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Members of the genus Brucella are intracellular alpha-Proteobacteria responsible for brucellosis, a chronic disease of humans and animals. Little is known about Brucella virulence mechanisms, but the abilities of these bacteria to invade and to survive within cells are decisive factors for causing disease. Transmission electron and fluorescence microscopy of infected nonprofessional phagocytic HeLa cells revealed minor membrane changes accompanied by discrete recruitment of F-actin at the site of Brucella abortus entry. Cell uptake of B. abortus was negatively affected to various degrees by actin, actin-myosin, and microtubule chemical inhibitors. Modulators of MAPKs and protein-tyrosine kinases hampered Brucella cell internalization. Inactivation of Rho small GTPases using clostridial toxins TcdB-10463, TcdB-1470, TcsL-1522, and TcdA significantly reduced the uptake of B. abortus by HeLa cells. In contrast, cytotoxic necrotizing factor from Escherichia coli, known to activate Rho, Rac, and Cdc42 small GTPases, increased the internalization of both virulent and non-virulent B. abortus. Expression of dominant-positive Rho, Rac, and Cdc42 forms in HeLa cells promoted the uptake of B. abortus, whereas expression of dominant-negative forms of these GTPases in HeLa cells hampered Brucella uptake. Cdc42 was activated upon cell contact by virulent B. abortus, but not by a noninvasive isogenic strain, as proven by affinity precipitation of active Rho, Rac, and Cdc42. The polyphasic approach used to discern the molecular events leading to Brucella internalization provides new alternatives for exploring the complexity of the signals required by intracellular pathogens for cell invasion.

The permeability of the outer membrane (OM) to hydrophobic probes and its susceptibility to bactericidal cationic peptides were investigated for natural rough Brucella ovis and for mutant rough Brucella abortus strains. The OM of B. ovis displayed an abrupt and faster kinetic profile than rough B. abortus during the uptake of the hydrophobic probe N-phenyl-naphthylamine. B. ovis was more sensitive than rough B. abortus to the action of cationic peptides. Bactenecins 5 and 7 induced morphological alterations on the OMs of both rough Brucella strains. B. ovis lipopolysaccharide (LPS) captured considerably more polymyxin B than LPSs from both rough and smooth B. abortus strains. Polymyxin B, poly-L-lysine, and poly-L-ornithine produced a thick coating on the surfaces of both strains, which was more evident in B. ovis than in rough B. abortus. The distinct functional properties of the OMs of these two rough strains correlate with some structural differences of their OMs and with their different biological behaviors in animals and culture cells.

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.

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.

In mammalian cells, Rho GTPases control the reorganisation of the actin cytoskeleton in response to growth factors. In the cytoplasm, the polymerisation of actin filaments and their organisation into complex architectures is orchestrated by numerous proteins which act either directly, by interacting with actin, or by producing secondary messengers which serve as mediators between signal transduction pathways and the microfilament organisation. We sought to determine whether the intracellular distribution of some of these regulatory components may be controlled by the Rho GTPase CDC42Hs. With this aim, we have established HeLa-derived human cell lines in which expression of a constitutively activated mutant of CDC42Hs is inducible. Morphological analysis by immunofluorescence labelling and confocal laser scanning microscopy revealed a massive reorganisation of F-actin in cortical microspikes as well as podosome-like structures located at the ventral face of the cells. Concomitantly, the cells became giant and multinucleate indicating that cytokinesis was impaired. The actin bundling protein T-plastin, the vasodilatator-stimulated phosphoprotein (VASP), a profilin ligand, as well as the 85 kDa regulatory subunit of the phosphoinosite 3-kinase redistributed with F-actin into the CDC42Hs-induced structures.

The highly differentiated plasma membrane of rabbit enterocytes constitutes an interesting model for membrane studies. Flow cytometry allows combined measurements of the size of membrane vesicles by light-scatter and fluorescence polarization at a single-particle level. The degree of fluorescence polarization of 1,6-diphenylhexa-1,3,5-triene was determined at 4, 18 and 37 degrees C in the brush-border and basolateral plasma membranes. The fluorescence polarization was considerably higher in brush-border than in basolateral membranes. After incubation with dimethyl sulphoxide, the membrane fluidity decreased in both types of membranes. Moreover, a time-effect study of dimethyl sulphoxide showed changes in fluorescence polarization. Only in brush-border membrane a temporary fluid phase was observed. The different properties of the two membrane domains in relation to the lipid-protein dynamics of enterocytes are discussed.

The processing of exogenous antigens and the association of peptides with class II molecules both occur within the endocytic pathway. 2A4 B lymphoma cells of the H-2k haplotype were grown in the presence or the absence of two different exogenous antigens (hen egg lysozyme and ribonuclease A) internalized by fluid-phase endocytosis. Using subcellular fractionation techniques, we demonstrate that, in the presence of hen egg lysozyme, newly synthesized SDS-stable class II molecules are detected in a dense endocytic compartment which does not have the characteristics of neither early and late endosomes nor lysosomes. In contrast, no SDS-stable class II molecules are observed between ribonuclease A and newly synthesized class II molecules. Interestingly, when class II molecules are analyzed at steady state, SDS-stable class II molecules induced by ribonuclease A are found in a compartment cosedimenting with late endosomes. These results suggest that the tight associations between ribonuclease A or hen egg lysozyme with class II molecules occur in distinct endocytic compartments and that these associations may depend on the sensitivity of antigens to proteolysis.

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.

Virulent and attenuated Brucella abortus strains attach to and penetrate nonprofessional phagocytic HeLa cells. Compared to pathogenic Brucella, the attenuated strain 19 hardly replicates within cells. The majority of the strain 19 bacteria colocalized with the lysosome marker cathepsin D, suggesting that Brucella-containing phagosomes had fused with lysosomes, in which they may have degraded. The virulent bacteria prevented lysosome-phagosome fusion and were found distributed in the perinuclear region within compartments resembling autophagosomes.

We have previously identified an intracellular compartment involved in the association between processed lysozyme and IAk major histocompatibility complex class II molecules (called the lysozyme-loading compartment (LLC)). Here, we show that the LLC polypeptide composition analyzed by two-dimensional gel electrophoresis shares similarities with that of early endosomes, but not with that of late endosomes. The transferrin receptor, a well known marker for both early and recycling endosomes, colocalizes with IAk molecules in LLC. Moreover, both transferrin and fluid-phase markers have access to LLC after 15 min of internalization. In the presence of concanamycin B, SDS-stable dimer formation and transport of class II molecules out of LLC are impaired. In contrast, nocodazole treatment has no effect. These results suggest that LLC is a specialized compartment of the recycling pathway involved in lysozyme loading and in the targeting of lysozyme-major histocompatibility class II complexes toward the cell surface.

Invariant chain associated with class II molecules is proteolytically processed in several distinct intermediates during its transport through the endocytic pathway. Using subcellular fractionation, early and late endosomal compartments were separated in human fibroblasts transfected with HLA-DR (4N5 cells) and supertransfected with invariant chain (4N5Ii cells) or invariant chain lacking most of the cytoplasmic tail (4N5 delta 20Ii cells). Early and late endosome membrane fractions were characterized by morphology and by analyzing the presence of the Rab5 and Rab7 GTPases as markers of early and late endosomes, respectively. The transfer of endocytosed horseradish peroxidase from early to late endosomes proceeded relatively rapid both in 4N5 and 4N5 delta 20Ii cells (t1/2 = 25 min), whereas this transfer was significantly delayed (t1/2 = 2 h) in 4N5Ii cells. Pulse-chase experiments showed that invariant chain and its degradation products were first observed in early endosomes and thereafter in late endosomes. Our results strongly suggest that invariant chain induces a retention mechanism in the endocytic pathway.

Membrane vesicle preparations are very appropriate material for studying the topology of glycoproteins integrated into specialized plasma membrane domains of polarized cells. Here we show that the flow cytometric measurement of fluorescence energy transfer used previously to study the relationship between surface components of isolated cells can be applied to membrane vesicles. The fluorescein and rhodamine derivatives of a monoclonal antibody (4H7.1) that recognized one common epitope of the rabbit and pig aminopeptidase N were used for probing the oligomerization and conformational states of the enzyme integrated into the brush border and basolateral membrane vesicles prepared from rabbit and pig enterocytes. The high fluorescent energy transfer observed in the case of pig enzyme integrated into both types of vesicles and in the case of the rabbit enzyme integrated into basolateral membrane vesicles agreed very well with the existence of a dimeric organization, which was directly demonstrated by cross-linking experiments. Although with the latter technique we observed that the rabbit aminopeptidase was also dimerized in the brush border membrane, no energy transfer was detected with the corresponding vesicles. This indicates that the relative positions of two associated monomers differ depending on whether the rabbit aminopeptidase is transiently integrated into the basolateral membrane or permanently integrated into the brush border membrane. Cross-linking of aminopeptidases solubilized by detergent and of their ectodomains liberated by trypsin showed that only interactions between anchor domains maintained the dimeric structure of rabbit enzyme whereas interactions between ectodomains also exist in the pig enzyme. This might explain why the noticeable change in the organization of the two ectodomains observed in the case of rabbit aminopeptidase N does not occur in the case of pig enzyme.