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.     

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.     

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.     

Mechanisms of outer membrane vesicle entry into host cells

Bacterial outer membrane vesicles (OMVs) are nano‐sized compartments consisting of a lipid bilayer that encapsulates periplasm‐derived, luminal content. OMVs, which pinch off of Gram‐negative bacteria, are now recognized as a generalized secretion pathway which provides a means to transfer cargo to other bacterial cells as well as eukaryotic cells. Compared with other secretion systems, OMVs can transfer a chemically extremely diverse range of cargo, including small molecules, nucleic acids, proteins, and lipids to proximal cells. Although it is well recognized that OMVs can enter and release cargo inside host cells during infection, the mechanisms of host association and uptake are not well understood. This review highlights existing studies focusing on OMV‐host cell interactions and entry mechanisms, and how these entry routes affect cargo processing within the host. It further compares the wide range of methods currently used to dissect uptake mechanisms, and discusses potential sources of discrepancy regarding the mechanism of OMV uptake across different studies.     

Salmonella entry into host cells: the work in concert of type III secreted effector proteins.

Upon contact with intestinal epithelial cells, Salmonella enterica serovar spp. inject a set of bacterial proteins into host cells via the bacterial SPI-1 type III secretion system. SopE, SopE2 and SopB, activate CDC42 and Rac to initiate actin cytoskeleton rearrangements. SipA and SipC, two Salmonella actin-binding proteins, directly modulate host actin dynamics to facilitate bacterial uptake. SptP promotes the recovery of the actin cytoskeleton rearrangements by antagonizing CDC42 and Rac. Therefore, Salmonella-induced reversible actin cytoskeleton rearrangements are the result of two coordinated steps: (i) stimulation of host signal transduction to indirectly promote actin rearrangements and (ii) direct modulation of actin dynamics.     

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.     

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.     

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.     

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.     

Identification of cytokeratins as accessory mediators of Salmonella entry into eukaryotic cells

Pathogenic Salmonella species initiate infection of a mammalian host by inducing their own uptake into intestinal M-cells. During the uptake process, the bacteria utilize an intrinsic secretion system to release proteins that enter host cells. The secreted invasion-mediating proteins subsequently interact with host cell components that induce alterations in the actin cytoskeleton. To identify potential cellular determinants of invasion, we employed a yeast two-hybrid system using the secreted Salmonella invasion protein (SipC) as the bait protein. This system identified cytokeratins, supportive components of the cytoskeletal matrix, as proteins that may physically interact with SipC. Transfection-based studies revealed an inhibition of Salmonella invasion when a dominant negative cytokeratin-18 was expressed. Immunofluorescent confocal microscopy studies revealed that Salmonella did not enter HEp-2 cells expressing the dominant negative cytokeratin-18. These results suggest that an interaction between SipC and cytokeratin-18 may occur as part of Salmonella invasion.     

Functional conservation of the Salmonella and Shigella effectors of entry into epithelial cells

A Salmonella typhi chromosomal locus composed of five adjacent genes, designated sipEBCDA , was identified by transposon mutagenesis as being essential for cell invasion. Products of the sip genes exhibit extensive sequence similarities to the effectors of Shigella entry into epithelial cells encoded by the virulence plasmid-borne ipa operon. Expression of sipE and sipB in a Shigella non-invasive ipaB mutant restored the ability to invade epithelial cells. The structural and functional conservation of the Sip and Ipa proteins suggests that Salmonella and Shigella entry processes are promoted by similar effectors.     

Identification of novel loci affecting entry of Salmonella enteritidis into eukaryotic cells.

There are an estimated 2 million cases of salmonellosis in the United States every year. Unlike the incidence of many infectious diseases, the incidence of salmonellosis in the United States and other developed countries has been rising steadily over the past 30 years, and the disease now accounts for 10 to 15% of all cases of acute gastroenteritis in the United States. The infecting organism is ingested and must traverse the intestinal epithelium to reach its preferred site for multiplication, the reticuloendothelial system. Despite several recent studies, the genetic basis of the invasion process is poorly understood. An emerging theme from these studies is that wild-type Salmonella organisms probably have several chromosomal loci that are required for the most efficient level of invasion. In this study, we have identified and characterized 13 TnphoA insertion mutants of Salmonella enteritidis CDC5 that exhibit altered invasion phenotypes. The mutants were identified by screening a bank of TnphoA insertions in S. enteritidis CDC5str for their invasion phenotype in three tissue culture cell lines (HEp-2, CHO, and MDCK). These 13 mutants were separated into six classes based on their invasive phenotypes in the tissue culture cell lines. Several mutants were defective for entry of some cell lines but not for others, while two mutants (SM6 and SM7) were defective for entry into all three tissue culture cell lines. This suggests that Salmonella spp. may express more than one invasion pathway. Southern analysis and chromosomal mapping indicated that as many as nine chromosomal loci may contribute to the invasion phenotype. It is becoming clear that the invasive phenotype of Salmonella spp. is multifactorial and more complex than that of some other invasive members of the family Enterobacteriaceae.     

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.