Mechanisms of Salmonella entry into host cells

Salmonella enterica is an enteric bacterial pathogen that causes a variety of food and water-borne diseases ranging from gastroenteritis to typhoid fever. Ingested bacteria colonize the intestinal epithelium by triggering their own phagocytosis, using a sophisticated array of effector proteins that are injected into the host cell cytoplasm through a type III secretion apparatus. The synergistic action of these secreted effectors leads to a dramatic reorganization of the host actin cytoskeleton, resulting in vigorous membrane protrusion and the engulfment of attached bacteria. Analysis of these effector proteins and identification of their cellular targets has provided insight into the molecular mechanisms by which bacteria can subvert the host signalling and cytoskeletal machinery for their own purposes. This review is intended to summarize our current understanding of the tools used by Salmonella to enter host cells, with a focus on effectors that modulate the actin cytoskeleton.     

Mechanisms of herpesvirus infection--virus entry into host cells and virus assembly

Herpesvirus entry into host cells occurs by recognition of specific cellular receptor(s) with viral envelope glycoproteins. Nucleocapsids formed in nucleus are released into cytoplasm, and acquire tegument proteins there. Nucleocapsids with tegument proteins bud into intracellular vesicles formed in infected cells, which are thought to be derived from Golgi apparatus, trans-Golgi network or endosomes. However, the precise mechanisms involved in virus final envelopment are poorly understood. Here, I review our current knowledge regarding herpesvirus entry into host cells and virus assembly.     

Bacterial adhesion and entry into host cells

Successful establishment of infection by bacterial pathogens requires adhesion to host cells, colonization of tissues, and in certain cases, cellular invasion-followed by intracellular multiplication, dissemination to other tissues, or persistence. Bacteria use monomeric adhesins/invasins or highly sophisticated macromolecular machines such as type III secretion systems and retractile type IV pili to establish a complex host/pathogen molecular crosstalk that leads to subversion of cellular functions and establishment of disease.     

Bartonella entry mechanisms into mammalian host cells

The Gram-negative genus Bartonella comprises arthropod-borne pathogens that typically infect mammals in a host-specific manner. Bartonella bacilliformis and Bartonella quintana are human-specific pathogens, while several zoonotic bartonellae specific for diverse animal hosts infect humans as an incidental host. Clinical manifestations of Bartonella infections range from mild symptoms to life-threatening disease. Following transmission by blood-sucking arthropods or traumatic contact with infected animals, bartonellae display sequential tropisms towards endothelial and possibly other nucleated cells and erythrocytes, the latter in a host-specific manner. Attachment to the extracellular matrix (ECM) and to nucleated cells is mediated by surface-exposed bacterial adhesins, in particular trimeric autotransporter adhesins (TAAs). The subsequent engulfment of the pathogen into a vacuolar structure follows a unique series of events whereby the pathogen avoids the endolysosomal compartments. For Bartonella henselae and assumingly most other species, the infection process is aided at different steps by Bartonella effector proteins (Beps). They are injected into host cells through the type IV secretion system (T4SS) VirB/D4 and subvert host cellular functions to favour pathogen uptake. Bacterial binding to erythrocytes is mediated by Trw, another T4SS, in a strictly host-specific manner, followed by pathogen-forced uptake involving the IalB invasin and subsequent replication and persistence within a membrane-bound intra-erythrocytic compartment.     

Mechanics of enveloped virus entry into host cells

Enveloped viruses such as HIV-1 enter their hosts by first establishing a contact region at the cell surface, which is stabilized by the formation of receptor-ligand complexes. We show that the favorable contact energy stemming from the formation of the receptor complexes in the interaction zone is sufficient to drive the engulfment of the virus by the cell. Using a continuum model, we show that the equilibrium engulfment depth and the force driving the engulfment are functions of the virus size and the complex formation energy. Resistance to engulfment is dominated by the elastic deformation of the cytoskeleton.     

On resistance to virus entry into host cells

In this article, we adopt a continuum model from Sun and Wirtz (2006. Biophys. J. 90:L10-L12) to show that, for the enveloped virus entry into host cells, the binding energy of the receptor-ligand complex can drive the engulfment of the viral particle to overcome the resistance alternatively dominated by the membrane deformation and cytoskeleton deformation at a different engulfing stage. This is contrary to the conclusions by Sun and Wirtz that the cytoskeleton deformation is always dominant. This discrepancy occurs because the energy of membrane deformation in their article is incorrect. Such an unfortunate small error has led to a severe underestimation of the contribution from membrane deformation to the total energy of the system, which then led them to improperly conclude that the cytoskeleton deformation plays the dominant role in the virus entry into host cell. By using the correct energy expression, our conclusion is justified by energy comparisons under a large range of virus sizes and Young's moduli of cytoskeleton. We even find that a critical radius of virus exists, beyond which the resistance to the virus engulfment becomes dominated by the membrane deformation during the whole stage, contrary to the point of view of Sun and Wirtz.     

Interaction of quorum signals with outer membrane lipids: insights into prokaryotic membrane vesicle formation

Bacteria have evolved elaborate communication strategies to co-ordinate their group activities, a process termed quorum sensing (QS). Pseudomonas aeruginosa is an opportunistic pathogen that utilizes QS for diverse activities, including disease pathogenesis. P. aeruginosa has evolved a novel communication system in which the signal molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas Quinolone Signal, PQS) is trafficked between cells via membrane vesicles (MVs). Not only is PQS packaged into MVs, it is required for MV formation. Although MVs are involved in important biological processes aside from signalling, the molecular mechanism of MV formation is unknown. To provide insight into the molecular mechanism of MV formation, we examined the interaction of PQS with bacterial lipids. Here, we show that PQS interacts strongly with the acyl chains and 4'-phosphate of bacterial lipopolysaccharide (LPS). Using PQS derivatives, we demonstrate that the alkyl side-chain and third position hydroxyl of PQS are critical for these interactions. Finally, we show that PQS stimulated purified LPS to form liposome-like structures. These studies provide molecular insight into P. aeruginosa MV formation and demonstrate that quorum signals serve important non-signalling functions.     

Molecular genetic bases of Salmonella entry into host cells

Salmonella spp. can enter into non-phagocytic cells, a property that is essential for their pathogenicity. Recently, considerable progress has been made in the understanding of the molecular genetic bases of this process. It is now evident that Salmonella entry functions are largely encoded on a 35–40 kb region of the Salmonella chromosome located at centisome 63. The majority of the loci in this region encode components of a type III or contact-dependent secretion system homologous to those described in a variety of animal and plant-pathogenic bacteria as well as a number of proteins that require this system for their export to the extracellular environment. A somewhat unexpected finding has been the remarkable homology between the Salmonella and Shigella proteins that mediate the entry of these organisms into cultured epithelial cells.     

Trypanosoma cruzi: mechanisms for entry into host cells

Infective trypomastigote stages of the obligate intracellular protozoan parasite Trypanosoma cruzi are capable of entering virtually any mammalian cell in vitro. Entry is a complex process, involving initial parasite attachment to surface moieties of the target cell, internalization of the parasite via formation of a vacuole, and finally disruption of the vacuolar membrane to permit access of the parasite to the host cell cytoplasm. Attachment requires parasite metabolic energy. At sites of parasite entry recruitment of host cell lysosomes may occur, and lysosomal membrane components contribute prominently to formation of the parasitophorous vacuole. Parasite escape from the vacuole depends upon vacuolar acidification and is mediated by the coordinated action of a parasite-derived neuramindase/trans-sialidase that is capable of desialylating host-derived vacuolar membrane constituents, and a parasite-derived trans-membrane pore-forming protein. Dissection of the entry process at both the organellar and molecular level is providing fundamental and complementary insights into microbial pathogenesis and cell biology.     

Francisella targets cholesterol-rich host cell membrane domains for entry into macrophages

Francisella tularensis is a pathogen optimally adapted to efficiently invade its respective host cell and to proliferate intracellularly. We investigated the role of host cell membrane microdomains in the entry of F. tularensis subspecies holarctica vaccine strain (F. tularensis live vaccine strain) into murine macrophages. F. tularensis live vaccine strain recruits cholesterol-rich lipid domains ("lipid rafts") with caveolin-1 for successful entry into macrophages. Interference with lipid rafts through the depletion of plasma membrane cholesterol, through induction of raft internalization with choleratoxin, or through removal of raft-associated GPI-anchored proteins by treatment with phosphatidylinositol phospholipase C significantly inhibited entry of Francisella and its intracellular proliferation. Lipid raft-associated components such as cholesterol and caveolin-1 were incorporated into Francisella-containing vesicles during entry and the initial phase of intracellular trafficking inside the host cell. These findings demonstrate that Francisella requires cholesterol-rich membrane domains for entry into and proliferation inside macrophages.     

Utilization of DC-SIGN for entry of feline coronaviruses into host cells

The entry and dissemination of viruses in several families can be mediated by C-type lectins such as DC-SIGN. We showed that entry of the serotype II feline coronavirus strains feline infectious peritonitis virus (FIPV) WSU 79-1146 and DF2 into nonpermissive mouse 3T3 cells can be rescued by the expression of human DC-SIGN (hDC-SIGN) and that infection of a permissive feline cell line (Crandall-Reese feline kidney) was markedly enhanced by the overexpression of hDC-SIGN. Treatment with mannan considerably reduced infection of feline monocyte-derived cells expressing DC-SIGN, indicating a role for FIPV infection in vivo.     

Phospholipid scramblase 1 mediates hepatitis C virus entry into host cells

Hepatitis C virus (HCV) infects human hepatocytes through several host factors. However, other prerequisite factors for viral entry remain to be identified. Using a yeast two-hybrid screen, we found that human phospholipid scramblase 1 interacts with HCV envelope proteins E1 and E2. These physical interactions were confirmed by co-immunoprecipitation and GST pull-down assays. Knocking down the expression of PLSCR1 inhibited the entry of HCV pseudoparticles. Moreover, PLSCR1 was required for the initial attachment of HCV onto hepatoma cells, where it specifically interacted with entry factor OCLN. We show that PLSCR1 is a novel attachment factor for HCV entry.     

Rho-dependent membrane folding causes Shigella entry into epithelial cells.

The small GTPase rho is functionally involved in the formation of cytoskeletal structures like stress fibers or focal adhesion plaques. Shigella entry into HeLa cells induces a blossom-like membrane structure at the bacterial entry site. We show here that this membrane-folding process is rho-dependent. The three rho isoforms were recruited into bacterial entry sites with differential localization relative to the membrane structure. A rho-specific inhibitor abolished Shigella-induced membrane folding and impaired bacterial entry accordingly. S1-myosin labeling indicated that rho was involved in Shigella-induced actin polymerization but not actin nucleation in the bacterial invasion site. This provides a major link in the signalization cascade allowing entry of a bacterial pathogen into a eukaryotic cell.     

Membrane rafts: a potential gateway for bacterial entry into host cells

Pathogenic bacteria have developed various mechanisms to evade host immune defense systems. Invasion of pathogenic bacteria requires interaction of the pathogen with host receptors, followed by activation of signal transduction pathways and rearrangement of the cytoskeleton to facilitate bacterial entry. Numerous bacteria exploit specialized plasma membrane microdomains, commonly called membrane rafts, which are rich in cholesterol, sphingolipids and a special set of signaling molecules which allow entry to host cells and establishment of a protected niche within the host. This review focuses on the current understanding of the raft hypothesis and the means by which pathogenic bacteria subvert membrane microdomains to promote infection.     

Clathrin-dependent entry of a gingipain adhesin peptide and Porphyromonas gingivalis into host cells

Porphyromonas gingivalis, a Gram-negative oral anaerobe, is associated with periodontitis, a disease that in some form affects up to 80% of the adult population in the USA. The organism interacts with gingival epithelium and surrounding tissue, and in this study we analysed interactions initiated by P. gingivalis and by a peptide derived from the adhesin domain of arg-gingipain A, a member of a family of surface cysteine proteinases. Recombinant peptide A44 blocked adherence of bacteria to host cell monolayers, and bound to components of the cell membrane fraction. In pull-down assays A44 associated with proteins involved in a clathrin-dependent endocytosis pathway. Inhibitor studies confirmed a role for clathrin, and confocal microscopy demonstrated that both A44-coated beads and intact bacteria colocalized with GFP-clathrin in host cells. Finally, we used siRNA to determine whether clathrin or caveolin-1 was involved in association of peptide and intact bacteria with host cells. Again, the results of these assays indicated that association of both A44 and P. gingivalis depended on the presence of clathrin, and support a working model in which A44 initiates a clathrin-dependent pathway that potentially leads to internalization of peptide or bacteria by host epithelial cells.     

Caveolae‐mediated entry of Salmonella typhimurium into senescent nonphagocytotic host cells

Elderly individuals have an increased susceptibility to microbial infections because of age‐related anatomical, physiological, and environmental factors. However, the mechanism of aging‐dependent susceptibility to infection is not fully understood. Here, we found that caveolae‐dependent endocytosis is elevated in senescent cells. Thus, we focused on the implications of caveolae‐dependent endocytosis using Salmonella typhimurium, which causes a variety of diseases in humans and animals by invading the eukaryotic host cell. Salmonella invasion increased in nonphagocytotic senescent host cells in which caveolin‐1 was also increased. When caveolae structures were disrupted by methyl‐β‐cyclodextrin or siRNA of caveolin‐1 in the senescent cells, Salmonellae invasion was reduced markedly compared to that in nonsenescent cells. In contrast, the over‐expression of caveolin‐1 led to increased Salmonellae invasion in nonsenescent cells. Moreover, in aged mice, caveolin‐1 was found to be highly expressed in Peyer’s patch and spleen, which are targets for infection by Salmonellae. These results suggest that high levels of caveolae and caveolin‐1 in senescent host cells might be related to the increased susceptibility of elderly individuals to microbial infections.     

Insights into outer membrane protein crystallization

Outer membrane proteins are structurally distinct from those that reside in the inner membrane and play important roles in bacterial pathogenicity and human metabolism. X-ray crystallography studies on >40 different outer membrane proteins have revealed that the transmembrane portion of these proteins can be constructed from either β-sheets or less commonly from α-helices. The most common architecture is the β-barrel, which can be formed from either a single anti-parallel sheet, fused at both ends to form a barrel or from multiple peptide chains. Outer membrane proteins exhibit considerable rigidity and stability, making their study through x-ray crystallography particularly tractable. As the number of structures of outer membrane proteins increases a more rational approach to their crystallization can be made. Herein we analyse the crystallization data from 53 outer membrane proteins and compare the results to those obtained for inner membrane proteins. A targeted sparse matrix screen for outer membrane protein crystallization is presented based on the present analysis.     

Mechanisms of protein import across the mitochondrial outer membrane

Mitochondria import the majority of their proteins from the cytosol. At the mitochondrial outer membrane, import is initiated through a series of reactions, which include preprotein recognition, unfolding, insertion and translocation. These processes are facilitated by a multisubunit complex, the TOM complex. Specific roles can now be assigned to several components of this complex. Although the import machinery of the outer membrane can insert and translocate a few proteins on its own, completion of translocation o f most preproteins is dependent upon coupling to both the membrane potential and mt-Hsp70/ATP-driven transport across the inner membrane, mediated by the TIM complex.