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.     

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.     

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.     

SopE, a secreted protein of Salmonelladublin, is translocated into the target eukaryotic cell via a sip-dependent mechanism and promotes bacterial entry

The entry of Salmonella into cultured epithelial cells is dependent on genes located in several adjacent chromosomal loci. One of these loci encodes the recently identified secretory proteins, denoted Sips ( Salmonella invasion proteins). SipB,C,D proteins are essential for the ability of the pathogen to invade epithelial cells. To examine if additional invasion-associated proteins were secreted by Salmonella dublin , the genes encoding already characterized secretory proteins were inactivated to facilitate this analysis. The proteins produced and secreted by a double fliM /polar sipB mutant of S. dublin were analysed; this revealed a set of novel secreted proteins. These proteins, which we denoted Sops ( Salmonella outer proteins), formed large filamentous aggregates in the medium of bacterial culture growing at 37°C. These aggregates contained five predominant proteins. Here we report the identification and characterization of one of these proteins, SopE, which is a novel invasion-associated secretory protein of S. dublin . A specific sopE mutant of S. dublin was found to be defective for invasion into epithelial cells. Upon interaction of Salmonella with HeLa cells, SopE was found to be translocated into the cytoplasm of the target cell by extracellular bacteria. The translocation of SopE was shown to be dependent on the Sip proteins because a polar sipB mutant did not translocate SopE across the HeLa cell membrane.     

Toxin entry: how bacterial proteins get into mammalian cells

Certain bacteria secrete protein toxins that catalytically modify and disrupt essential processes in mammalian cells, often leading to cell death. As the substrates modified by these toxins are located in the mammalian cell cytosol, a catalytically active toxin polypeptide must reach this compartment in order to act. The toxins bind to receptors on the surface of susceptible cells and enter them by endocytic uptake. Endocytosed toxins initially accumulate in endosomes, where some of these proteins take advantage of the acidic environment within these organelles to form, or contribute to the formation of, protein-conducting channels through which the catalytic polypeptide is able to translocate into the cytosol. Other toxins are unable to respond to low pH in this way and must undergo intracellular vesicular transport to reach a compartment where pre-existing protein-conducting channels occur and can be exploited for membrane translocation — the endoplasmic reticulum. In this way, cell entry by this second group of toxins demonstrates that the secretory pathway of mammalian cells is completely reversible.     

Role of sipA in the early stages of Salmonella typhimurium entry into epithelial cells

Salmonella virulence depends on an ability to invade host cells, which is in turn dependent on a type III protein secretion system encoded in Salmonella pathogenicity island 1 (SPI1). Several protein targets of the SPI1‐encoded secretion system are translocated into host cells, where they subvert cellular processes that contribute to bacterial invasion, actin rearrangement, membrane ruffling and other aspects of virulence. We examined the role of sipA (encoding the translocated protein SipA) and found that a sipA mutant was significantly less invasive in Madin–Darby canine kidney (MDCK) cells than in its parental strain at the earliest stages of infection (5 min). The invasion defect associated with sipA was no longer apparent after 15 min of infection. Confocal microscopy of F‐actin in tetramethyl rhodamine isothiocyanate (TRITC)–phalloidin‐stained MDCK cells revealed no difference in either the frequency or the morphology of membrane ruffles induced by wild‐type and sipA mutant strains of S. typhimurium. Time‐lapse phase‐contrast microscopy of membrane ruffle propagation in live cells confirmed that the sipA mutant induced membrane ruffles as efficiently as the wild‐type bacteria. These studies also revealed that, after ruffle propagation, individual sipA mutant S. typhimurium either invaded more slowly than wild‐type bacteria or failed to invade at all. Furthermore, although wild‐type S. typhimurium typically maintained a position central to the developing membrane ruffle, sipA mutant bacteria frequently moved initially to the periphery of the spreading ruffle and were sometimes observed to detach from it. A wild‐type pattern of invasion was restored to the sipA mutant after the introduction of sipA on a plasmid. Together, these data indicate that loss of sipA significantly decreases the efficiency of S. typhimurium invasion at the early stages of infection without affecting its ability to induce membrane ruffles. It thus appears that the secreted effector protein SipA promotes invasion by a previously unrecognized mechanism separate from the induction of membrane ruffling per se.     

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.     

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.     

A myosin heavy-chain-like polypeptide is associated with the nuclear envelope in higher eukaryotic cells

A high molecular weight polypeptide, identified as an ATPase subunit by direct ultraviolet photoaffinity labeling, has been shown to be a component of nuclear envelope-enriched fractions prepared from a variety of higher eukaryotes (Berrios, M., G. Blobel, and P. A. Fisher, 1983, J. Biol. Chem., 258:4548-4555). In rat liver as well as Drosophila melanogaster embryos, this polypeptide appears to be a form of myosin heavy chain. This conclusion is based on both immunochemical and immunocytochemical data, as well as on the results of CNBr and chymotryptic peptide map analyses. In Drosophila, the identification of this myosin heavy chain-like polypeptide as a nuclear envelope component has been corroborated in situ by indirect immunofluorescence analyses using permeabilized whole cells, mechanically extruded nuclei, and cryosections obtained from a number of larval tissues. Localization appears to be restricted to the nuclear periphery in a manner similar to that observed for the nuclear lamins and the pore complex glycoprotein. Antibodies directed against the Drosophila nuclear envelope ATPase have also been shown to decorate mammalian and higher plant cell nuclei in situ. Implications for intracellular nuclear mobility and for nucleocytoplasmic exchange of macromolecules in vivo are discussed.     

Kinetic measurement of bacterial adherence to eukaryotic cells

We developed a kinetic assay using a monolayer of differentiated respiratory epithelium in culture to assess bacterial adherence. Mean residence time of bacteria in the tissue culture chamber was estimated from a model-independent (moment) analysis of the rate of bacterial washout from perfused Rose chambers. Results with this method compared favorably with visual assessment of adherence and double radiolabel method with H. influenzae. Adherence was assessed with low inoculae of H. influenzae, P. cepacia and P. aeruginosa avoiding cytotoxic effects seen when large inoculae are added to eukaryotic cells. This method will provide a means of assessing adherence of pathogenic respiratory bacteria to their cellular target at low inoculae.     

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.     

Modulation of bacterial entry into epithelial cells by association between vinculin and the Shigella IpaA invasin.

Shigella flexneri is the causative agent of bacillary dysentery in humans. Shigella invasion of epithelial cells is characterized by cytoskeletal rearrangements and formation of cellular projections engulfing the bacterium in a macropinocytic process. We show here that vinculin, a protein involved in linking actin filaments to the plasma membrane, is a direct target of Shigella during cell invasion. IpaA, a Shigella protein secreted upon cell contact, rapidly associates with vinculin during bacterial invasion. Although defective for cell entry, an ipaA mutant is still able to induce foci of actin polymerization, but differs from wild-type Shigella in its ability to recruit vinculin and alpha-actinin. Presumably, IpaA-vinculin interaction initiates the formation of focal adhesion-like structures required for efficient invasion.     

真核细胞内膜泡运输的分子机制

真核细胞内一些蛋白质需靠膜泡进行定向运输,膜泡是在外衣蛋白的作用下形成的,根据外衣蛋白的不同,膜泡分为笼蛋白,COPⅠ和COPⅡ外衣膜泡,这些外衣膜泡分别在细胞内不同供膜（donor membrane)处形成,因为被运输蛋白具有分选信号可与供膜上相应的受体结合,所以能被包裹在特异的膜泡之中,在膜泡形成过程中,外衣蛋白在“芽生”膜泡的细胞质侧组装成笼状外衣,帮助“芽生”膜泡从供膜处脱落,一旦笼状外衣膜泡脱离供膜,笼状外衣蛋白便发生解聚而成为无衣膜泡,无衣膜泡在Rab蛋白的调控下可定向运输蛋白质,而解聚后的外衣蛋白可重新介导新的外衣膜泡形成。     

A rapid microplate method for quantifying inhibition of bacterial adhesion to eukaryotic cells

Adhesion of bacteria to the mucosal epithelial cell surface is the first step in infection, and studies have shown that inhibition of this step may be useful therapeutically. To test compounds that may prevent bacterial binding to a number of epithelial cell lines, we have developed a high-throughput adhesion assay using a microtitre plate system and bacteria that have been modified to express firefly luciferase. This method has proved to be a sensitive, rapid, and reproducible system for screening antiadhesive agents for their effects on bacterial adhesion.     

Early steps of polyomavirus entry into cells

The mechanism by which murine polyomavirus penetrates cells and arrives at the nucleus, the site of viral replication, is not well understood. Simian virus 40 and JC virus, two closely related members of the polyomavirus subfamily, use caveola- and clathrin-mediated uptake pathways for entry, respectively. The data presented here indicate that compounds that block endocytosis of both caveola- and clathrin-derived vesicles have no effect on polyomavirus infectivity. Polyomavirus does not appear to colocalize with either clathrin light chain or caveolin-1 by immunofluorescence microscopy. Additionally, expression of a dominant-negative form of dynamin I has no effect on polyomavirus uptake and infectivity. Therefore, polyomavirus uptake occurs through a class of uncoated vesicles in a clathrin-, caveolin-1-, and dynamin I-independent manner.