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Biomedical subjects

B B Finlay

Publications and source records attributed to B B Finlay.

At least 19 recordsLinked to original sources
PubMed2007

The role of dynamin 3 in the testis.

We report here that dynamin 3 in the testis is associated with structures termed tubulobulbar complexes that internalize intact intercellular junctions during sperm release and turnover of the blood-testis barrier. The protein lies adjacent to an actin-Arp2/3 network that cuffs the double plasma membrane tubular invagination at the core of each complex. To explore the possible relationship between dynamin 3 and nectin-based adhesion junctions, we transiently transfected DsRed-tagged dynamin 3 into MDCK cells stably transfected with eGFP-tagged nectin 2, one of the adhesion molecules known to be expressed in Sertoli cells at adhesion junctions. Cells transfected with the dynamin 3 construct had less uniformly distributed nectin 2 at intercellular contacts when compared to control cells expressing only nectin 2 or transfected with the DsRed plasmid alone. Significantly, tubular extensions positive for nectin 2 were visible projecting into the cells from regions of intercellular contact. Our findings are consistent with the conclusion that dynamin 3 is involved with tubulobulbar morphogenesis. Dynamin 3 also occurs in concentrated deposits around the capitulum and striated columns in the connecting piece of sperm tails suggesting that the protein in these cells may function to stabilize the base of the tail or serve as a reservoir for use during or after fertilization.

Actin-Related Protein 2-3 Complex↗
PubMed2006

Salmonella, the host and disease: a brief review.

Salmonella species cause substantial morbidity, mortality and burden of disease globally. Infections with Salmonella species cause multiple clinical syndromes. Central to the pathophysiology of all human salmonelloses is the induction of a strong host innate immune/inflammatory response. Whether this ultimately reflects an adaptive advantage to the host or pathogen is not clear. However, it is evident that both the host and pathogen have evolved mechanisms of triggering host responses that are detrimental to the other. In this review, we explore some of the host and pathogenic mechanisms mobilized in the two predominant clinical syndromes associated with infection with Salmonella enterica species: enterocolitis and typhoid.

Animals↗
PubMed2005

Bacterial virulence strategies that utilize Rho GTPases.

The ability to modify central host cellular functions is a major advantage to many bacterial pathogens that use such strategies as part of their virulence mechanisms. Small GTPases, including Rho GTPases, make particularly attractive targets for pathogens because of their central roles in modulating cellular functions such as cytoskeletal control. Such modifications of these GTPases can include direct chemical modification of the GTPase or interfacing with some of the regulatory elements associated with GTPase control. Pathogens use these alterations in GTPase functions for a variety of functions, including killing the host cell, mediating bacterial uptake into the host cell (invasion), reprogramming actin to form a lesion in host cells underlying adherent bacteria, to mediate intracellular survival by affecting intracellular trafficking, or to provide polymerized actin mechanisms to propel microbes around inside host cells and into adjacent cells. Collectively, these examples represent many key microbial virulence mechanisms that have led to a much deeper understanding of both microbial pathogens and GTPase functions.

Animals↗
PubMed2004

Pathogens: bacterial needles ruled to length and specificity.

The mechanisms behind length regulation of prokaryotic surface structures has long eluded microbiologists. The recent identification of a protein that functions as a 'molecular ruler' to determine the physical length of a bacterial extracellular needle advances our understanding of surface structure biogenesis.

Bacterial Proteins↗
PubMed2002

Microbial pathogenesis: new niches for salmonella.

Salmonella occupies a vacuolar compartment inside cells of its host. Recent studies have shown that the fate of this vacuole is different in various cell types, and that the outcome of colonization is determined by both the infecting bacterium and defense mechanisms of the host cell.

Animals↗
PubMed2001

A synaptojanin-homologous region of Salmonella typhimurium SigD is essential for inositol phosphatase activity and Akt activation.

The Ser-Thr kinase Akt is activated in epithelial cells by Salmonella enterica serovar typhimurium. The bacterial effector SigD, which is translocated into host cells via the specialized type III secretion system, is essential for Akt activation. Here, we investigated the inositol phospholipid substrate preferences of SigD. Recombinant SigD preferentially dephosphorylated phosphatidylinositol 3,5-biphosphate and phosphatidylinositol 3,4,5-triphosphate over other phosphatidylinositol lipids. Phosphatidylinositol 3-phosphate was not a substrate, suggesting the 5' phosphate moiety is one of the preferred substrates. Database searches revealed that SigD bears a small region of homology to the mammalian type II inositol 5-phosphatase synaptojanin. Mutation of two conserved residues in this region, Lys527 and Lys530, decreased or abrogated phosphatase activity, respectively. The Shigella flexneri SigD homologue, IpgD, displayed a similar activity in vitro and also activated Akt when used to complement a DeltasigD Salmonella strain. A mutation in IpgD at Lys507, analogous to Lys530 of SigD, also failed to activate Akt. Thus, we have characterized a region near the carboxyl-terminus of SigD which is important for phosphatase activity. We discuss how dephosphorylation of inositol phospholipids by SigD in vivo might contribute to the activation of Akt.

Amino Acid Sequence↗
PubMed2001

Bacterial pathogenesis: the answer to virulence is in the pore.

A wide variety of Gram-negative bacterial pathogens use a 'type III' protein secretion system to deliver bacterial virulence factors into host cells. Recent results suggest that Gram-positive pathogens may employ similar methods to deliver virulence factors into host cells.

Bacterial Adhesion↗
PubMed2001

Enteropathogenic Escherichia coli mediates antiphagocytosis through the inhibition of PI 3-kinase-dependent pathways.

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 Fc gamma 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. Phagocytosis 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.

Actins↗
PubMed2001

Characterization of Salmonella-induced filaments (Sifs) reveals a delayed interaction between Salmonella-containing vacuoles and late endocytic compartments.

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.

Animals↗
PubMed2001

Pathogenic trickery: deception of host cell processes.

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.

Animals↗
PubMed2001

Enteropathogenic E. coli Tir binds Nck to initiate actin pedestal formation in host cells.

Enteropathogenic Escherichia coli (EPEC) is a bacterial pathogen that causes infantile diarrhea worldwide. EPEC injects a bacterial protein, translocated intimin receptor (Tir), into the host-cell plasma membrane where it acts as a receptor for the bacterial outer membrane protein, intimin. The interaction of Tir and intimin triggers a marked rearrangement of the host actin cytoskeleton into pedestals beneath adherent bacteria. On delivery into host cells, EPEC Tir is phosphorylated on tyrosine 474 of the intracellular carboxy-terminal domain, an event that is required for pedestal formation. Despite its essential role, the function of Tir tyrosine phosphorylation has not yet been elucidated. Here we show 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. Cells with null alleles of both mammalian Nck genes are resistant to the effects of EPEC on the actin cytoskeleton. These results implicate Nck adaptors as host-cell determinants of EPEC virulence.

Actins↗
PubMed2001

Structural and biochemical characterization of the type III secretion chaperones CesT and SigE.

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.

Amino Acid Sequence↗
PubMed2001

Enterohaemorrhagic and enteropathogenic Escherichia coli use a different Tir-based mechanism for pedestal formation.

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.

Amino Acid Sequence↗
PubMed2001

SifA permits survival and replication of Salmonella typhimurium in murine macrophages.

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.

Amino Acid Sequence↗
PubMed2001

Tir tyrosine phosphorylation and pedestal formation are delayed in enteropathogenic Escherichia coli sepZ::TnphoA mutant 30-5-1(3).

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.

Bacterial Adhesion↗
PubMed2001

Enteropathogenic Escherichia coli infection induces expression of the early growth response factor by activating mitogen-activated protein kinase cascades in epithelial cells.

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.

Animals↗
PubMed2001

Locus of enterocyte effacement from Citrobacter rodentium: sequence analysis and evidence for horizontal transfer among attaching and effacing pathogens.

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.

Adhesins, Bacterial↗