Showing papers by "B. Brett Finlay published in 2001"
TL;DR: It is shown that tyrosine 474 of Tir directly binds the host-cell adaptor protein Nck, and that Nck is required for the recruitment of both neural Wiskott–Aldrich-syndrome protein (N-WASP) and the actin-related protein (Arp)2/3 complex to the EPEC pedestal, directly linking Tir to the cytoskeleton.
Abstract: Enteropathogenic E. coli Tir binds Nck to initiate actin pedestal formation in host cells
377 citations
TL;DR: The results indicate that the full DNA sequence of C.rodentium LEE has been acquired by C. rodentium and A/E E.coli strains independently during evolution.
Abstract: The family of attaching and effacing (A/E) bacterial pathogens, which includes diarrheagenic enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC), remains a significant threat to human and animal health. These bacteria intimately attach to host intestinal cells, causing the effacement of brush border microvilli. The genes responsible for this phenotype are encoded in a pathogenicity island called the locus of enterocyte effacement (LEE). Citrobacter rodentium is the only known murine A/E pathogen and serves as a small animal model for EPEC and EHEC infections. Here we report the full DNA sequence of C. rodentium LEE and provide a comparative analysis with the published LEEs from EPEC, EHEC, and the rabbit diarrheagenic E. coli strain RDEC-1. Although C. rodentium LEE shows high similarities throughout the entire sequence and shares all 41 open reading frames with the LEE from EPEC, EHEC, and RDEC-1, it is unique in its location of the rorf1 and rorf2/espG genes and the presence of several insertion sequences (IS) and IS remnants. The LEE of EPEC and EHEC is inserted into the selC tRNA gene. In contrast, the Citrobacter LEE is flanked on one side by an operon encoding an ABC transport system, and an IS element and sequences homologous to Shigella plasmid R100 and EHEC pO157 flank the other. The presence of plasmid sequences next to C. rodentium LEE suggests that the prototype LEE resided on a horizontally transferable plasmid. Additional sequence analysis reveals that the 3-kb plasmid in C. rodentium is nearly identical to p9705 in EHEC O157:H7, suggesting that horizontal plasmid transfer among A/E pathogens has occurred. Our results indicate that the LEE has been acquired by C. rodentium and A/E E. coli strains independently during evolution.
223 citations
TL;DR: It is demonstrated that although EPEC and EHEC recruit similar cytoskeletal proteins, there are also significant differences in pedestal composition.
Abstract: Enteropathogenic Escherichia coli (EPEC) is a human pathogen that attaches to intestinal epithelial cells and causes chronic watery diarrhea. A close relative, enterohemorrhagic E. coli (EHEC), causes severe bloody diarrhea and hemolytic-uremic syndrome. Both pathogens insert a protein, Tir, into the host cell plasma membrane where it binds intimin, the outer membrane ligand of EPEC and EHEC. This interaction triggers a cascade of signaling events within the host cell and ultimately leads to the formation of an actin-rich pedestal upon which the pathogen resides. Pedestal formation is critical in mediating EPEC- and EHEC-induced diarrhea, yet very little is known about its composition and organization. In EPEC, pedestal formation requires Tir tyrosine 474 phosphorylation. In EHEC Tir is not tyrosine phosphorylated, yet the pedestals appear similar. The composition of the EPEC and EHEC pedestals was analyzed by examining numerous cytoskeletal, signaling, and adapter proteins. Of the 25 proteins examined, only two, calpactin and CD44, were recruited to the site of bacterial attachment independently of Tir. Several others, including ezrin, talin, gelsolin, and tropomyosin, were recruited to the site of EPEC attachment independently of Tir tyrosine 474 phosphorylation but required Tir in the host membrane. The remaining proteins were recruited to the pedestal in a manner dependent on Tir tyrosine phosphorylation or were not recruited at all. Differences were also found between the EPEC and EHEC pedestals: the adapter proteins Grb2 and CrkII were recruited to the EPEC pedestal but were absent in the EHEC pedestal. These results demonstrate that although EPEC and EHEC recruit similar cytoskeletal proteins, there are also significant differences in pedestal composition.
182 citations
TL;DR: The findings suggest that SifA is an effector of the SPI‐2 type III secretion system and allows colonization of murine macrophages, the host niche exploited during systemic phases of disease in these animals.
Abstract: SifA was originally identified as a virulence factor required for formation of Salmonella-induced filaments (Sifs), elongated tubules rich in lysosomal glycoproteins that extend from the Salmonella-containing vacuole in infected epithelial cells. Here, we demonstrate that deletion mutants of ssaR, a component of the SPI-2 type III secretion system, do not form Sifs in HeLa epithelial cells. This suggests that SifA is a translocated effector of this system, acting within host cells to form Sifs. In support of this hypothesis, transfection of HeLa cells with a vector encoding SifA fused to the green fluorescent protein caused extensive vacuolation of LAMP-1-positive compartments. Filamentous tubules that closely resembled Sifs were also observed in transfected cells, demonstrating that SifA is sufficient to initiate alteration of host cell endosomal structures. deltasifA mutants were impaired in their ability to survive/replicate in RAW 264.7 murine macrophages, a phenotype similar to ssaR mutants. Our findings suggest that SifA is an effector of the SPI-2 type III secretion system and allows colonization of murine macrophages, the host niche exploited during systemic phases of disease in these animals. A family of SifA-related proteins and their importance to Salmonella pathogenesis is also discussed.
182 citations
TL;DR: Isothermal titration calorimetry studies of Tir–CesT and enzymatic activity profiles of SigD–SigE indicate that the effector proteins are not globally unfolded in the presence of their cognate chaperones, and variable, delocalized hydrophobic surfaces observed on the chaperone homodimers are responsible for specific binding to a particular effector protein.
Abstract: Several Gram-negative bacterial pathogens have evolved a type III secretion system to deliver virulence effector proteins directly into eukaryotic cells, a process essential for disease This specialized secretion process requires customized chaperones specific for particular effector proteins The crystal structures of the enterohemorrhagic Escherichia coli O157:H7 Tir-specific chaperone CesT and the Salmonella enterica SigD-specific chaperone SigE reveal a common overall fold and formation of homodimers Site-directed mutagenesis suggests that variable, delocalized hydrophobic surfaces observed on the chaperone homodimers are responsible for specific binding to a particular effector protein Isothermal titration calorimetry studies of Tir-CesT and enzymatic activity profiles of SigD-SigE indicate that the effector proteins are not globally unfolded in the presence of their cognate chaperones
155 citations
TL;DR: A large number of pathogens have developed virulence factors that interact with host molecules, often usurping normal cellular processes, and mediate events as diverse as bacterial invasion, antiphagocytosis, and intracellular parastism.
Abstract: Microbial pathogens cause a spectrum of diseases in humans. Although the disease mechanisms vary considerably, most pathogens have developed virulence factors that interact with host molecules, often usurping normal cellular processes, including cytoskeletal dynamics and vesicle targeting. These virulence factors often mimic host molecules, and mediate events as diverse as bacterial invasion, antiphagocytosis, and intracellular parastism.
152 citations
TL;DR: The results show that EPEC blocks its uptake by inhibiting a PI 3‐kinase‐mediated pathway, and translocates effectors other than Tir to interfere with actin‐driven host cell processes, which constitutes a novel mechanism of phagocytosis avoidance by an extracellular pathogen.
Abstract: The extracellular pathogen enteropathogenic Escherichia coli (EPEC) uses a type III secretion system to inhibit its uptake by macrophages. We show that EPEC antiphagocytosis is independent of the translocated intimin receptor Tir and occurs by preventing F‐actin polymerization required for bacterial uptake. EPEC–macrophage contact triggered activation of phosphatidylinositol (PI) 3‐kinase, which was subsequently inhibited in a type III secretion‐dependent manner. Inhibition of PI 3‐kinase significantly reduced uptake of a secretion‐deficient mutant, without affecting antiphagocytosis by the wild type, suggesting that EPEC blocks a PI 3‐kinase‐dependent phagocytic pathway. EPEC specifically inhibited Fcγ receptor‐ but not CR3‐receptor mediated phagocytosis of opsonized zymosan. We showed that EPEC inhibits PI 3‐kinase activity rather than its recruitment to the site of bacterial contact. Phago cytosis of a secretion mutant correlated with the association of PI 3‐kinase with tyrosine‐phosphorylated proteins, which wild‐type EPEC prevented. These results show that EPEC blocks its uptake by inhibiting a PI 3‐kinase‐mediated pathway, and translocates effectors other than Tir to interfere with actin‐driven host cell processes. This constitutes a novel mechanism of phagocytosis avoidance by an extracellular pathogen.
145 citations
TL;DR: It is demonstrated that Sif formation involves fusion of late endocytic compartments with the Salmonella‐containing vacuole, and suggested that S ifA modulates this event.
Abstract: Salmonella typhimurium is a facultative intracellular pathogen that colonizes host cells throughout the course of infection. A unique feature of this pathogen is its ability to enter into (invade) epithelial cells and elongate the vacuole within which it resides into tubular structures called Salmonella-induced filaments (Sifs). In this study we sought to characterize the mechanism of Sif formation by immunofluorescence analysis using subcellular markers. The late endosomal lipid lysobisphosphatidic acid associated in a punctate pattern with the Salmonella-containing vacuole, starting 90 min after infection and increasing thereafter. Lysobisphosphatidic acid-rich vesicles were also found to interact with Sifs, at numerous sites along the tubules. Similarly, cholesterol-rich vesicles were also found in association with intracellular bacteria and Sifs. The lysosomal hydrolase cathepsin D was present in Sifs, both in a punctate pattern and, at later times, predominantly in an uninterrupted linear pattern. Rab7 associated with Sifs and expression of the N125I dominant negative mutant of this GTPase inhibited Sif formation. Transfection of HeLa cells with a vector encoding SifA fused to the green fluorescent protein caused swelling and aggregation of lysobisphosphatidic acid-containing compartments, suggesting that this virulence factor directs membrane fusion events involving late endosomes. Our findings demonstrate that Sif formation involves fusion of late endocytic compartments with the Salmonella-containing vacuole, and suggest that SifA modulates this event.
129 citations
TL;DR: A region near the carboxyl‐terminus of SigD is characterized which is important for phosphatase activity, and how dephosphorylation of inositol phospholipids by SigD in vivo might contribute to the activation of Akt is discussed.
124 citations
TL;DR: Differences in the mechanisms of pedestal formation by these closely related pathogens are highlighted and indicate that EPEC and EHEC modulate different signalling pathways to affect the host actin cytoskeleton.
Abstract: Enterohaemorrhagic Escherichia coli (EHEC) adheres to the host intestinal epithelium, resulting in the formation of actin pedestals beneath adhering bacteria. EHEC and a related pathogen, enteropathogenic E. coli (EPEC), insert a bacterial receptor, Tir, into the host plasma membrane, which is required for pedestal formation. An important difference between EPEC and EHEC Tir is that EPEC but not EHEC Tir is tyrosine phosphorylated once delivered into the host. In this study, we assessed the role of Tir tyrosine phosphorylation in pedestal formation by EPEC and EHEC. In EPEC, pedestal formation is absolutely dependent on Tir tyrosine phosphorylation and is not complemented by EHEC Tir. The protein sequence surrounding EPEC Tir tyrosine 474 is critical for Tir tyrosine phosphorylation and pedestal formation by EPEC. In contrast, Tir tyrosine phosphorylation is not required for pedestal formation by EHEC. EHEC forms pedestals with both wild-type EPEC Tir and the non-tyrosine-phosphorylatable EPEC Tir Y474F. Pedestal formation by EHEC requires the type III delivery of additional EHEC factors into the host cell. These findings highlight differences in the mechanisms of pedestal formation by these closely related pathogens and indicate that EPEC and EHEC modulate different signalling pathways to affect the host actin cytoskeleton.
123 citations
TL;DR: The manipulation of apoptosis by Salmonella is described and the advantages that such actions may confer to the bacteria, and its implications in resistance to disease are discussed.
TL;DR: Gen array technology was used to study epithelial cell responses to EPEC infection at the level of gene expression and found that EPEC induces the expression of several genes in infected HeLa cells by a lipopolysaccharide-independent mechanism, including cytokines and early growth response factor 1 (Egr-1).
Abstract: Enteropathogenic Escherichia coli (EPEC) is an extracellular bacterial pathogen that infects the human intestinal epithelium and is a major cause of infantile diarrhea in developing countries. EPEC belongs to the group of attaching and effacing (A/E) pathogens. It uses a type III secretion system to deliver proteins into the host cell that mediate signal transduction events in host cells. We used gene array technology to study epithelial cell responses to EPEC infection at the level of gene expression. We found that EPEC induces the expression of several genes in infected HeLa cells by a lipopolysaccharide (LPS)-independent mechanism, including cytokines and early growth response factor 1 (Egr-1). The transcription factor Egr-1 is an immediate-early-induced gene that is activated in most cell types in response to stress. EPEC-induced upregulation of egr-1 is mediated by the activation of the MEK/extracellular signal-regulated kinase signal transduction pathway and is dependent on the type III secretion system. egr-1 is also induced during infection of mice by the A/E pathogen Citrobacter rodentium, suggesting that both Egr-1 and the activation of this mitogen-activated protein kinase signal transduction pathway may play a role in disease.
TL;DR: This multidisciplinary research will enhance the understanding of the basic mechanisms of bacterial infections and immunity; it also provides new approaches towards therapeutic and control measures.
TL;DR: In this paper, the authors used a naturally occurring weaned rabbit infection model to determine physiological levels of apoptosis in rabbit ileum and ileal Peyer's patches (PP) and compared them to those found after infection with adherent rabbit EPEC (REPEC O103).
Abstract: Significant changes occur in intestinal epithelial cells after infection with enteropathogenic Escherichia coli (EPEC). However, it is unclear whether this pathogen alters rates of apoptosis. By using a naturally occurring weaned rabbit infection model, we determined physiological levels of apoptosis in rabbit ileum and ileal Peyer's patches (PP) and compared them to those found after infection with adherent rabbit EPEC (REPEC O103). Various REPEC O103 strains were first tested in vitro for characteristic virulence features. Rabbits were then inoculated with the REPEC O103 strains that infected cultured cells the most efficiently. After experimental infection, intestinal samples were examined by light and electron microscopy. Simultaneously, ileal apoptosis was assessed by using terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) and caspase 3 assays and by apoptotic cell counts based on morphology (hematoxylin-and-eosin staining). The highest physiological apoptotic indices were measured in PP germinal centers (median = 14.7%), followed by PP domed villi (8.1%), tips of absorptive villi (3.8%), and ileal crypt regions (0.5%). Severe infection with REPEC O103 resulted in a significant decrease in apoptosis in PP germinal centers (determined by TUNEL assay; P = 0.01), in the tips of ileal absorptive villi (determined by HE P = 0.04), and in whole ileal cell lysates (determined by caspase 3 assay; P = 0.001). We concluded that REPEC O103 does not promote apoptosis. Furthermore, we cannot rule out the possibility that REPEC O103, in fact, decreases apoptotic levels in the rabbit ileum.
TL;DR: Recent insights into macrophage and epithelial cell responses to Salmonella infection garnered from array studies are outlined and used as a basis to discuss various future research directions using gene arrays that will advance the field of cellular microbiology.
01 Dec 2001
TL;DR: In this paper, the authors focus on Salmonella interactions with its host cells, both in tissue culture (in vitro) and in relevant animal models (in vivo), and compare results obtained using these different models.
Abstract: Salmonellosis (diseases caused by Salmonella species) have several clinical manifestations, ranging from gastroenteritis (food poisoning) to typhoid (enteric) fever and bacteraemia. Salmonella species (especially Salmonella typhimurium) also represent organisms that can be readily used to investigate the complex interplay that occurs between a pathogen and its host, both in vitro and in vivo. The ease with which S. typhimurium can be cultivated and genetically manipulated, in combination with the availability of tissue culture models and animal models, has made S. typhimurium a desirable organism for such studies. In this review, we focus on Salmonella interactions with its host cells, both in tissue culture (in vitro) and in relevant animal models (in vivo), and compare results obtained using these different models. The recent advent of sophisticated imaging and molecular genetic tools has facilitated studying the events that occur in disease, thereby confirming tissue culture results, yet identifying new questions that need to be addressed in relevant disease settings.
TL;DR: A new mouse model of listeriosis is discussed that mimics the oral infection route of humans and confirms the part played by the bacterial surface protein, internalin, in enabling the bacteria to enter gut epithelial cells of the host.
Abstract: Listeriosis is a serious food-borne bacterial disease that causes death in newborns and the elderly and can induce late-term miscarriages in pregnant women. In a Perspective, [Finlay][1] discusses a new mouse model of listeriosis ([ Lecuit et al .][2]) that mimics the oral infection route of humans and confirms the part played by the bacterial surface protein, internalin, in enabling the bacteria to enter gut epithelial cells of the host.
[1]: http://www.sciencemag.org/cgi/content/full/292/5522/1665
[2]: http://www.sciencemag.org/cgi/content/short/292/5522/1722
TL;DR: Enteropathogenic Escherichia coli strain 30-5-1(3), which has a transposon insertion within thesepZ gene, forms wild-type A/E lesions including Tir tyrosine phosphorylation, but at a slower rate.
Abstract: Enteropathogenic Escherichia coli (EPEC) strain 30-5-1(3) has been reported to form attaching and effacing (A/E) lesions without Tir tyrosine phosphorylation. In this study, we show that 30-5-1(3), which has a transposon insertion within the sepZ gene, forms wild-type A/E lesions including Tir tyrosine phosphorylation, but at a slower rate. A/E lesion formation by 30-5-1(3) occurs without detectable secretion of Tir or other EPEC Esp secreted proteins.
TL;DR: A wide variety of Gram-negative bacterial pathogens use a 'type III' protein secretion system to deliver bacterial virulence factors into host cells, and recent results suggest that Gram-positive pathogens may employ similar methods.