Internalization of Bordetella pertussis adenylate cyclase-haemolysin into endocytic vesicles contributes to macrophage cytotoxicity.

Bordetella pertussis adenylate cyclase-haemolysin is a critical virulence factor in the murine model of intranasal infection, where it is required for several pathological effects, including macrophage apoptosis. Based on biochemical and immunological properties, it was proposed that the toxin was delivered directly to the cytoplasm of eukaryotic cells without trafficking through the endocytic pathway. In the present study, we analysed the cellular distribution of the adenylate cyclase-haemolysin during intoxication of macrophages. We showed that, shortly after its initial binding to the plasma membrane of macrophages, the toxin gains access to intracellular compartments that become progressively positive for the endosomal marker transferrin, but not for the lysosomal membrane protein CD107a/Lamp1. Importantly, the vesicular trafficking of the adenylate cyclase-haemolysin appears to be required for its ability to induce macrophage death. Inhibitors of actin polymerization and of macropinocytosis, as well as depletion of plasma membrane cholesterol and disruption of the Golgi network, reduce the toxin's ability to kill macrophages. Altogether, these results suggest that internalization of the adenylate cyclase-haemolysin into endocytic vesicles, at least partly through macropinocytosis, contributes to cytotoxicity.

Internalin B activates nuclear factor-kappa B via Ras, phosphoinositide 3-kinase, and Akt.

Internalin B (InlB), a 630-amino acid protein loosely attached to the surface of Listeria monocytogenes, participates in the entry of the bacterium into mammalian cells. This process requires the activation of phosphoinositide (PI) 3-kinase by InlB. Previously, we demonstrated that InlB activates the transcription factor Nuclear Factor-kappaB in murine J774 macrophage-like cells, an event that also requires PI 3-kinase. Here we have further investigated this phenomenon. InlB activated the small G-protein Ras in J774 cells. Inhibition of Ras with the farnesyltransferase inhibitor manumycin A inhibited NF-kappaB activation and the recruitment of the p85 subunit of PI 3-kinase, implying that Ras is required for PI 3-kinase activation. InlB also activated the PI 3-kinase downstream effector, Akt, as assessed by increased phosphorylation of Akt on serine 473. Transfection of Hep2 cells with dominant negative Ras N17 or dominant negative Akt inhibited the induction of a reporter gene linked to the interleukin-8 promoter by InlB. Furthermore, the Ras inhibitor manumycin A, the PI 3-kinase inhibitor LY294002, and an Akt inhibitor all blocked the induction of interleukin-8 by InlB. Our study is the first report of a bacterial product activating a pathway involving Ras, PI 3-kinase, and Akt, which leads to NF-kappaB activation. This process could be involved in host defense or the inhibition of apoptosis during infection.

Phagocytosis of wild-type Legionella pneumophila occurs through a wortmannin-insensitive pathway.

Wild-type Legionella pneumophila grows in human macrophages within a replicative phagosome, avoiding lysosomal fusion, while nonreplicative mutants are killed in lysosomes. Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, blocks phagocytosis of an avirulent mutant, but not of wild-type L. pneumophila, without affecting membrane ruffling and actin polymerization. These results show that wild-type and mutant Legionella strains use different entry pathways. They suggest that PI3Ks are involved in phagocytosis of an avirulent L. pneumophila mutant and regulate the ability of microorganisms to generate a replicative phagosome.

The adenylate cyclase toxin of Bordetella pertussis binds to target cells via the alpha(M)beta(2) integrin (CD11b/CD18).

The adenylate cyclase toxin (CyaA) of Bordetella pertussis is a major virulence factor required for the early phases of lung colonization. It can invade eukaryotic cells where, upon activation by endogenous calmodulin, it catalyzes the formation of unregulated cAMP levels. CyaA intoxication leads to evident toxic effects on macrophages and neutrophils. Here, we demonstrate that CyaA uses the alpha(M)beta(2) integrin (CD11b/CD18) as a cell receptor. Indeed, the saturable binding of CyaA to the surface of various hematopoietic cell lines correlated with the presence of the alpha(M)beta(2) integrin on these cells. Moreover, binding of CyaA to various murine cell lines and human neutrophils was specifically blocked by anti-CD11b monoclonal antibodies. The increase of intracellular cAMP level and cell death triggered by CyaA intoxication was also specifically blocked by anti-CD11b monoclonal antibodies. In addition, CyaA bound efficiently and triggered intracellular cAMP increase and cell death in Chinese hamster ovary cells transfected with alpha(M)beta(2) (CD11b/CD18) but not in cells transfected with the vector alone or with the alpha(X)beta(2) (CD11c/CD18) integrin. Thus, the cellular distribution of CD11b, mostly on neutrophils, macrophages, and dendritic and natural killer cells, supports a role for CyaA in disrupting the early, innate antibacterial immune response.

Enrichment of acyl coenzyme A:cholesterol O-acyltransferase near trans-golgi network and endocytic recycling compartment.

Acyl coenzyme A:cholesterol O-acyltransferase (ACAT) is the enzyme responsible for cholesterol esterification in macrophages leading to foam cell formation. The determination of its localization is a critical step in understanding its regulation by cholesterol. Using immunofluorescence and confocal microscopy, we previously showed that the enzyme colocalized with markers of the endoplasmic reticulum, but in addition, ACAT was found in an unidentified paranuclear site. In the present study, we further define the localization of paranuclear ACAT. First, we found that ACAT does not colocalize with sorting endosomes or late endosomes labeled with fluorescent alpha(2)-macroglobulin. The paranuclear ACAT is close to the endocytic recycling compartment labeled with fluorescent transferrin. We also show that the paranuclear structure containing ACAT is very close to TGN38, a membrane protein of the trans-Golgi network (TGN), but farther from Gos28, a marker of cis, medial, and trans Golgi. After treatment with nocodazole, the central localization of ACAT did not colocalize with markers of the TGN. These data indicate that a significant fraction of ACAT resides in membranes that may be a subcompartment of the endoplasmic reticulum in proximity to the TGN and the endocytic recycling compartment. Because the TGN and the endocytic recycling compartment are engaged in extensive membrane traffic with the plasma membrane, esterification of cholesterol in these membranes may play an important role in macrophage foam cell formation during atherogenesis.

A sustained increase in the endogenous level of cAMP reduces the retinoid concentration required for APL cell maturation to near physiological levels.

cAMP-dependent signal transduction co-operates with retinoids to induce acute promyelocytic leukaemia (APL) cell maturation. The rationale of this work was to determine whether signal cross-talk could be used to decrease the pharmacological doses of retinoids in the treatment of APL. When only the basal level of adenylate-cyclase (AC) activity is present in NB4 cells, up to 1 microM concentration of all-trans retinoic acid (RA) is required for full maturation (100%). In these conditions, with only 10 nM RA less than 20% of cells will differentiate. Although the use of membrane receptor agonists to activate AC has been proved to synergize with RA treatment, these agents were never as potent as cell permeant cAMP analogues. Analogues have disadvantages since cleavage by serum and cellular phosphodiesterases generates metabolites which interfere in cellular response. In the present study, we observed cell maturation by engrafting an autonomous Bordetella pertussis AC which steadily delivers natural cAMP into the cell. The enzyme alone had no effect on cell maturation. Importantly, cell maturation was increased in a dose-dependent manner when the bacterial AC (1 ng/ml to 1 microg/ml) was used to potentiate the effects of low doses RA (10 nM). More than 50% of cells matured with only 10 nM of RA and 200 ng/ml of B. pertussis AC. The maturation response was significantly increased when lower amounts of enzyme were repetitively added to the culture to compensate for enzymatic decay. These results indicate that a sustained AC activity enhanced cell maturation. We were able to reduce to 3 nM the RA requirement, provided that a minimal amount (20 ng/ml) of B. pertussis AC was added every 12 h in culture. Membrane signalling maintaining high the level of cAMP substantially improved the efficacy of APL cell maturation by retinoids. Therefore, therapeutic benefits are expected by lowering the concentration of RA towards physiological (nanomolar) levels, thus reducing the side-effects of the drug. cAMP-elevating drugs that act on a post-cyclase target (cyclic-nucleotide phosphodiesterases) or cell-targeted drug carriers (cAMP and RA loaded liposomes) should be evaluated as maturation therapies combining the activation of multiple signalling pathways.

Immunolocalization of acyl-coenzyme A:cholesterol O-acyltransferase in macrophages.

Macrophages in atherosclerotic lesions accumulate large amounts of cholesteryl-fatty acyl esters ("foam cell" formation) through the intracellular esterification of cholesterol by acyl-coenzyme A:cholesterol O-acyltransferase (ACAT). In this study, we sought to determine the subcellular localization of ACAT in macrophages. Using mouse peritoneal macrophages and immunofluorescence microscopy, we found that a major portion of ACAT was in a dense reticular cytoplasmic network and in the nuclear membrane that colocalized with the luminal endoplasmic reticulum marker protein-disulfide isomerase (PDI) and that was in a similar distribution as the membrane-bound endoplasmic reticulum marker ribophorin. Remarkably, another portion of the macrophage ACAT pattern did not overlap with PDI or ribophorin, but was found in as yet unidentified cytoplasmic structures that were juxtaposed to the nucleus. Compartments containing labeled beta-very low density lipoprotein, an atherogenic lipoprotein, did not overlap with the ACAT label, but rather were embedded in the dense reticular network of ACAT. Furthermore, cell-surface biotinylation experiments revealed that freshly harvested, non-attached macrophages, but not those attached to tissue culture dishes, contained approximately 10-15% of ACAT on the cell surface. In summary, ACAT was found in several sites in macrophages: a cytoplasmic reticular/nuclear membrane site that overlaps with PDI and ribophorin and has the characteristics of the endoplasmic reticulum, a perinuclear cytoplasmic site that does not overlap with PDI or ribophorin and may be another cytoplasmic structure or possibly a unique subcompartment of the endoplasmic reticulum, and a cell-surface site in non-attached macrophages. Understanding possible physiological differences of ACAT in these locations may reveal an important component of ACAT regulation and macrophage foam cell formation.

Identification of Btr-regulated genes using a titration assay. Search for a role for this transcriptional regulator in the growth and virulence of Bordetella pertussis.

Bordetella pertussis is the causative agent of the respiratory disease pertussis or whopoping cough. Btr, an oxygen-responsive transcriptional regulator of B. pertussis, is homologous to the FNR protein of E. coli. Using a murine respiratory model, we observed in the present study that Btr is important in growth and survival of B. pertussis in vivo. A titration assay was developed that identified genes containing Btr binding sites including B. pertussis sodB and btr, E. coli aspA and a new B. pertussis gene, brg1. The brg1 gene encodes a protein similar to the LysR family of transcriptional regulators and its expression is activated threefold by Btr under anaerobic growth conditions but unaffected by Btr aerobically. The nucleotide sequence flanking brg1 encodes proteins with similarity to various metabolic enzymes. Putative overlapping promoters and a Btr binding site (FNR box) were identified in the DNA sequence between brg1 and the adjacent genes. These intervening sequences may represent sites for regulation by Btr and Brg1.

In vivo and in vitro analysis of Bordetella pertussis catalase and Fe-superoxide dismutase mutants.

Bordetella pertussis produces a catalase and a Fe-superoxide dismutase. The importance of these enzymes in virulence was investigated, in vitro as well as in vivo, by using mutants deficient in their production. The catalase-deficient mutant survived within polymorphonuclear leukocytes, killed J774A.1 macrophages through apoptosis, and behaved as the parental strain in a murine respiratory infection model. These results suggest no direct role for catalase in B. pertussis virulence. The absence of expression of Fe-superoxide dismutase had profound effects on the bacterium including a reduced ability to express adenylate cyclase-hemolysin and pertactin, two factors important for B. pertussis pathogenesis. The Fe-superoxide dismutase-deficient mutant also had decreased abilities to colonize and persist in the murine respiratory infection model.

Induction of macrophage apoptosis by Bordetella pertussis adenylate cyclase-hemolysin.

We previously demonstrated that Bordetella pertussis induced macrophage apoptosis and that adenylate cyclase-hemolysin expression is required for this phenomenon. In order to determine a role for other B. pertussis factors, such as pertussis toxin or the adhesins, pertactin and filamentous hemagglutinin, we compared mutants deficient in adhesin expression or purified proteins for their ability to induce cell death. We showed that only purified adenylate cyclase-hemolysin, but neither adhesins, nor pertussis toxin, killed macrophages through apoptosis. We also demonstrated that all activities of adenylate cyclase-hemolysin were required. Moreover, our results indicate that B. pertussis does not have to invade cells to be lethal.

Characterization of murine lung inflammation after infection with parental Bordetella pertussis and mutants deficient in adhesins or toxins.

Bordetella pertussis expresses factors such as filamentous hemagglutinin, agglutinogens, pertactin, and pertussis toxin, which participate in bacterial adhesion; pertussis toxin, dermonecrotic toxin, lipopolysaccharide, and tracheal cytotoxin, which are responsible for toxic effects; and adenylate cyclase-hemolysin, which is required to initiate infection. By using a murine respiratory model, we showed that the RGD sequences of filamentous hemagglutinin and pertactin are important for bacterial persistence. However, mutants deficient in filamentous hemagglutinin and agglutinogens or in pertactin and the RGD sequence of filamentous hemagglutinin behaved as did wild-type B. pertussis, i.e., induced bronchopneumonia, alveolitis, and an influx of macrophages, lymphocytes, and polymorphonuclear leukocytes into bronchoalveolar lavage fluids. These results suggest that these adhesins are not involved in the induction of pulmonary lesions following infection. The intensity of inflammation was markedly reduced after infection with mutants deficient in either hemolytic activity or pertussis toxin expression, whereas a mutant devoid of adenylate cyclase activity behaved as did the avirulent mutant. Pertussis toxin and adenylate cyclase-hemolysin may act indirectly by altering immune cell functions and thus allowing other factors, such as filamentous hemagglutinin, agglutinogens, and pertactin, to trigger adhesion and lipopolysaccharide, dermonecrotic toxin, and tracheal cytotoxin to induce their toxic effects. However, it is possible that pertussis toxin is also responsible for the induction of some pulmonary alterations.

A phase variant of Bordetella pertussis with a mutation in a new locus involved in the regulation of pertussis toxin and adenylate cyclase toxin expression.

A novel nonhemolytic phase variant of Bordetella pertussis was characterized. This strain is strongly impaired in the transcription of the pertussis and adenylate cyclase toxins, whereas other known virulence-related factors such as the filamentous hemagglutinin, the fimbriae, and the outer membrane protein pertactin are expressed and regulated normally. Complementation and allelic exchange experiments demonstrated that the mutation is localized neither in the bvg locus involved in virulence regulation nor in the genes responsible for synthesis and transport of the toxins pertussis and adenylate cyclase. Instead, the mutation impairing transcription of at least the two toxin genes is located in a new genetic locus, which acts together with the BvgA/S two-component regulatory system on the expression of a subset of virulence genes. Further analysis suggested that most presumably the mutation affects a sequence-specific DNA-binding protein which contributes to transcriptional activation. The mutant was nonlethal in a murine respiratory model, which corresponds well with the lack of expression of the toxins. However, the clearing rate of this mutant from the lungs of mice was much lower than that of a bvg mutant, suggesting that factors other than the toxins may play a role in the persistence of the bacteria in the respiratory tract of mice.

Bordetella pertussis induces apoptosis in macrophages: role of adenylate cyclase-hemolysin.

Bordetella pertussis, the causative agent of whooping cough, has been shown recently to enter and survive in epithelial cells and macrophages in vitro. In the present study, we show that B. pertussis is cytotoxic for J774A.1 cells, a monocyte-macrophage cell line, and for murine alveolar macrophages. We demonstrate that cell cytotoxicity mediated by B. pertussis occurred through apoptosis, as shown by changes in nuclear morphology and by host cell DNA fragmentation. Parental strains and a mutant deficient in pertussis toxin expression are able to induce apoptosis, whereas avirulent mutant or adenylate cyclase-hemolysin-deficient mutants are not cytotoxic. Both adenylate cyclase and hemolytic activities are required for programmed cell death. These results show that induction of apoptosis is dependent on the expression of adenylate cyclase-hemolysin. The infection of murine alveolar macrophages in primary culture with B. pertussis leads to apoptosis, suggesting that this process might be relevant in vivo. The ability of B. pertussis to promote cell death may be important for the initiation of infection, bacterial survival, and escape of the host immune response.

Bordetella pertussis and Bordetella parapertussis: two immunologically distinct species.

Bordetella pertussis and Bordetella parapertussis are closely related species. Both are responsible for outbreaks of whooping cough in humans and produce similar virulence factors, with the exception of pertussis toxin, specific to B. pertussis. Current pertussis whole-cell vaccine will soon be replaced by acellular vaccines containing major adhesins (filamentous hemagglutinin and pertactin) and major toxin (pertussis toxin). All of these factors are antigens that stimulate a protective immune response in the murine respiratory model and in clinical assays. In the present study, we examined the protective efficacies of these factors, and that of adenylate cyclase-hemolysin, another B. pertussis toxin, against B. parapertussis infection in a murine respiratory model. As expected, pertussis toxin did not protect against B. parapertussis infection, since this bacterium did not express this protein, but the surprising result was that none of the other factors were protective against B. parapertussis infection. Furthermore, B. parapertussis adenylate cyclase-hemolysin, although it protected against B. parapertussis infection, did not protect against B. pertussis infection. Despite a high degree of homology between both B. pertussis and B. parapertussis species, no cross-protection was observed. Our results outline the fact that, as in other gram-negative bacteria, Bordetella surface proteins vary immunologically.

Both adenylate cyclase and hemolytic activities are required by Bordetella pertussis to initiate infection.

Among virulence factors synthesized and secreted by Bordetella pertussis, pertussis toxin (PTX) and the bifunctional adenylate cyclase-hemolysin (AC-Hly) are able to invade mammalian cells and to impair intracellular functions. Moreover, both proteins are protective antigens in murine intracerebral and respiratory models. In order to study their in vivo properties, different B. pertussis mutants, deficient in AC-Hly expression or secretion, or producing modified AC-Hly devoid of either adenylate cyclase or hemolytic activities, were constructed and examined. The in vivo properties of the mutants were compared to PTX deficient strains, using the murine respiratory model. We show that lack of PTX as well as adenylate cyclase or hemolytic activities results in avirulence. Furthermore, we show that mutants lacking adenylate cyclase or hemolytic activities were unable to multiply as fast as the parental strains and PTX mutants during the first 5 days following infection. Thus, both adenylate cyclase and hemolytic activities are required by B. pertussis to initiate infection.