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Biomedical subjects

J E Galán

Publications and source records attributed to J E Galán.

At least 19 recordsLinked to original sources
PubMed2001

Induction of specific CD8+ memory T cells and long lasting protection following immunization with Salmonella typhimurium expressing a lymphocytic choriomeningitis MHC class I-restricted epitope.

Numerous studies have shown the potential of Salmonella typhimurium as a vector for delivery of heterologous proteins for vaccination against other pathogens. Earlier studies showed that the inefficient elicitation of MHC class I-restricted responses could limit the use of S. typhimurium as a heterologous antigen delivery vector for vaccination. We recently developed an approach to overcome this limitation by using a bacterial-encoded specialized protein secretion system, termed type III, to deliver proteins into the class I antigen presenting pathways. Thus, peptides of interest fused to proteins bearing the type III secretion signal, which can elicit protective CTL responses. Because protective immunity is usually assessed a few weeks after vaccination, there is a paucity of information regarding duration of protective immunity induced by this system. We show here that mice immunized orally with S. typhimurium vectors expressing a MHC class I-restricted epitope of the lymphocytic choriomeningitis virus (LCMV) nucleoprotein developed specific antiviral CTL responses. CD8+ T cells were found to be necessary for this CTL activity against targets presenting the LCMV epitope. The survival of mice challenged with lethal doses of LCMV 60 or 135 days after vaccination was as complete as the survival of mice challenged 2 weeks after immunization with the same vectors. By demonstrating their ability to induce prolonged protective immunity after oral delivery, S. typhimurium vectors have met an essential requirement in support of their development as vectors for heterologous vaccination.

Animals↗
PubMed2001

Maintenance of an unfolded polypeptide by a cognate chaperone in bacterial type III secretion.

Many bacterial pathogens use a type III protein secretion system to deliver virulence effector proteins directly into the host cell cytosol, where they modulate cellular processes. A requirement for the effective translocation of several such effector proteins is the binding of specific cytosolic chaperones, which typically interact with discrete domains in the virulence factors. We report here the crystal structure at 1.9 A resolution of the chaperone-binding domain of the Salmonella effector protein SptP with its cognate chaperone SicP. The structure reveals that this domain is maintained in an extended, unfolded conformation that is wound around three successive chaperone molecules. Short segments from two different SptP molecules are juxtaposed by the chaperones, where they dimerize across a hydrophobic interface. These results imply that the chaperones associated with the type III secretion system maintain their substrates in a secretion-competent state that is capable of engaging the secretion machinery to travel through the type III apparatus in an unfolded or partially folded manner.

Bacterial Proteins↗
PubMed2001

Structural mimicry in bacterial virulence.

An important mechanism underlying the strategies used by microbial pathogens to manipulate cellular functions is that of functional mimicry of host activities. In some cases, mimicry is achieved through virulence factors that are direct homologues of host proteins. In others, convergent evolution has produced new effectors that, although having no obvious amino-acid sequence similarity to host factors, are revealed by structural studies to display mimicry at the molecular level.

Adhesins, Bacterial↗
PubMed2001

Characterization of the mutS-proximal region of the Salmonella typhimurium SPI-1 identifies a group of pathogenicity island-associated genes.

The virulence properties of Salmonella enterica are largely encoded within a set of horizontally acquired gene blocks termed pathogenicity islands. One such pathogenicity island, SPI-1, located at centisome 63 of the Salmonella chromosome between the mutS and fhlA genes, encodes a type III protein secretion system and an iron uptake system. We have characterized the mutS-proximal border of this pathogenicity island and have identified two sets of genes, pigAB and pigCD. All four genes have homologs of unknown function in several bacteria that share the ability to establish an intimate association with higher eukaryotic hosts. The expression of at least two of these genes, pigA and pigB, is controlled by SprA, a transcription factor encoded within SPI-1 that controls the expression of genes associated with the type III secretion system of this island. In addition, we found that homologs of the pig genes are also found at different locations of the S. enterica chromosome in association with segments of DNA that exhibit features of pathogenicity islands. The presence of several apparently functional copies of these genes argues for an important role in the biology of this bacterial pathogen. Furthermore, they constitute a valuable tool to identify potential pathogenicity islands.

Animals↗
PubMed2001

A Salmonella inositol polyphosphatase acts in conjunction with other bacterial effectors to promote host cell actin cytoskeleton rearrangements and bacterial internalization.

A central feature of Salmonella pathogenicity is the bacterium's ability to enter into non-phagocytic cells. Bacterial internalization is the consequence of cellular responses characterized by Cdc42- and Rac-dependent actin cytoskeleton rearrangements. These responses are triggered by the co-ordinated function of bacterial proteins delivered into the host cell by a specialized protein secretion system termed type III. We report here that SopB, a Salmonella inositol polyphosphatase delivered to the host cell by this secretion system, mediates actin cytoskeleton rearrangements and bacterial entry in a Cdc42-dependent manner. SopB exhibits overlapping functions with two other effectors of bacterial entry, the Rho family GTPase exchange factors SopE and SopE2. Thus, Salmonella strains deficient in any one of these proteins can enter into cells at high efficiency, whereas a strain lacking all three effectors is completely defective for entry. Consistent with an important role for inositol phosphate metabolism in Salmonella-induced cellular responses, a catalytically defective mutant of SopB failed to stimulate actin cytoskeleton rearrangements and bacterial entry. Furthermore, bacterial infection of intestinal cells resulted in a marked increase in Ins(1,4,5,6)P4, a consumption of InsP5 and the activation of phospholipase C. In agreement with the in vivo findings, purified SopB specifically dephosphorylated InsP5 to Ins(1,4,5,6)P4 in vitro. Surprisingly, the inositol phosphate fluxes induced by Salmonella were not caused exclusively by SopB. We show that the SopB-independent inositol phosphate fluxes are the consequence of the SopE-dependent activation of an endogenous inositol phosphatase. The ability of Salmonella to stimulate Rho GTPases signalling and inositol phosphate metabolism through alternative mechanisms is an example of the remarkable ability of this bacterial pathogen to manipulate host cellular functions.

Actins↗
PubMed2001

Role of tyrosine kinases and the tyrosine phosphatase SptP in the interaction of Salmonella with host cells.

Salmonella has evolved an intimate functional interface with its host. Central to this interface is a battery of bacterial proteins delivered into host cells via a specialized organelle termed the type III secretion system. A subset of these bacterial proteins stimulates cellular responses by activating the Rho family GTPases Cdc42 and Rac. Stimulation of these responses leads to actin cytoskeleton reorganization and the activation of cellular transcription factors that result in bacterial uptake and proinflammatory cytokine production. Remarkably, the cellular responses stimulated by Salmonella are quickly reversed by another bacterial protein, SptP, which exerts its function as a GTPase-activating protein (GAP) for Cdc42 and Rac. In addition to its GAP activity located within its amino-terminus, the carboxy-terminal domain of SptP possesses potent tyrosine phosphatase activity. We show here that the tyrosine phosphatase activity of SptP is involved in reversing the MAP kinase activation that results from Salmonella infection. We also demonstrate an important role for tyrosine kinases, including ACK, in the cellular responses induced by Salmonella. We also found that a potential target for the tyrosine phosphatase activity of SptP is the intermediate filament protein vimentin, which is recruited to the membrane ruffles stimulated by Salmonella.

Actins↗
PubMed2001

CdtA, CdtB, and CdtC form a tripartite complex that is required for cytolethal distending toxin activity.

Campylobacter jejuni encodes a cytolethal distending toxin (CDT) that causes cells to arrest in the G(2)/M transition phase of the cell cycle. Highly related toxins are also produced by other important bacterial pathogens. CDT activity requires the function of three genes: cdtA, cdtB, and cdtC. Recent studies have established that CdtB is the active subunit of CDT, exerting its effect as a nuclease that damages the DNA and triggers cell cycle arrest. Microinjection of CdtB into target cells led to G(2)/M arrest and cytoplasmic distention, in a manner indistinguishable from that caused by CDT treatment. Despite this progress, nothing is known about the composition of the CDT holotoxin or the function of CdtA and CdtC. We show here that, when applied individually, purified CdtA, CdtB, or CdtC does not exhibit toxic activity. In contrast, CdtA, CdtB, and CdtC when combined, interact with one another to form an active tripartite holotoxin that exhibits full cellular toxicity. CdtA has a domain that shares similarity with the B chain of ricin-related toxins. We therefore proposed that CDT is a tripartite toxin composed of CdtB as the enzymatically active subunit and of CdtA and CdtC as the heterodimeric B subunit required for the delivery of CdtB.

Animals↗
PubMed2001

Genetic analysis of assembly of the Salmonella enterica serovar Typhimurium type III secretion-associated needle complex.

Several pathogenic bacteria have evolved a specialized protein secretion system termed type III to secrete and deliver effector proteins into eukaryotic host cells. Salmonella enterica serovar Typhimurium uses one such system to mediate entry into nonphagocytic cells. This system is composed of more than 20 proteins which are encoded within a pathogenicity island (SPI-1) located at centisome 63 of its chromosome. A subset of these components form a supramolecular structure, termed the needle complex, that resembles the flagellar hook-basal body complex. The needle complex is composed of a multiple-ring cylindrical base that spans the bacterial envelope and a needle-like extension that protrudes from the bacterial outer surface. Although the components of this structure have been identified, little is known about its assembly. In this study we examined the effect of loss-of-function mutations in each of the type III secretion-associated genes encoded within SPI-1 on the assembly of the needle complex. This analysis indicates that the assembly of this organelle occurs in discrete, genetically separable steps. A model for the assembly pathway of this important organelle is proposed that involves a sec-dependent step leading to the assembly of the base substructure followed by a sec-independent process resulting in the assembly of the needle portion.

Bacterial Proteins↗
PubMed2001

In vitro transposition system for efficient generation of random mutants of Campylobacter jejuni.

Campylobacter jejuni is the most common cause of food-borne illnesses in the United States. Despite the fact that the entire nucleotide sequence of its genome has recently become available, its mechanisms of pathogenicity are poorly understood. This is in part due to the lack of an efficient mutagenesis system. Here we describe an in vitro transposon mutagenesis system based on the Staphylococcus aureus transposable element Tn552 that allows the efficient generation of insertion mutants of C. jejuni. Insertions occur randomly and throughout the entire bacterial genome. We have tested this system in the isolation of nonmotile mutants of C. jejuni. Demonstrating the utility of the system, six nonmotile mutants from a total of nine exhibited insertions in genes known to be associated with motility. An additional mutant had an inactivating insertion in sigma 54, implicating this transcription factor in flagellum regulation. The availability of this efficient system will greatly facilitate the study of the mechanisms of pathogenesis of this important pathogen.

Base Sequence↗
PubMed2001

Salmonella interactions with host cells: type III secretion at work.

The bacterial pathogen Salmonella enterica has evolved a very sophisticated functional interface with its vertebrate hosts. At the center of this interface is a specialized organelle, the type III secretion system, that directs the translocation of bacterial proteins into the host cell. Salmonella spp. encode two such systems that deliver a remarkable array of bacterial proteins capable of modulating a variety of cellular functions, including actin cytoskeleton dynamics, nuclear responses, and endocytic trafficking. Many of these bacterial proteins operate by faithful mimicry of host proteins, in some cases representing the result of extensive molecular tinkering and convergent evolution. The coordinated action of these type III secreted proteins secures the replication and survival of the bacteria avoiding overt damage to the host. The study of this remarkable pathogen is not only illuminating general paradigms in microbial pathogenesis but is also providing valuable insight into host cell functions.

Actins↗
PubMed2000

A bacterial toxin that controls cell cycle progression as a deoxyribonuclease I-like protein.

Many bacterial pathogens encode a multisubunit toxin, termed cytolethal distending toxin (CDT), that induces cell cycle arrest, cytoplasm distention, and, eventually, chromatin fragmentation and cell death. In one such pathogen, Campylobacter jejuni, one of the subunits of this toxin, CdtB, was shown to exhibit features of type I deoxyribonucleases. Transient expression of this subunit in cultured cells caused marked chromatin disruption. Microinjection of low amounts of CdtB induced cytoplasmic distention and cell cycle arrest. CdtB mutants with substitutions in residues equivalent to those required for catalysis or magnesium binding in type I deoxyribonucleases did not cause chromatin disruption. CDT holotoxin containing these mutant forms of CdtB did not induce morphological changes or cell cycle arrest.

Amino Acid Sequence↗
PubMed2000

Biophysical characterization of SipA, an actin-binding protein from Salmonella enterica.

An essential step in the pathogenesis of Salmonella enterica infections is bacterial entry into non-phagocytic cells of the intestinal epithelium. Proteins injected by Salmonella into host cells stimulate cellular responses that lead to extensive actin cytoskeleton reorganization and subsequent bacterial uptake. One of these proteins, SipA, modulates actin dynamics by directly binding to F-actin. We have biophysically characterized a C-terminal fragment, SipA(446-684), which has previously been shown to retain activity. Our results show that SipA(446-684) exhibits an elongated shape with a predominantly helical conformation and predict the existence of a coiled-coil domain. We suggest that the protein is able to span two adjacent actin monomers in a filament and propose a model that is consistent with the observed effects of SipA(446-684) on actin dynamics and F-actin stability and morphology.

Actins↗
PubMed2000

Molecular characterization and assembly of the needle complex of the Salmonella typhimurium type III protein secretion system.

Many bacterial pathogens of plants and animals have evolved a specialized protein-secretion system termed type III to deliver bacterial proteins into host cells. These proteins stimulate or interfere with host cellular functions for the pathogen's benefit. The Salmonella typhimurium pathogenicity island 1 encodes one of these systems that mediates this bacterium's ability to enter nonphagocytic cells. Several components of this type III secretion system are organized in a supramolecular structure termed the needle complex. This structure is made of discrete substructures including a base that spans both membranes and a needle-like projection that extends outward from the bacterial surface. We demonstrate here that the type III secretion export apparatus is required for the assembly of the needle substructure but is dispensable for the assembly of the base. We show that the length of the needle segment is determined by the type III secretion associated protein InvJ. We report that InvG, PrgH, and PrgK constitute the base and that PrgI is the main component of the needle of the type III secretion complex. PrgI homologs are present in type III secretion systems from bacteria pathogenic for animals but are absent from bacteria pathogenic for plants. We hypothesize that the needle component may establish the specificity of type III secretion systems in delivering proteins into either plant or animal cells.

Amino Acid Sequence↗
PubMed2000

Developmental control of endocytosis in dendritic cells by Cdc42.

Dendritic cells (DCs) developmentally regulate antigen uptake by controlling their endocytic capacity. Immature DCs actively internalize antigen. However, mature DCs are poorly endocytic, functioning instead to present antigens to T cells. We have found that endocytic downregulation reflects a decrease in endocytic activity controlled by Rho family GTPases, especially Cdc42. Blocking Cdc42 function by Toxin B treatment or injection of dominant-negative inhibitors of Cdc42 abrogates endocytosis in immature DCs. In mature DCs, injection of constitutively active Cdc42 or microbial delivery of a Cdc42 nucleotide exchange factor reactivates endocytosis. DCs regulate endogenous levels of Cdc42-GTP with activated Cdc42 detectable only in immature cells. We conclude that DCs developmentally regulate endocytosis at least in part by controlling levels of activated Cdc42.

Animals↗
PubMed2000

Modulation of host signaling by a bacterial mimic: structure of the Salmonella effector SptP bound to Rac1.

Salmonella spp. utilize a specialized protein secretion system to deliver a battery of effector proteins into host cells. Several of these effectors stimulate Cdc42- and Rac1-dependent cytoskeletal changes that promote bacterial internalization. These potentially cytotoxic alterations are rapidly reversed by the effector SptP, a tyrosine phosphatase and GTPase activating protein (GAP) that targets Cdc42 and Rac1. The 2.3 A resolution crystal structure of an SptP-Rac1 transition state complex reveals an unusual GAP architecture that mimics host functional homologs. The phosphatase domain possesses a conserved active site but distinct surface properties. Binding to Rac1 induces a dramatic stabilization in SptP of a four-helix bundle that makes extensive contacts with the Switch I and Switch II regions of the GTPase.

Aluminum Compounds↗
PubMed2000

The flagellar sigma factor FliA (sigma(28)) regulates the expression of Salmonella genes associated with the centisome 63 type III secretion system.

One of the essential features of all pathogenic strains of Salmonella enterica is the ability to enter into nonphagocytic cells. This pathogenic property is mediated by the Salmonella pathogenicity island 1 (SPI-1)-encoded type III secretion system. Expression of components and substrates of this system is subject to complex regulatory mechanisms. These mechanisms include a number of specific and global transcriptional regulatory proteins. In this study we have compared in S. enterica serovars Typhimurium and Typhi the effect of mutations in flagellar genes on the phenotypes associated with the SPI-1 type III protein secretion system. We found that serovar Typhi strains carrying a null mutation in either of the flagellar regulatory genes flhDC or fliA were severely deficient in entry into cultured epithelial cells and macrophage cytotoxicity. This defect could not be reversed by applying a mild centrifugal force, suggesting that the effects of the mutations were not due to the absence of motility. In contrast, the same mutations had no significant effect on the ability of serovar Typhimurium to enter into cultured Henle-407 cells or to induce macrophage cell death. Consistent with these observations, we found that the mutations in the flagellar regulatory proteins significantly reduced the expression of components of the SPI-1-encoded type III system in serovar Typhi but had a marginal effect in serovar Typhimurium. Our results therefore indicate that there is an overlap between regulatory mechanisms that control flagellar and type III secretion gene expression in Salmonella serovar Typhi.

Bacterial Proteins↗