Ralph R. Isberg

Multiple β1 chain integrins are receptors for invasin, a protein that promotes bacterial penetration into mammalian cells

Abstract Mammalian cell receptors that promote entry of intracellular bacteria into nonphagocytic cells have not been identified. We show here that multiple members of the integrin superfamily of cell adhesion receptors bind the Y. pseudotuberculosis invasin protein prior to bacterial penetration into mammalian cells. Affinity chromatography of crude detergent extracts demonstrated that integrins containing the subunit structures α 3 β 1 , α 4 β 1 , α 5 β 1 , and α 6 β 1 bound to immobilized invasin. Furthermore, phospholipid vesicles containing isolated integrin proteins were able to attach to invasin. Specificity for invasin binding to the identified integrin receptors was also demonstrated, as immunoprobing and phospholipid reconstitution studies showed that the α 2 β 1 integrin, β 2 chain integrins, and vitronectin receptor ( α v β 3 ) were not involved in cellular attachment to invasin.

Identification of invasin: A protein that allows enteric bacteria to penetrate cultured mammalian cells

Bacterial strains harboring the Yersinia pseudotuberculosis inv locus were analyzed in order to investigate the mechanism of host cell penetration by an invasive pathogen. The inv locus was found to be necessary for Y. pseudotuberculosis to enter HEp-2 cells and sufficient to convert E. coli into a microorganism able to penetrate cultured cells. Both E. coli and Y. pseudotuberculosis strains harboring inv mutations were defective for entry into HEp-2 cells. Furthermore, molecular clones containing inv, and little additional DNA, converted E. coli into a microorganism that was indistinguishable from the parental Yersinia strain with regard to the entry of cultured cells. Data from in vitro protein synthesis indicated that a 103 kd protein was synthesized from inv, saturating the coding capacity of the locus. The nucleotide sequence shows an open reading frame corresponding to a protein of similar size. This protein, called invasin, is necessary for the microorganisms to penetrate HEp-2 cells, and is compartmentalized on the outer surface of the bacterium.

Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila

Legionella pneumophila mutants specifically defective for intracellular replication were isolated using an intracellular thymineless death enrichment strategy. Mutants belonging to two distinct phenotypic classes were unable to grow in macrophage‐like cultured cells. One class of mutants was defective for both inhibition of phagosome–lysosome fusion and association of host cell organelles with bacteria‐containing phagosomes (‘recruitment’). Another class of mutants was defective only for organelle recruitment, suggesting that recruitment may be necessary for intracellular growth. Recombinant clones were identified that complemented the intracellular growth defects of these mutants. A single genetic locus, designated dot (for defect in organelle trafficking), restored wild‐type phenotypes for intracellular growth, organelle recruitment, and inhibition of phagosome–lysosome fusion to mutants belonging to both phenotypic classes.

The Legionella pneumophila replication vacuole: making a cosy niche inside host cells

The pathogenesis of Legionella pneumophila is derived from its growth within lung macrophages after aerosols are inhaled from contaminated water sources. Interest in this bacterium stems from its ability to manipulate host cell vesicular-trafficking pathways and establish a membrane-bound replication vacuole, making it a model for intravacuolar pathogens. Establishment of the replication compartment requires a specialized translocation system that transports a large cadre of protein substrates across the vacuolar membrane. These substrates regulate vesicle traffic and survival pathways in the host cell. This Review focuses on the strategies that L. pneumophila uses to establish intracellular growth and evaluates why this microorganism has accumulated an unprecedented number of translocated substrates that are targeted at host cells.

LEGIONELLA PNEUMOPHILA DOTA PROTEIN IS REQUIRED FOR EARLY PHAGOSOME TRAFFICKING DECISIONS THAT OCCUR WITHIN MINUTES OF BACTERIAL UPTAKE

Numerous intracellular bacterial pathogens modulate the nature of the membrane‐bound compartment in which they reside, although little is known about the molecular basis for this control. Legionella pneumophila is a bacterial pathogen able to grow within human alveolar macrophages and residing in a phagosome that does not fuse with lysosomes. This study demonstrates that the dotA product is required to regulate trafficking of the L. pneumophila phagosome. Phagosomes containing L. pneumophila dotA+ bacteria exhibited differential trafficking profiles when compared with isogenic dotA mutants. Phagosomes containing dotA mutants showed rapid accumulation of the lysosomal glycoprotein LAMP‐1 as early as 5 min after uptake, whereas the majority of wild‐type L. pneumophila phagosomes did not acquire LAMP‐1. The association of LAMP‐1 with phagosomes containing dotA mutant bacteria was concomitant with the appearance of the small GTP‐binding protein Rab7 on the vacuolar membrane. These data demonstrate that phagosomes containing replication‐competent L. pneumophila evade early endocytic fusion events. In contrast, the kinetics of LAMP‐1 and Rab7 association indicate that the dotA mutants are routed along a well‐characterized endocytic pathway leading to fusion with lysosomes. Genetic studies show that L. pneumophila requires DotA expression before macrophage uptake in order to establish an intracellular site for replication. However, the bacteria do not appear to require continuous expression of the DotA protein to maintain a replicative phagosome. These data indicate that DotA is one factor that plays a fundamental role in regulating initial phagosome trafficking decisions either upon or immediately after macrophage uptake.

The Legionella pneumophila LidA protein: a translocated substrate of the Dot/Icm system associated with maintenance of bacterial integrity

Legionella pneumophila establishes a replication vacuole within phagocytes that requires the bacterial Dot/Icm apparatus for its formation. This apparatus is predicted to translocate effectors into host cells. We hypothesized that some translocated proteins also function to maintain the integrity of the Dot/Icm translocator. Mutations that destroy this function are predicted to result in a Dot/Icm complex that poisons the bacterium, resulting in reduced viability. To identify such mutants, strains were isolated (called lid – ) that showed reduced viability on bacteriological medium in the presence of an intact Dot/Icm apparatus, but which had high viability in the absence of the translocator. Several such mutants were analysed in detail to identify candidate strains that may have lost the ability to synthesize a translocated substrate of Dot/Icm. Two such strains had mutations in the lidA gene. The LidA protein exhibits properties expected for a translocated substrate of Dot/Icm that is important for maintenance of bacterial cell integrity: it associates with the phagosomal surface, promotes replication vacuole formation, and is important for both efficient intracellular growth and high viability on bacteriological media after introduction of a plasmid that allows high level expression of the dotA gene.

Identification of the integrin binding domain of the Yersinia pseudotuberculosis invasin protein.

The invasin protein of the pathogenic Yersinia pseudotuberculosis mediates entry of the bacterium into cultured mammalian cells by binding several beta 1 chain integrins. In this study, we identified the region of invasin responsible for cell recognition. Thirty‐two monoclonal antibodies directed against invasin were isolated, and of those, six blocked cell attachment to invasin. These six antibodies recognized epitopes within the last 192 amino acids of invasin. Deletion mutants of invasin and maltose‐binding protein (MBP)‐‐invasin fusion proteins were generated and tested for cell attachment. All of the invasin derivatives that carried the carboxyl‐terminal 192 amino acids retained cell binding activity. One carboxyl‐terminal invasin fragment and seven MBP‐‐invasin fusion proteins were purified. The purified derivatives that retained binding activity inhibited bacterial entry into cultured mammalian cells. These results indicated that the carboxyl‐terminal 192 amino acids of invasin contains the integrin‐binding domain, even though this region does not contain the tripeptide sequence Arg‐Gly‐Asp.

Intracellular Growth of Legionella pneumophila in Dictyostelium discoideum, a System for Genetic Analysis of Host-Pathogen Interactions

ABSTRACT Conditions were established in which Legionella pneumophila, an intracellular bacterial pathogen, could replicate within the unicellular organism Dictyostelium discoideum. By several criteria, L. pneumophila grew by the same mechanism within D. discoideum as it does in amoebae and macrophages. Bacteria grew within membrane-bound vesicles associated with rough endoplasmic reticulum, and L. pneumophila dot/icm mutants, blocked for growth in macrophages and amoebae, also did not grow in D. discoideum. Internalized L. pneumophila avoided degradation by D. discoideum and showed evidence of reduced fusion with endocytic compartments. The ability of L. pneumophila to grow within D. discoideum depended on the growth state of the cells. D. discoideum grown as adherent monolayers was susceptible toL. pneumophila infection and to contact-dependent cytotoxicity during high-multiplicity infections, whereas D. discoideum grown in suspension was relatively resistant to cytotoxicity and did not support intracellular growth. Some knownD. discoideum mutants were examined for their effect on growth of L. pneumophila. The coronin mutant and themyoA/B double myosin I mutant were more permissive than wild-type strains for intracellular growth. Growth of L. pneumophila in a Gβ mutant was slightly reduced compared to the parent strain. This work demonstrates the usefulness of the L. pneumophila-D. discoideum system for genetic analysis of host-pathogen interactions.

The Legionella Effector RavZ Inhibits Host Autophagy Through Irreversible Atg8 Deconjugation

Axing Autophagy When intracellular pathogens like Legionella pneumophila take up residence in mammalian host cells, they must combat the efforts of the host cell to attack them. Autophagy is a process by which cells digest their own constituents, often involved in response to starvation or pathogen attack. Choy et al. (p. 1072, published online 25 October) now describe how L. pneumophila can inhibit the autophagy pathway in eukaryotic cells, and provide a detailed description of the biochemical mechanism. A Legionella effector protein, RavZ, acts as a very potent enzyme that specifically deconjugates a key autophagy protein, Atg8, from autophagosomal membranes, thus blocking autophagy. An intracellular pathogen disrupts autophagy by targeting an essential host protein on the early autophagosome. Eukaryotic cells can use the autophagy pathway to defend against microbes that gain access to the cytosol or reside in pathogen-modified vacuoles. It remains unclear if pathogens have evolved specific mechanisms to manipulate autophagy. Here, we found that the intracellular pathogen Legionella pneumophila could interfere with autophagy by using the bacterial effector protein RavZ to directly uncouple Atg8 proteins attached to phosphatidylethanolamine on autophagosome membranes. RavZ hydrolyzed the amide bond between the carboxyl-terminal glycine residue and an adjacent aromatic residue in Atg8 proteins, producing an Atg8 protein that could not be reconjugated by Atg7 and Atg3. Thus, intracellular pathogens can inhibit autophagy by irreversibly inactivating Atg8 proteins during infection.

Binding and internalization of microorganisms by integrin receptors.

Many microbial pathogens bind host-cell integrin receptors. These interactions are promoted either by a host protein binding the microorganism or by a surface-localized ligand encoded by the pathogen. Attachment facilitates extracellular adhesion of the microorganism or internalization by the host cell.