Hélène Ohayon

E-Cadherin Is the Receptor for Internalin, a Surface Protein Required for Entry of L. monocytogenes into Epithelial Cells

We report the first identification of a cellular receptor mediating entry of a gram-positive bacterium into nonphagocytotic cells. By an affinity chromatography approach, we identified E-cadherin as the ligand for internalin, an L. monocytogenes protein essential for entry into epithelial cells. Expression of the chicken homolog of E-cadherin (L-CAM) in transfected fibroblasts dramatically increases entry of L. monocytogenes and promotes that of a recombinant L. innocua strain expressing internalin but does not promote entry of the wild-type noninvasive L. innocua or that of an internalin-deficient mutant of L. monocytogenes. Furthermore, L-CAM-specific antibodies block internalin-mediated entry. In contrast to Salmonella, Listeria enters cells by a mechanism of induced phagocytosis occurring without membrane ruffling. This work reveals a novel type of heterophilic interactions for E-cadherin.

L. monocytogenes-induced actin assembly requires the actA gene product, a surface protein

The intracellular pathogenic bacterium L. monocytogenes can spread directly from cell to cell without leaving the cytoplasm. The mechanism of this movement, generated through bacterially induced actin polymerization, is not understood. By analyzing an avirulent Tn917-lac mutant defective for actin polymerization, we have identified a bacterial component involved in this process. The transposon had inserted in actA, the second gene of an operon. Gene disruption of downstream genes and transformation of the mutant strain with actA showed that the actA gene encodes a surface protein necessary for bacterially induced actin assembly. Our results indicate that it is a 610 amino acid protein with an apparent molecular weight of 90 kd.

THE INLB PROTEIN OF LISTERIA MONOCYTOGENES IS SUFFICIENT TO PROMOTE ENTRY INTO MAMMALIAN CELLS

InlB is one of the two Listeria monocytogenes invasion proteins required for bacterial entry into mammalian cells. Entry into human epithelial cells such as Caco‐2 requires InlA, whereas InlB is needed for entry into cultured hepatocytes and some epithelial or fibroblast cell lines such as Vero, HEp‐2 and HeLa cells. InlB‐mediated entry requires tyrosine phosphorylation, cytoskeletal rearrangements and activation of the host protein phosphoinositide (PI) 3‐kinase, probably in response to engagement of a receptor. In this study, we demonstrate for the first time that InlB is sufficient to promote internalization. Indeed, coating of normally non‐invasive bacteria or inert latex beads with InlB leads to internalization into mammalian cells. In addition, a soluble form of InlB also appears to promote uptake of non‐invasive bacteria, albeit at a very low level. Similar to entry of L. monocytogenes, uptake of InlB‐coated beads required tyrosine phosphorylation in the host cell, PI 3‐kinase activity and cytoskeletal reorganization. Taken together, these data indicate that InlB is sufficient for entry of L. monocytogenes into host cells and suggest that this protein is an effector of host cell signalling pathways.

Fate of germinated Bacillus anthracis spores in primary murine macrophages

We investigated the fate of germinated Bacillus anthracis spores after their germination in Swiss murine peritoneal macrophages and in the cell line RAW264.7. We found that the lethal toxin and the oedema toxin are germ‐associated factors that are essential for the survival of the vegetative form in host cells. We also found that pX02 is not involved in this complex pathogenic process. By transmission electron microscopy, we showed the tight interaction between the exosporium of the spore and the phagosomal membrane of the macrophage. Our data strongly suggest that the B. anthracis toxinogenic, unencapsulated Sterne strain (7702) does not multiply within macrophages. These results contributed to reveal the strategies used by B. anthracis to survive within the host and to reach the external medium where they proliferate.

CD44 binds to the Shigella IpaB protein and participates in bacterial invasion of epithelial cells

Shigella entry into epithelial cells is characterized by a transient reorganization of the host cell cytoskeleton at the site of bacterial interaction with the cell membrane, which leads to bacterial engulfment in a macropinocytic process. Using affinity chromatography on HeLa cell extracts, we show here that the hyaluronan receptor CD44 associates with IpaB, a Shigella protein that is secreted upon cell contact. Overlay and solid‐phase assays indicated that IpaB binds directly to the extracellular domain of CD44; binding is saturable and inhibitable, with a half‐ maximal inhibitory concentration of 175 nM. Immunoprecipitation experiments showed that IpaB associates with CD44 during Shigella entry. CD44 is recruited at bacterial entry sites and localizes at the plasma membrane of cellular extensions induced by Shigella. Pretreatment of cells with an anti‐CD44 monoclonal antibody resulted in inhibition of Shigella‐induced cytoskeletal reorganization, as well as inhibition of bacterial entry, whereas transfection of CD44 in cells that are deficient for CD44 results in increased bacterial binding to cells and internalization. The IpaB–CD44 interaction appears to be required for Shigella invasion by initiating the early steps of the entry process.

V. Functions of S‐layers

Although S-layers are being increasingly identified on Bacteria and Archaea, it is enigmatic that in most cases S-layer function continues to elude us. In a few instances, S-layers have been shown to be virulence factors on pathogens (e.g. Campylobacter fetus ssp. fetus and Aeromonas salmonicida), protective against Bdellovibrio, a depository for surface-exposed enzymes (e.g. Bacillus stearothermophilus), shape-determining agents (e.g. Thermoproteus tenax) and nucleation factors for fine-grain mineral development (e.g. Synechococcus GL 24). Yet, for the vast majority of S-layered bacteria, the natural function of these crystalline arrays continues to be evasive. The following review up-dates the functional basis of S-layers and describes such diverse topics as the effect of S-layers on the Gram stain, bacteriophage adsorption in lactobacilli, phagocytosis by human polymorphonuclear leukocytes, the adhesion of a high-molecular-mass amylase, outer membrane porosity, and the secretion of extracellular enzymes of Thermoanaerobacterium. In addition, the functional aspect of calcium on the Caulobacter S-layer is explained.

The secreted IpaB and IpaC invasins and their cytoplasmic chaperone IpgC are required for intercellular dissemination of Shigella flexneri.

Invasion of epithelial cells by Shigella flexneri involves entry and dissemination. The main effectors of entry, IpaB and IpaC, are also required for contact haemolytic activity and escape from the phagosome in infected macrophages. These proteins are stored in the cytoplasm in association with the chaperone IpgC, before their secretion by a type III secretion apparatus is activated by host cells. We used a His‐tagged IpgC protein to purify IpgC‐containing complexes and showed that only IpaB and IpaC are associated with IpgC. Plasmids expressing His6‐IpgC either alone or together with IpaB or IpaC under the control of an IPTG‐inducible lac promoter were introduced into ipgC, ipaB or ipaC mutants. Induction of expression of the recombinant plasmid‐encoded proteins by IPTG allowed bacteria to enter epithelial cells, and the role of these proteins in dissemination was investigated by incubating infected cells in either the absence or the presence of IPTG. The size of plaques produced by recombinant strains on cell monolayers was regulated by IPTG, indicating that IpgC, IpaB and IpaC were each required for efficient dissemination. Electron microscopy analysis of infected cells indicated that these proteins were necessary for lysis of the membrane of the protrusions during cell‐to‐cell spread.

Internalin must be on the bacterial surface to mediate entry of Listeria monocytogenes into epithelial cells

Entry of Listeria monocytogenes into cultured epithelial cells requires production of internalin, a protein with features characteristic of some Gram‐positive bacterial surface proteins, in particular an LPXTG motif preceding a hydrophobic sequence and a few basic residues at its C‐terminal end. By immunofluorescence and immunogold labelling, we show that in wild‐type L. monocytogenes, internalin is present on the cell surface and has a polarized distribution similar to that of ActA, another surface protein of L. monocytogenes involved in actin assembly. Through a genetic analysis, we establish that the C‐terminal region of internalin is necessary for cell‐surface association, and that although internalin is partially released in the culture medium, its location on the bacterial surface is required to promote entry. Finally, using a‘domain‐swapping’strategy ‐ replacement of the cell wall anchor of InIA by the membrane anchor of ActA ‐ we show that the reduced ability to adhere and enter cells of strains expressing InIA‐ActA correlates with a lower amount of surface‐exposed internalin. Taken together, these results suggest that internalin exposed on the bacterial surface mediates direct contact between the bacterium and the host cell.

Phage infection of the obligate intracellular bacterium, Chlamydia psittaci strain guinea pig inclusion conjunctivitis.

The infectious cycle of phiCPG1, a bacteriophage that infects the obligate intracellular pathogen, Chlamydia psittaci strain Guinea Pig Inclusion Conjunctivitis, was observed using transmission electron microscopy of phage-hyperinfected, Chlamydia-infected HeLa cells. Phage attachment to extracellular, metabolically dormant, infectious elementary bodies and cointernalisation are demonstrated. Following entry, phage infection takes place as soon as elementary bodies differentiate into metabolically active reticulate bodies. Phage-infected bacteria follow an altered developmental path whereby cell division is inhibited, producing abnormally large reticulate bodies, termed maxi-reticulate bodies, which do not mature to elementary bodies. These forms eventually lyse late in the chlamydial developmental cycle, releasing abundant phage progeny in the inclusion and, upon lysis of the inclusion membrane, into the cytosol of the host cell. Structural integrity of the hyperinfected HeLa cell is markedly compromised at late stages. Released phage particles attach avidly to the outer leaflet of the outer membranes of lysed and unlysed Chlamydiae at different stages of development, suggesting the presence of specific phage receptors in the outer membrane uniformly during the chlamydial developmental cycle. A mechanism for phage infection is proposed, whereby phage gains access to replicating chlamydiae by attaching to the infectious elementary body, subsequently subverting the chlamydial developmental cycle to its own replicative needs. The implications of phage infection in the context of chlamydial infection and disease are discussed.

The use of nocodazole in cell cycle analysis and parasite purification from Theileria parva-infected B cells

Theileria parasites transform bovine leukocytes and induce uncontrolled lymphoproliferation only in the macroschizont stage of their life cycle. The isolation of highly purified stage-specific parasite RNA and proteins is an essential prerequisite when studying the Theileria-host relationship. We therefore improved a protocol based on the cytolytic bacterial toxin aerolysin by taking advantage of the microtubule inhibitor nocodazole. In this report we describe that nocodazole-mediated separation of the parasite from the host cell microtubule network was used with success to improve quantity and quality of purified parasites. We furthermore show that nocodazole is a useful tool to study cell cycle checkpoints due to its capacity to induce reversible cell cycle arrest in Theileria-infected B cells.