Most notably, they are found in up to 50% of cystitis cases in children and 30% of pyelonephritis in pregnant women. Afa/Dr adhesins are capped surface fibrils that mediate recognition of the host and subsequent bacterial internalization. Using the newly solved three-dimensional structure of the minimal invasive complex (AfaDE) combined with biochemical and cellular assays, we reveal the architecture of the fibrillar cap and identify a novel mode of synergistic integrin recognition.


Subject(s)
Adhesins, Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Adhesins, Escherichia coli/chemistry , Adhesins, Escherichia coli/genetics , Amino Acid Sequence , Bacterial Adhesion/genetics , Bacterial Adhesion/physiology , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Fimbriae, Bacterial/genetics , Fimbriae, Bacterial/metabolism , Magnetic Resonance Spectroscopy , Microscopy, Fluorescence , Models, Molecular , Molecular Sequence Data , Protein Binding
15.
J Biol Chem ; 281(39): 29120-30, 2006 Sep 29.
Article in English | MEDLINE | ID: mdl-16882658

ABSTRACT

Escherichia coli expressing the Dr family of adhesins adheres to epithelial cells by binding to decay-accelerating factor (DAF) and carcinoembryonic antigen (CEA)-related cell surface proteins. The attachment of bacteria expressing Dr adhesins to DAF induces clustering of DAF around bacterial cells and also recruitment of CEA-related cell adhesion molecules. CEA, CEACAM1, and CEACAM6 have been shown to serve as receptors for some Dr adhesins (AfaE-I, AfaE-III, DraE, and DaaE). We demonstrate that AfaE-I, AfaE-V, DraE, and DaaE adhesins bind to the N-domain of CEA. To identify the residues involved in the N-CEA/DraE interaction, we performed SPR binding analyses of naturally occurring variants and a number of randomly generated mutants in DraE and N-CEA. Additionally, we used chemical shift mapping by NMR to determine the surface of DraE involved in N-CEA binding. These results show a distinct CEA binding site located primarily in the A, B, E, and D strands of the Dr adhesin. Interestingly, this site is located opposite to the beta-sheet encompassing the previously determined binding site for DAF, which implies that the adhesin can bind simultaneously to both receptors on the epithelial cell surface. The recognition of CEACAMs from a highly diverse DrCEA subfamily of Dr adhesins indicates that interaction with these receptors plays an important role in niche adaptation of E. coli strains expressing Dr adhesins.


Subject(s)
Adhesins, Bacterial/physiology , CD55 Antigens/physiology , Carcinoembryonic Antigen/chemistry , Escherichia coli/metabolism , Adhesins, Bacterial/chemistry , Animals , CD55 Antigens/chemistry , CHO Cells , Cell Adhesion , Cricetinae , Fimbriae, Bacterial/metabolism , Humans , Protein Binding , Protein Structure, Tertiary
16.
J Cell Sci ; 117(Pt 7): 1033-45, 2004 Mar 01.
Article in English | MEDLINE | ID: mdl-14970261

ABSTRACT

Infection of host cells by Salmonella enterica serovar Typhimurium (S. typhimurium) leads to the formation of specialised membrane-bound compartments called Salmonella-containing vacuoles (SCVs). Bacteria remain enclosed by the vacuolar membrane as they divide, and by translocating effector proteins across the vacuolar membrane through the SPI-2 type III secretion system, they interfere with host cell processes in ways that promote bacterial growth. One such effector is SifA, which is required to maintain the integrity of the vacuolar membrane and for the formation in epithelial cells of long tubular structures called Sifs that are connected to SCVs. Unknown effector(s) mediate the assembly of a meshwork of F-actin around SCVs. We report that intracellular bacteria also cause a dramatic accumulation of microtubules around S. typhimurium microcolonies in both epithelial cells and macrophages. Although this process appears to be independent of SPI-2-mediated F-actin assembly, it does require bacterial protein synthesis. In epithelial cells, microtubule accumulation is accompanied by the recruitment of both kinesin and dynein. Inhibition of the activity of either motor prevented both Sif formation and the loss of vacuolar membrane from sifA mutant bacteria. It also resulted in morphologically abnormal vacuoles enclosing wild-type bacteria, and impaired their replication. Our experiments indicate that recruitment of dynein to SCVs is dependent on Rab7 activity. We show that the recently described Rab7 effector RILP is also recruited to SCVs in a Rab7-dependent manner. However, overexpression of RILP did not restore dynein recruitment to SCVs in cells expressing dominant negative Rab7, suggesting that RILP requires a functional Rab7 to be activated at the SCV membrane, or that dynein recruitment is mediated by an effector other than RILP. Together, these experiments indicate that microtubule motors play important roles in regulating vacuolar membrane dynamics during intracellular replication of S. typhimurium.


Subject(s)
Microtubules/physiology , Molecular Motor Proteins/physiology , Salmonella typhimurium/pathogenicity , Vacuoles/microbiology , Vacuoles/physiology , Actins/physiology , Adaptor Proteins, Signal Transducing , Animals , Bacterial Proteins/biosynthesis , Carrier Proteins/genetics , Carrier Proteins/physiology , Cell Line , Dyneins/antagonists & inhibitors , Dyneins/physiology , HeLa Cells , Humans , Kinesins/antagonists & inhibitors , Kinesins/physiology , Mice , Molecular Motor Proteins/antagonists & inhibitors , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Salmonella typhimurium/growth & development , Salmonella typhimurium/physiology , Transfection , rab GTP-Binding Proteins/physiology , rab7 GTP-Binding Proteins
17.
EMBO Rep ; 5(1): 104-10, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14710194

ABSTRACT

While remaining extracellular, enteropathogenic Escherichia coli (EPEC) establish direct links with the cytoskeleton of the target epithelial cell leading to the formation of actin-rich pedestals underneath attached bacteria. The translocated adaptor protein Tir forms the transmembrane bridge between the cytoskeleton and the bacterium; the extracellular domain of Tir functions as a receptor for the bacterial adhesin intimin, while the intracellular amino and carboxy termini interact with a number of focal adhesion and other cytoskeletal proteins; and recruitment of some is dependent on phosphorylation of Tyr 474. Using Tir as bait and HeLa cell cDNA library as prey in a yeast two-hybrid screen, we identified cytokeratin 18 as a novel Tir partner protein. Cytokeratin 18 is recruited to the EPEC-induced pedestal and has a direct role in actin accretion and cytoskeleton reorganization. This study is the first to implicate intermediate filaments in microfilament reorganization following EPEC infection.


Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/pathogenicity , Keratins/metabolism , Receptors, Cell Surface/metabolism , Actins/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Infections/microbiology , Escherichia coli Proteins/genetics , HeLa Cells , Humans , Immunohistochemistry , Immunoprecipitation , Phosphorylation , Plasmids , RNA, Small Interfering/genetics , Receptors, Cell Surface/genetics , Transfection
18.
Cell Microbiol ; 5(10): 681-93, 2003 Oct.
Article in English | MEDLINE | ID: mdl-12969374

ABSTRACT

The afa operons from Escherichia coli associated with extra-intestinal and intestinal infections have been characterized and the AfaD protein has been shown to be involved in the low internalization of laboratory strains expressing the afa-3 operon. The aim of this study was to determine the role of the AfaD invasin during the interaction of pathogenic E. coli with epithelial cells. We show that AfaD is implicated in the entry of a clinical isolate into both HeLa and undifferentiated Caco-2 cells. Once in the cytoplasm of these cells, the bacteria formed inclusions in which they were able to survive for at least 72 h. Internalization assays using polystyrene beads coated with His6-tagged purified AfaD (rAfaD) demonstrated that this invasin mediates entry into cells derived from various tissues (intestine and urothelium) that are targets for afa-positive strains. Consistent with the previous observation that an antibody blockade involving anti-alpha5beta1 integrin abolishes bacterial internalization, we show here that the entry of rAfaD-coated beads was dependent on the production and accessibility of beta1 integrins on the cells. The AfaD proteins belong to a family of invasins that are at least 45% identical. Despite their differences, the recombinant rAfaD-III and rAfaD-VIII proteins both bound to beta1 integrins. Our results suggest that beta1 integrin is a common receptor for AfaD invasins and that additional AfaD-type-specific receptors exist.


Subject(s)
Adhesins, Escherichia coli/genetics , Adhesins, Escherichia coli/metabolism , Escherichia coli Infections , Escherichia coli/pathogenicity , Integrin beta1/metabolism , Adhesins, Escherichia coli/isolation & purification , Animals , Cell Line , Cell Line, Tumor , Colony Count, Microbial , Cricetinae , Dogs , Epithelial Cells/cytology , Epithelial Cells/microbiology , Epithelial Cells/ultrastructure , Gene Deletion , Genes, Bacterial , Genetic Complementation Test , Humans , Immunoassay , Inclusion Bodies/microbiology , Integrin beta1/genetics , Integrin beta1/immunology , Mice , Mutagenesis, Insertional/genetics , Recombinant Proteins/metabolism
19.
Infect Immun ; 71(4): 1774-83, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12654791

ABSTRACT

Ulcerative colitis and Crohn's disease are inflammatory bowel diseases thought to involve strains of Escherichia coli. We report here that two wild-type Afa/Dr diffusely adhering E. coli (DAEC) strains, C1845 and IH11128, which harbor the fimbrial F1845 adhesin and the Dr hemagglutinin, respectively, and the E. coli laboratory strain HB101, transformed with the pSSS1 plasmid to produce Afa/Dr F1845 adhesin, all induced interleukin-8 (IL-8) production and transepithelial migration of polymorphonuclear leukocytes (PMNL) in polarized monolayers of the human intestinal cell line T84 grown on semipermeable filters. We observed that after PMNL migration, expression of decay-accelerating factor (DAF, or CD55), the brush border-associated receptor for Afa/Dr adhesins, was strongly enhanced, increasing the adhesion of Afa/Dr DAEC bacteria. When examining the mechanism by which DAF expression was enhanced, we observed that the PMNL transepithelial migration induced epithelial synthesis of tumor necrosis factor alpha and IL-1beta, which in turn promoted the upregulation of DAF.


Subject(s)
Adhesins, Escherichia coli/metabolism , Bacterial Adhesion , CD55 Antigens/metabolism , Escherichia coli/pathogenicity , Neutrophil Infiltration , Up-Regulation , Adhesins, Escherichia coli/genetics , Adhesins, Escherichia coli/physiology , Cell Polarity , Cytokines/metabolism , Escherichia coli/genetics , Escherichia coli/immunology , Escherichia coli/physiology , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , HeLa Cells , Hemagglutinins/genetics , Hemagglutinins/metabolism , Humans , Microscopy, Electron , Neutrophils/immunology , Tumor Cells, Cultured
20.
Infect Immun ; 71(3): 1068-74, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12595416

ABSTRACT

Afa/Dr diffusely adhering Escherichia coli (Afa/Dr DAEC) strains cause symptomatic urinary tract and intestinal infections. The proinflammatory effects of Afa/Dr DAEC strains in vitro have been not investigated to date. In the present study, we used confluent polarized monolayers of intestinal cell line T84 to evaluate the consequences of epithelial infection by Afa/Dr DAEC strains in terms of proinflammatory response. Polymorphonuclear leukocyte (PMNL) migration across the epithelial barrier was induced after incubation of the T84 monolayers with the wild-type Afa/Dr DAEC strain C1845 harboring the fimbrial F1845 adhesin and strain IH11128 harboring the Dr hemagglutinin, and the E. coli laboratory strain HB101 was transformed with the pSSS1 plasmid, producing Afa/Dr F1845 adhesin. PMNL migrations were correlated with a basolateral secretion of interleukin-8 by T84 cells and were abolished after incubation of epithelial cells with an anti-decay accelerating factor (DAF) antibody that recognized the short consensus repeat 3 domain of DAF (monoclonal antibody 1H4). Moreover, Afa/Dr DAEC strains induced tyrosine phosphorylation of several T84 proteins and activated the mitogen-activated protein kinases (ERK1/2 mitogen-activated protein, P38, and Jun-C kinases). These data demonstrated for the first time that, in vitro, Afa/Dr DAEC strains exert a proinflammatory signal in intestinal epithelial cells.


Subject(s)
Adhesins, Escherichia coli/physiology , Bacterial Adhesion , Escherichia coli/physiology , Interleukin-8/biosynthesis , Mitogen-Activated Protein Kinases/metabolism , Neutrophils/physiology , Cell Movement , Cell Polarity , Cells, Cultured , Enzyme Activation , Humans , Intestinal Mucosa/microbiology , Phosphorylation , Signal Transduction , Tyrosine/metabolism
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