Murine AML12 hepatocytes allow Salmonella Typhimurium T3SS1-independent invasion and intracellular fate.

Numerous studies have demonstrated the key role of the Salmonella Pathogenicity Island 1-encoded type III secretion system (T3SS1) apparatus as well as its associated effectors in the invasion and intracellular fate of Salmonella in the host cell. Several T3SS1 effectors work together to control cytoskeleton networks and induce massive membrane ruffles, allowing pathogen internalization. Salmonella resides in a vacuole whose maturation requires that the activity of T3SS1 subverts early stages of cell signaling. Recently, we identified five cell lines in which Salmonella Typhimurium enters without using its three known invasion factors: T3SS1, Rck and PagN. The present study investigated the intracellular fate of Salmonella Typhimurium in one of these models, the murine hepatocyte cell line AML12. We demonstrated that both wild-type Salmonella and T3SS1-invalidated Salmonella followed a common pathway leading to the formation of a Salmonella containing vacuole (SCV) without classical recruitment of Rho-GTPases. Maturation of the SCV continued through an acidified phase that led to Salmonella multiplication as well as the formation of a tubular network resembling Salmonella induced filaments (SIF). The fact that in the murine AML12 hepatocyte, the T3SS1 mutant induced an intracellular fate resembling to the wild-type strain highlights the fact that Salmonella Typhimurium invasion and intracellular survival can be completely independent of T3SS1.

Remodelling of the actin cytoskeleton is essential for replication of intravacuolar Salmonella.

Maturation and maintenance of the intracellular vacuole in which Salmonella replicates is controlled by virulence proteins including the type III secretion system encoded by Salmonella pathogenicity island 2 (SPI-2). Here, we show that, several hours after bacterial uptake into different host cell types, Salmonella induces the formation of an F-actin meshwork around bacterial vacuoles. This structure is assembled de novo from the cellular G-actin pool in close proximity to the Salmonella vacuolar membrane. We demonstrate that the phenomenon does not require the Inv/Spa type III secretion system or cognate effector proteins, which induce actin polymerization during bacterial invasion, but does require a functional SPI-2 type III secretion system, which plays an important role in intracellular replication and systemic infection in mice. Treatment with actin-depolymerizing agents significantly inhibited intramacrophage replication of wild-type Salmonella typhimurium. Furthermore, after this treatment, wild-type bacteria were released into the host cell cytoplasm, whereas SPI-2 mutant bacteria remained within vacuoles. We conclude that actin assembly plays an important role in the establishment of an intracellular niche that sustains bacterial growth.

Unusual intracellular trafficking of Salmonella typhimurium in human melanoma cells.

Salmonella spp. are enterobacteria capable of invading and replicating in both professional and non-professional phagocytes. Here, we investigate the fate of S. typhimurium in human melanoma MelJuSo cells. The bacterium entered MelJuSo cells by a trigger mechanism and resided within a unique organelle, the Salmonella-containing vacuole (SCV). The SCV acquired early endosomal markers transiently and then underwent a series of membrane modifications. In HeLa cells, vacuole maturation is characterized by the simultaneous acquisition of the lysosomal membrane glycoproteins (Lgps) Lamp1, CD63 and vacuolar (v)-ATPase; in MelJuSo cells, however, acquisition of CD63 and v-ATPase preceded that of Lamp1. A very striking event in MelJuSo cells was the arrest of bacterial septation starting from 8 h after infection. Bacteria nevertheless continued to elongate, remained morphologically intact and viable and were eventually exocytosed. This original feature was observed in several skin-related cells including melanocytes, suggesting that it may provide the basis for an efficient host defence mechanism against Salmonella infection.

Maturation steps of the Salmonella-containing vacuole.

After uptake, Salmonella resides within a unique organelle, the Salmonella-containing vacuole (SCV) in which it eventually replicates. Here we recapitulate the knowledge about how Salmonella controls SCV maturation and the different steps of its intracellular trafficking.

Salmonella maintains the integrity of its intracellular vacuole through the action of SifA.

A method based on the Competitive Index was used to identify Salmonella typhimurium virulence gene interactions during systemic infections of mice. Analysis of mixed infections involving single and double mutant strains showed that OmpR, the type III secretion system of Salmonella pathogenicity island 2 (SPI-2) and SifA [required for the formation in epithelial cells of lysosomal glycoprotein (lgp)-containing structures, termed Sifs] are all involved in the same virulence function. sifA gene expression was induced after Salmonella entry into host cells and was dependent on the SPI-2 regulator ssrA. A sifA(-) mutant strain had a replication defect in macrophages, similar to that of SPI-2 and ompR(-) mutant strains. Whereas wild-type and SPI-2 mutant strains reside in vacuoles that progressively acquire lgps and the vacuolar ATPase, the majority of sifA(-) bacteria lost their vacuolar membrane and were released into the host cell cytosol. We propose that the wild-type strain, through the action of SPI-2 effectors (including SpiC), diverts the Salmonella-containing vacuole from the endocytic pathway, and subsequent recruitment and maintenance of vacuolar ATPase/lgp-containing membranes that enclose replicating bacteria is mediated by translocation of SifA.

Controlling the maturation of pathogen-containing vacuoles: a matter of life and death.

Once considered to be contained, infectious diseases of bacterial origin are now making a comeback. A lack of innovative therapies and the appearance of drug-resistant pathogens are becoming increasingly serious problems. A better understanding of pathogen-host interactions at the cellular and molecular levels is necessary to define new targets in our fight against microorganisms. In the past few years, the merging of cell biology and microbiology has started to yield critical and often surprising new information on the interactions that occur between various pathogens and their mammalian host cells. Here we focus on the intracellular routing of vacuoles containing microorganisms, as well as on the bacterial effectors and their host-cell targets that control vacuole maturation. We also describe new approaches for isolating microorganism-containing vacuoles and analysing their molecular composition, which will help researchers to define the molecules and mechanisms governing vacuole biogenesis.

The rab7 GTPase controls the maturation of Salmonella typhimurium-containing vacuoles in HeLa cells.

Following entry into non-phagocytic HeLa cells, the facultative pathogen Salmonella typhimurium survives and replicates within a membrane-bound vacuole. Preceding the initiation of intracellular replication there is a lag phase, during which the bacteria modulate their environment. This phase is characterized by the rapid recycling of early endosomal proteins present on the nascent vacuole followed by the acquisition of a subset of lysosomal proteins. To gain a better understanding of the mechanism of intracellular survival, we have followed the biogenesis of the S. typhimurium-containing vacuole (SCV) in HeLa cells expressing different mutant forms of the small GTPase rab7. We demonstrate that the SCV recruits pre-existing lysosomal glycoproteins (Lgps) in a rab7-dependent manner, without directly interacting with lysosomes. We also show the transient accumulation, in the vicinity of the SCV, of novel rab7- and Lgp-containing vesicles containing very low amounts of cathepsin D. The size of these vesicles is dependent on rab7 activity, suggesting a role for rab7 in their homotypic fusion. Taken together, these results indicate that rab7 regulates SCV biogenesis during the phase characterized by the rapid acquisition of lysosomal proteins. We propose that SCV maturation involves its interaction with rab7/Lgp-containing vesicles which are possible intermediate cargo components of the late endocytic pathway.

Interaction of Brucella abortus lipopolysaccharide with major histocompatibility complex class II molecules in B lymphocytes.

Lipopolysaccharide (LPS), a major amphiphilic molecule located at the outer membrane of gram-negative bacteria, is a potent antigen known to induce specific humoral immune responses in infected mammals. LPS has been described as a polyclonal activator of B lymphocytes, triggering the secretion of antibodies directed against distinct sugar epitopes of the LPS chain. But, how LPS is handled by B cells remains to be fully understood. This task appears to be essential for a better knowledge of the anti-LPS humoral immune response. In this study, we examine the internalization of LPS and its interaction with antigen-presenting major histocompatibility complex (MHC) class II molecules in murine and human B-cell lines. By use of immunofluorescence, we observe that structurally different LPSs from Brucella and Shigella strains accumulate in an intracellular compartment enriched in MHC class II molecules. By use of immunoprecipitation, we illustrate that only Brucella abortus LPS associates with MHC class II molecules in a haplotype-independent manner. Taken together, these results raise the possibility that B. abortus LPS may play a role in T-cell activation.

Trafficking of Shigella lipopolysaccharide in polarized intestinal epithelial cells.

Bacterial lipopolysaccharide (LPS) at the apical surface of polarized intestinal epithelial cells was previously shown to be transported from the apical to the basolateral pole of the epithelium (Beatty, W.L., and P.J. Sansonetti. 1997. Infect. Immun. 65:4395-4404). The present study was designed to elucidate the transcytotic pathway of LPS and to characterize the endocytic compartments involved in this process. Confocal and electron microscopic analyses revealed that LPS internalized at the apical surface became rapidly distributed within endosomal compartments accessible to basolaterally internalized transferrin. This compartment largely excluded fluid-phase markers added at either pole. Access to the basolateral side of the epithelium subsequent to trafficking to basolateral endosomes occurred via exocytosis into the paracellular space beneath the intercellular tight junctions. LPS appeared to exploit other endocytic routes with much of the internalized LPS recycled to the original apical membrane. In addition, analysis of LPS in association with markers of the endocytic network revealed that some LPS was sent to late endosomal and lysosomal compartments.

Impaired recruitment of the small GTPase rab7 correlates with the inhibition of phagosome maturation by Leishmania donovani promastigotes.

We have shown recently that one of the survival strategies used by Leishmania donovani promastigotes during the establishment of infection in macrophages consists in inhibiting phagosome-endosome fusion. This inhibition requires the expression of lipophosphoglycan (LPG), the predominant surface glycoconjugate of promastigotes, as parasites expressing truncated forms of LPG reside in phagosomes that fuse extensively with endocytic organelles. In the present study, we developed a single-organelle fluorescence analysis approach to study and analyse the intracellular trafficking of 'fusogenic' and 'low-fusogenic' phagosomes induced by an LPG repeating unit-defective mutant (Ipg2 KO) or by wild-type L. donovani promastigotes respectively. The results obtained indicate that phagosomes containing mutant parasites fuse extensively with endocytic organelles and transform into phagolysosomes by losing the early endosome markers EEA1 and transferrin receptor, and acquiring the late endocytic and lysosomal markers rab7 and LAMP1. In contrast, a majority of 'low-fusogenic' phagosomes containing wild-type L. donovani promastigotes do not acquire rab7, wheres they acquire LAMP1 with slower kinetics. These results suggest that L. donovani parasites use LPG to restrict phagosome-endosome fusion at the onset of infection in order to prevent phagosome maturation. This is likely to permit the transformation of hydrolase-sensitive promastigotes into hydrolase-resistant amastigotes within a hospitable vacuole not displaying the harsh environment of phagolysosomes.

Biogenesis of Salmonella typhimurium-containing vacuoles in epithelial cells involves interactions with the early endocytic pathway.

In epithelial cells, the intracellular pathogen Salmonella typhimurium resides and replicates within a unique cytoplasmic organelle, the Salmonella-containing vacuole (SCV). In vitro studies have shown that the SCV is a dynamic organelle that selectively acquires lysosomal glycoproteins (Igps) without fusing directly with lyosomes. Here, we have investigated early events in SCV biogenesis using immunofluorescence microscopy and epitope-specific flow cytometry. We show that proteins specific to the early endocytic pathway, EEA1 and transferrin receptor (TR), are present on early SCVs. The association of these proteins with SCVs is transient, and both proteins are undetectable at later time points when Igp and vATPase are acquired. Analysis of the fraction of SCVs containing both TR and lamp-1 showed that TR is lost from SCVs as the Igp is acquired, and that these processes occur progressively and not as the result of a single fusion/fission event. These experiments reveal a novel mechanism of SCV biogenesis, involving previously undetected initial interactions with the early endocytic pathway followed by the sequential delivery of Igp. The pathway does not involve interactions with the late endosome/prelysosome and is distinct from traditional phagocytic and endocytic pathways. Our study indicates that intracellular S. typhimurium occupies a unique niche, branching away from the traditional endocytic pathway between the early and late endosomal compartments.

Brucella abortus transits through the autophagic pathway and replicates in the endoplasmic reticulum of nonprofessional phagocytes.

Brucella abortus is an intracellular pathogen that replicates within a membrane-bounded compartment. In this study, we have examined the intracellular pathway of the virulent B. abortus strain 2308 (S2308) and the attenuated strain 19 (S19) in HeLa cells. At 10 min after inoculation, both bacterial strains are transiently detected in phagosomes characterized by the presence of early endosomal markers such as the early endosomal antigen 1. At approximately 1 h postinoculation, bacteria are located within a compartment positive for the lysosome-associated membrane proteins (LAMPs) and the endoplasmic reticulum (ER) marker sec61beta but negative for the mannose 6-phosphate receptors and cathepsin D. Interestingly, this compartment is also positive for the autophagosomal marker monodansylcadaverin, suggesting that S2308 and S19 are located in autophagic vacuoles. At 24 h after inoculation, attenuated S19 is degraded in lysosomes, while virulent S2308 multiplies within a LAMP- and cathepsin D-negative but sec61beta- and protein disulfide isomerase-positive compartment. Furthermore, treatment of infected cells with the pore-forming toxin aerolysin from Aeromonas hydrophila causes vacuolation of the bacterial replication compartment. These results are compatible with the hypothesis that pathogenic B. abortus exploits the autophagic machinery of HeLa cells to establish an intracellular niche favorable for its replication within the ER.

Flow cytometric sorting and biochemical characterization of the late endosomal rab7-containing compartment.

Rab7 is a small molecular weight GTPase that is known to be associated with late endocytic compartments. Studies in which wild-type or mutant forms of this protein have been overexpressed in mammalian cells have indicated that rab7 plays a role in controlling membrane transport between late endocytic compartments. However, both the precise site(s) of action and localization of rab7 remain unclear. In the present study, we have used density-gradient centrifugation in combination with a new epitope-specific flow cytometric sorting method to isolate rab7-containing vesicles from baby hamster kidney (BHK) cells. Electron-micrographs of sorted elements showed a homogeneous population of vesicles that resembles late endosomes. The polypeptide composition of rab7-containing vesicles was then analyzed by two-dimensional (2-D) gel electrophoresis. Rab7-containing vesicles were enriched in the cation-independent mannose 6-phosphate receptor and especially in the precursor forms of cathepsin D. Taken together, these results show that the rab7-containing vesicles are a component of the endocytic pathway that connects late endosomes and lysosomes and in which precursor forms of lysosomal hydrolases, segregated from their receptor, might be included.

The rab7 GTPase resides on a vesicular compartment connected to lysosomes.

Rab GTPases belong to the Ras GTPase superfamily and are key regulators of membrane traffic. Among them, rab7 has been localized on late endosomes of NRK cells but its function remains unknown. In order to investigate its role, we generated stable HeLa cell lines that express either wild type or a GTPase-defective mutant of rab7 in an inducible manner. A morphological analysis of the intracellular localization of these proteins was performed by confocal laser microscopy. Here we show that, in HeLa cells, rab7 is present on a vesicular compartment that extends from the perinuclear area to the cell periphery and shows only a partial colocalization with the cation-independent mannose 6-phosphate receptor, a marker for late endosomes. The topology of this compartment is dependent on the microtubule network since nocodazole treatment results in its scattering throughout the cytoplasm. In addition, we observed that, in contrast to the wild-type protein, a rab7 mutant with a reduced GTPase activity is in part associated with lysosomal membranes. This observation was confirmed by subcellular fractionation in a Percoll gradient. Our data implicate rab7 as the first GTPase functioning on terminal endocytic structures in mammalian cells.

Phosphorylation of the cation-independent mannose 6-phosphate receptor is closely associated with its exit from the trans-Golgi network.

We have previously shown that two serine residues present in two conserved regions of the bovine cation-independent mannose 6-phosphate receptor (CI-MPR) cytoplasmic domain are phosphorylated in vivo (residues 2421 and 2492 of the full length bovine CI-MPR precursor). In this study, we have used CHO cells to investigate the phosphorylation state of these two serines along the different steps of the CI-MPR exocytic and endocytic recycling pathways. Transport and phosphorylation of the CI-MPR in the biosynthetic pathway were examined using deoxymannojirimycin (dMM), a specific inhibitor of the cis-Golgi processing enzyme alpha-mannosidase I which leads to the accumulation of N-linked high mannose oligosaccharides on glycoproteins. Upon removal of dMM, normal processing to complex-type oligosaccharides (galactosylation and then sialylation) occurs on the newly synthesized glycoproteins, including the CI-MPR which could then be purified and analyzed on lectin affinity columns. Phosphorylation of the newly synthesized CI-MPR was concomitant with the sialylation of its oligosaccharides and appeared as a major albeit transient modification. Phosphorylation of the cell surface CI-MPR was examined during its endocytosis as well as its return to the Golgi using antibody tagging and exogalactosylation. The cell surface CI-MPR was not phosphorylated when it entered clathrin-coated pits or when it moved to the early and late endosomes. In contrast, the surface CI-MPR was phosphorylated when it had been resialylated upon its return to the trans-Golgi network. Subcellular fractionation experiments showed that the phosphorylated CI-MPR and the corresponding kinase were found in clathrin-coated vesicles. Collectively, these results indicate that phosphorylation of the two serines in the CI-MPR cytoplasmic domain is associated with a single step of transport of its recycling pathways and occurs when this receptor is in the trans-Golgi network and/or has left this compartment via clathrin-coated vesicles.

Phosphorylation of the cytoplasmic domain of the bovine cation-independent mannose 6-phosphate receptor. Serines 2421 and 2492 are the targets of a casein kinase II associated to the Golgi-derived HAI adaptor complex.

A kinase activity of purified bovine brain clathrin-coated vesicles phosphorylates the bovine cation-independent mannose 6-phosphate receptor (CI-MPR) with high efficiency (Km approximately 50-100 nM). The kinase copurifies in gel filtration, adsorption on hydroxylapatite, and ion exchange chromatography with the HAI assembly proteins which are part of the coat of Golgi-derived clathrin-coated vesicles. The kinase is associated to the 47-kDa subunit of the complex and exhibits properties similar to a casein kinase II: it uses either ATP or GTP as substrate and its activity is stimulated by poly-L-lysine and inhibited by heparin. Using different domains of the CI-MPR as potential substrates, we show that the phosphorylation is restricted to its cytoplasmic domain. Inhibition studies using synthetic peptides and two-dimensional mapping of the tryptic phosphopeptides indicate that this posttranslational modification occurs on serines 2421 and 2492 of the full-length bovine CI-MPR precursor, residues which are located in typical casein-kinase II recognition sequences. Labeling of Madin-Darby bovine kidney cells followed by immunoprecipitation of the CI-MPR and analysis of the corresponding tryptic phosphopeptides shows that the same serines are phosphorylated in vivo.

[Results of surgery in 144 cases for aneurysms of the infra-renal abdominal aorta in patients over 75 years of age]. / Résultats de 114 interventions pour des anévrysmes de l'aorte abdominale sous-rénale chez des patients âgés de 75 ans et plus.

114 patients aged over 75 underwent surgery for aneurysm of the infra-renal abdominal aorta in the University Teaching Hospitals in Nantes and Angers between 1979 and 1988. A retrospective study of these patients was performed to evaluate the immediate and long-term results. The mean age of the patients was 79 (+/- 4) years, the oldest being 94. 70% were men. Half of the patients underwent emergency or semi-emergency surgery (52 cases). Cardiovascular factors (in particular coronary insufficiency in 17% of cases) were the most common risk factors. In all cases grafting after laying open the aneurysm was performed, with an aorto-aortic graft in 32% of patients, an aorto-iliac graft in 37% or an aorto-bifemoral graft in 27% of patients. Combined intestinal revascularisation was performed in 10% of case either involving the inferior mesenteric artery or at least one internal iliac artery; renal revascularisation was performed in 3.5% of cases. 75% of patients underwent simple grafting. The mean duration of hospitalisation was 14 days (+/- 6), including a mean period of 7 days in ICU. 36 patients (31%) died in the first post-operative month. The mortality rate in patients who underwent emergency surgery for a complication of the aneurysm (essentially rupture) was 61% versus 6' for elective surgery. 96% of the patients who survived the first post-operative month were independent at the end of the study or at the time of their death.(ABSTRACT TRUNCATED AT 250 WORDS)

An easier, reproducible, and mass-production method to study the blood-brain barrier in vitro.

To provide an "in vitro" system for studying brain capillary function, we have developed a process of coculture that closely mimics the "in vivo" situation by culturing brain capillary endothelial cells on one side of a filter and astrocytes on the other. Under these conditions, endothelial cells retain all the endothelial cell markers and the characteristics of the blood-brain barrier, including tight junctions and gamma-glutamyl transpeptidase activity. The average electric resistance for the monolayers was 661 omega cm2. The system is impermeable to inulin and sucrose but allows the transport of leucine. Arabinose treatment increases transcellular transport flux by 70%. The relative ease with which such monolayers can be produced in large quantities would facilitate the "in vitro" study of brain capillary functions.

Bovine brain endothelial cells express tight junctions and monoamine oxidase activity in long-term culture.

The passage of substances across the blood-brain barrier is regulated by cerebral capillaries which possess certain distinctly different morphological and enzymatic properties compared to capillaries of other organs. Investigations of the functional characteristics of brain capillaries have been facilitated by the use of cultured brain endothelial cells, but in most studies a number of characteristics of the in vivo system are lost. To provide an in vitro system for studies of brain capillary functions, we developed a method of isolating and producing a large number of bovine brain capillary endothelial cells. These cells, absolutely free of pericyte contamination, are subcultured, at the split ratio of 1:20 (20-fold increase of the cultured surface), with no apparent changes in cell morphology up to the fiftieth generation (10 passages). Retention of endothelial-specific characteristics (factor VIII-related antigen, angiotensin-converting enzyme, and nonthrombogenic surface) is shown for brain capillary-derived endothelial cells up to passage 10, even after frozen storage at passage 3. Furthermore, we showed that bovine brain capillary endothelial cells retain, up to the fiftieth generation, some of the characteristics of the blood-brain barrier: occurrence of tight junctions, paucity of pinocytotic vesicles, and monoamine oxidase activity.

Interactions of high-density lipoprotein 3 with brain capillary endothelial cells.

High-density lipoprotein 3 (HDL3) binds to capillary endothelial cells when their lumen surfaces are exposed to 125I-HDL3 by post-mortem perfusion of whole brain. Kinetic studies of binding of HDL3 to isolated membranes show that HDL3 binds only to endothelial membranes with high affinity (Kd = 7 micrograms/ml). Trypsin treatment of membranes abolishes HDL3 binding. High-affinity binding sites for HDL3 were recovered when endothelial cells from bovine brain capillaries were maintained in culture (Kd = 13 micrograms/ml HDL3 protein). The characteristics of the binding were preserved up to the 6th passage. Competition experiments using isolated luminal membranes or cultured endothelial cells indicate that only HDL3 and not LDL or methylated LDL, are able to compete binding of 125I-HDL3. Furthermore, the inhibition of 125I-HDL3 binding by lipoprotein A-I and lipoprotein A-I:A-II strongly suggests that apolipoprotein A-I is implicated in the formation of HDL3-receptor complexes. The binding is increased by loading cells with free cholesterol or LDL cholesterol. In addition, surface-bound 125I-HDL3 remains sensitive to mild trypsin treatment after subsequent incubation of BBCE at 37 degrees C. HDL3 bound to the cell surface is not endocytosed, but rather rapidly released into the medium after binding (t1/2 = 5 min).