Genomic erosion and horizontal gene transfer shape functional differences of the ExlA toxin in Pseudomonas spp.

Two-partner secretion (TPS) is widespread in the bacterial world. The pore-forming TPS toxin ExlA of Pseudomonas aeruginosa is conserved in pathogenic and environmental Pseudomonas. While P. chlororaphis and P. entomophila displayed ExlA-dependent killing, P. putida did not cause damage to eukaryotic cells. ExlA proteins interacted with epithelial cell membranes; however, only ExlA Pch induced the cleavage of the adhesive molecule E-cadherin. ExlA proteins participated in insecticidal activity toward the larvae of Galleria mellonella and the fly Drosophila melanogaster. Evolutionary analyses demonstrated that the differences in the C-terminal domains are partly due to horizontal movements of the operon within the genus Pseudomonas. Reconstruction of the evolutionary history revealed the complex horizontal acquisitions. Together, our results provide evidence that conserved TPS toxins in environmental Pseudomonas play a role in bacteria-insect interactions and discrete differences in CTDs may determine their specificity and mode of action toward eukaryotic cells.

ExlA Pore-Forming Toxin: Localization at the Bacterial Membrane, Regulation of Secretion by Cyclic-Di-GMP, and Detection In Vivo.

ExlA is a highly virulent pore-forming toxin that has been recently discovered in outlier strains from Pseudomonas aeruginosa. ExlA is part of a two-partner secretion system, in which ExlA is the secreted passenger protein and ExlB the transporter embedded in the bacterial outer membrane. In previous work, we observed that ExlA toxicity in a host cell was contact-dependent. Here, we show that ExlA accumulates at specific points of the outer membrane, is likely entrapped within ExlB pore, and is pointing outside. We further demonstrate that ExlA is maintained at the membrane in conditions where the intracellular content of second messenger cyclic-di-GMP is high; lowering c-di-GMP levels enhances ExlB-dependent ExlA secretion. In addition, we set up an ELISA to detect ExlA, and we show that ExlA is poorly secreted in liquid culture, while it is highly detectable in broncho-alveolar lavage fluids of mice infected with an exlA+ strain. We conclude that ExlA translocation is halted at mid-length in the outer membrane and its secretion is regulated by c-di-GMP. In addition, we developed an immunological test able to quantify ExlA in biological samples.

The bacterial toxin ExoU requires a host trafficking chaperone for transportation and to induce necrosis.

Pseudomonas aeruginosa can cause nosocomial infections, especially in ventilated or cystic fibrosis patients. Highly pathogenic isolates express the phospholipase ExoU, an effector of the type III secretion system that acts on plasma membrane lipids, causing membrane rupture and host cell necrosis. Here, we use a genome-wide screen to discover that ExoU requires DNAJC5, a host chaperone, for its necrotic activity. DNAJC5 is known to participate in an unconventional secretory pathway for misfolded proteins involving anterograde vesicular trafficking. We show that DNAJC5-deficient human cells, or Drosophila flies knocked-down for the DNAJC5 orthologue, are largely resistant to ExoU-dependent virulence. ExoU colocalizes with DNAJC5-positive vesicles in the host cytoplasm. DNAJC5 mutations preventing vesicle trafficking (previously identified in adult neuronal ceroid lipofuscinosis, a human congenital disease) inhibit ExoU-dependent cell lysis. Our results suggest that, once injected into the host cytoplasm, ExoU docks to DNAJC5-positive secretory vesicles to reach the plasma membrane, where it can exert its phospholipase activity.

Inflammasome activation by Pseudomonas aeruginosa's ExlA pore-forming toxin is detrimental for the host.

During acute Pseudomonas aeruginosa infection, the inflammatory response is essential for bacterial clearance. Neutrophil recruitment can be initiated following the assembly of an inflammasome within sentinel macrophages, leading to activation of caspase-1, which in turn triggers macrophage pyroptosis and IL-1ß/IL-18 maturation. Inflammasome formation can be induced by a number of bacterial determinants, including Type III secretion systems (T3SSs) or pore-forming toxins, or, alternatively, by lipopolysaccharide (LPS) via caspase-11 activation. Surprisingly, previous studies indicated that a T3SS-induced inflammasome increased pathogenicity in mouse models of P. aeruginosa infection. Here, we investigated the immune reaction of mice infected with a T3SS-negative P. aeruginosa strain (IHMA879472). Virulence of this strain relies on ExlA, a secreted pore-forming toxin. IHMA879472 promoted massive neutrophil infiltration in infected lungs, owing to efficient priming of toll-like receptors, and thus enhanced the expression of inflammatory proteins including pro-IL-1ß and TNF-α. However, mature-IL-1ß and IL-18 were undetectable in wild-type mice, suggesting that ExlA failed to effectively activate caspase-1. Nevertheless, caspase-1/11 deficiency improved survival following infection with IHMA879472, as previously described for T3SS+ bacteria. We conclude that the detrimental effect associated with the ExlA-induced inflammasome is probably not due to hyperinflammation, rather it stems from another inflammasome-dependent process.

Structural and Functional Characterization of the Type Three Secretion System (T3SS) Needle of Pseudomonas aeruginosa.

The type three secretion system (T3SS) is a macromolecular protein nano-syringe used by different bacterial pathogens to inject effectors into host cells. The extracellular part of the syringe is a needle-like filament formed by the polymerization of a 9-kDa protein whose structure and proper localization on the bacterial surface are key determinants for efficient toxin injection. Here, we combined in vivo, in vitro, and in silico approaches to characterize the Pseudomonas aeruginosa T3SS needle and its major component PscF. Using a combination of mutagenesis, phenotypic analyses, immunofluorescence, proteolysis, mass spectrometry, atomic force microscopy, electron microscopy, and molecular modeling, we propose a model of the P. aeruginosa needle that exposes the N-terminal region of each PscF monomer toward the outside of the filament, while the core of the fiber is formed by the C-terminal helix. Among mutations introduced into the needle protein PscF, D76A, and P47A/Q54A caused a defect in the assembly of the needle on the bacterial surface, although the double mutant was still cytotoxic on macrophages in a T3SS-dependent manner and formed filamentous structures in vitro. These results suggest that the T3SS needle of P. aeruginosa displays an architecture that is similar to that of other bacterial needles studied to date and highlight the fact that small, targeted perturbations in needle assembly can inhibit T3SS function. Therefore, the T3SS needle represents an excellent drug target for small molecules acting as virulence blockers that could disrupt pathogenesis of a broad range of bacteria.

Functional Consequences of Calcium Influx Promoted by Bacterial Pore-Forming Toxins.

Bacterial pore-forming toxins induce a rapid and massive increase in cytosolic Ca2+ concentration due to the formation of pores in the plasma membrane and/or activation of Ca2+-channels. As Ca2+ is an essential messenger in cellular signaling, a sustained increase in Ca2+ concentration has dramatic consequences on cellular behavior, eventually leading to cell death. However, host cells have adapted mechanisms to protect against Ca2+ intoxication, such as Ca2+ efflux and membrane repair. The final outcome depends upon the nature and concentration of the toxin and on the cell type. This review highlights the repercussions of Ca2+ overload on the induction of cell death, repair mechanisms, cellular adhesive properties, and the inflammatory response.

CLIQ-BID: A method to quantify bacteria-induced damage to eukaryotic cells by automated live-imaging of bright nuclei.

Pathogenic bacteria induce eukaryotic cell damage which range from discrete modifications of signalling pathways, to morphological alterations and even to cell death. Accurate quantitative detection of these events is necessary for studying host-pathogen interactions and for developing strategies to protect host organisms from bacterial infections. Investigation of morphological changes is cumbersome and not adapted to high-throughput and kinetics measurements. Here, we describe a simple and cost-effective method based on automated analysis of live cells with stained nuclei, which allows real-time quantification of bacteria-induced eukaryotic cell damage at single-cell resolution. We demonstrate that this automated high-throughput microscopy approach permits screening of libraries composed of interference-RNA, bacterial strains, antibodies and chemical compounds in ex vivo infection settings. The use of fluorescently-labelled bacteria enables the concomitant detection of changes in bacterial growth. Using this method named CLIQ-BID (Cell Live Imaging Quantification of Bacteria Induced Damage), we were able to distinguish the virulence profiles of different pathogenic bacterial species and clinical strains.

Pseudomonas aeruginosa ExlA and Serratia marcescens ShlA trigger cadherin cleavage by promoting calcium influx and ADAM10 activation.

Pore-forming toxins are potent virulence factors secreted by a large array of bacteria. Here, we deciphered the action of ExlA from Pseudomonas aeruginosa and ShlA from Serratia marcescens on host cell-cell junctions. ExlA and ShlA are two members of a unique family of pore-forming toxins secreted by a two-component secretion system. Bacteria secreting either toxin induced an ExlA- or ShlA-dependent rapid cleavage of E-cadherin and VE-cadherin in epithelial and endothelial cells, respectively. Cadherin proteolysis was executed by ADAM10, a host cell transmembrane metalloprotease. ADAM10 activation is controlled in the host cell by cytosolic Ca2+ concentration. We show that Ca2+ influx, induced by ExlA or ShlA pore formation in the plasma membrane, triggered ADAM10 activation, thereby leading to cadherin cleavage. Our data suggest that ADAM10 is not a cellular receptor for ExlA and ShlA, further confirming that ADAM10 activation occurred via Ca2+ signalling. In conclusion, ExlA- and ShlA-secreting bacteria subvert a regulation mechanism of ADAM10 to activate cadherin shedding, inducing intercellular junction rupture, cell rounding and loss of tissue barrier integrity.

Pseudomonas aeruginosa Exolysin promotes bacterial growth in lungs, alveolar damage and bacterial dissemination.

Exolysin (ExlA) is a recently-identified pore-forming toxin secreted by a subset of Pseudomonas aeruginosa strains identified worldwide and devoid of Type III secretion system (T3SS), a major virulence factor. Here, we characterized at the ultrastructural level the lesions caused by an ExlA-secreting strain, CLJ1, in mouse infected lungs. CLJ1 induced necrotic lesions in pneumocytes and endothelial cells, resulting in alveolo-vascular barrier breakdown. Ectopic expression of ExlA in an exlA-negative strain induced similar tissue injuries. In addition, ExlA conferred on bacteria the capacity to proliferate in lungs and to disseminate in secondary organs, similar to bacteria possessing a functional T3SS. CLJ1 did not promote a strong neutrophil infiltration in the alveoli, owing to the weak pro-inflammatory cytokine reaction engendered by the strain. However, CLJ1 was rapidly eliminated from the blood in a bacteremia model, suggesting that it can be promptly phagocytosed by immune cells. Together, our study ascribes to ExlA-secreting bacteria the capacity to proliferate in the lung and to damage pulmonary tissues, thereby promoting metastatic infections, in absence of substantial immune response exacerbation.

Chronic Treatment with Minoxidil Induces Elastic Fiber Neosynthesis and Functional Improvement in the Aorta of Aged Mice.

Normal arterial aging processes involve vascular cell dysfunction associated with wall stiffening, the latter being due to progressive elastin and elastic fiber degradation, and elastin and collagen cross-linking by advanced glycation end products (AGEs). These processes progressively lead to cardiovascular dysfunction during aging. Elastin is only synthesized during late gestation and childhood, and further degradation occurring throughout adulthood cannot be physiologically compensated by replacement of altered material. However, the ATP-dependent K+ channel opener minoxidil has been shown to stimulate elastin expression in vitro and in vivo in the aorta of young adult rats. Therefore, we have studied the effect of a 10-week chronic oral treatment with minoxidil (120 mg/L in drinking water) on the aortic structure and function in aged 24-month-old mice. Minoxidil treatment increased tropoelastin, fibulin-5, and lysyl-oxidase messenger RNA levels, reinduced a moderate expression of elastin, and lowered the levels of AGE-related molecules. This was accompanied by the formation of newly synthesized elastic fibers, which had diverse orientations in the wall. A decrease in the glycation capacity of aortic elastin was also produced by minoxidil treatment. The ascending aorta also underwent a minoxidil-induced increase in diameter and decrease in wall thickness, which partly reversed the age-associated thickening and returned the wall thickness value and strain-stress relation closer to those of younger adult animals. In conclusion, our results suggest that minoxidil presents an interesting potential for arterial remodeling in an antiaging perspective, even when treating already aged animals.

Pseudomonas aeruginosa Pore-Forming Exolysin and Type IV Pili Cooperate To Induce Host Cell Lysis.

Clinical strains of Pseudomonas aeruginosa lacking the type III secretion system genes employ a toxin, exolysin (ExlA), for host cell membrane disruption. Here, we demonstrated that ExlA export requires a predicted outer membrane protein, ExlB, showing that ExlA and ExlB define a new active two-partner secretion (TPS) system of P. aeruginosa In addition to the TPS signals, ExlA harbors several distinct domains, which include one hemagglutinin domain, five arginine-glycine-aspartic acid (RGD) motifs, and a C-terminal region lacking any identifiable sequence motifs. However, this C-terminal region is important for the toxic activity, since its deletion abolishes host cell lysis. Using lipid vesicles and eukaryotic cells, including red blood cells, we demonstrated that ExlA has a pore-forming activity which precedes cell membrane disruption of nucleated cells. Finally, we developed a high-throughput cell-based live-dead assay and used it to screen a transposon mutant library of an ExlA-producing P. aeruginosa clinical strain for bacterial factors required for ExlA-mediated toxicity. The screen resulted in the identification of proteins involved in the formation of type IV pili as being required for ExlA to exert its cytotoxic activity by promoting close contact between bacteria and the host cell. These findings represent the first example of cooperation between a pore-forming toxin of the TPS family and surface appendages in host cell intoxication. IMPORTANCE: The course and outcome of acute, toxigenic infections by Pseudomonas aeruginosa clinical isolates rely on the deployment of one of two virulence strategies: delivery of effectors by the well-known type III secretion system or the cytolytic activity of the recently identified two-partner secreted toxin, exolysin. Here, we characterize several features of the mammalian cell intoxication process mediated by exolysin. We found that exolysin requires the outer membrane protein ExlB for export into extracellular medium. Using in vitro recombinant protein and ex vivo assays, we demonstrated a pore-forming activity of exolysin. A cellular cytotoxicity screen of a transposon mutant library, made in an exolysin-producing clinical strain, identified type IV pili as bacterial appendages required for exolysin toxic function. This work deciphers molecular mechanisms underlying the activity of novel virulence factors used by P. aeruginosa clinical strains lacking the type III secretion system, including a requirement for the toxin-producing bacteria to be attached to the targeted cell to induce cytolysis, and defines new targets for developing antivirulence strategies.

Phenotype and toxicity of the recently discovered exlA-positive Pseudomonas aeruginosa strains collected worldwide.

We recently identified a hypervirulent strain of Pseudomonas aeruginosa, differing significantly from the classical strains in that it lacks the type 3 secretion system (T3SS), a major determinant of P. aeruginosa virulence. This new strain secretes a novel toxin, called ExlA, which induces plasma membrane rupture in host cells. For this study, we collected 18 other exlA-positive T3SS-negative strains, analyzed their main virulence factors and tested their toxicity in various models. Phylogenetic analysis revealed two groups. The strains were isolated on five continents from patients with various pathologies or in the environment. Their proteolytic activity and their motion abilities were highly different, as well as their capacity to infect epithelial, endothelial, fibroblastic and immune cells, which correlated directly with ExlA secretion levels. In contrast, their toxicity towards human erythrocytes was limited. Some strains were hypervirulent in a mouse pneumonia model and others on chicory leaves. We conclude that (i) exlA-positive strains can colonize different habitats and may induce various infection types, (ii) the strains secreting significant amounts of ExlA are cytotoxic for most cell types but are poorly hemolytic, (iii) toxicity in planta does not correlate with ExlA secretion.

Pseudomonas aeruginosa Transmigrates at Epithelial Cell-Cell Junctions, Exploiting Sites of Cell Division and Senescent Cell Extrusion.

To achieve systemic infection, bacterial pathogens must overcome the critical and challenging step of transmigration across epithelial barriers. This is particularly true for opportunistic pathogens such as Pseudomonas aeruginosa, an agent which causes nosocomial infections. Despite extensive study, details on the mechanisms used by this bacterium to transmigrate across epithelial tissues, as well as the entry sites it uses, remain speculative. Here, using real-time microscopy and a model epithelial barrier, we show that P. aeruginosa employs a paracellular transmigration route, taking advantage of altered cell-cell junctions at sites of cell division or when senescent cells are expelled from the cell layer. Once a bacterium transmigrates, it is followed by a cohort of bacteria using the same entry point. The basal compartment is then invaded radially from the initial penetration site. Effective transmigration and propagation require type 4 pili, the type 3 secretion system (T3SS) and a flagellum, although flagellum-deficient bacteria can occasionally invade the basal compartment from wounded areas. In the basal compartment, the bacteria inject the T3SS toxins into host cells, disrupting the cytoskeleton and focal contacts to allow their progression under the cells. Thus, P. aeruginosa exploits intrinsic host cell processes to breach the epithelium and invade the subcellular compartment.

ExsB is required for correct assembly of the Pseudomonas aeruginosa type III secretion apparatus in the bacterial membrane and full virulence in vivo.

Pseudomonas aeruginosa is responsible for high-morbidity infections of cystic fibrosis patients and is a major agent of nosocomial infections. One of its most potent virulence factors is a type III secretion system (T3SS) that injects toxins directly into the host cell cytoplasm. ExsB, a lipoprotein localized in the bacterial outer membrane, is one of the components of this machinery, of which the function remained elusive until now. The localization of the exsB gene within the exsCEBA regulatory gene operon suggested an implication in the T3SS regulation, while its similarity with yscW from Yersinia spp. argued in favor of a role in machinery assembly. The present work shows that ExsB is necessary for full in vivo virulence of P. aeruginosa. Furthermore, the requirement of ExsB for optimal T3SS assembly and activity is demonstrated using eukaryotic cell infection and in vitro assays. In particular, ExsB promotes the assembly of the T3SS secretin in the bacterial outer membrane, highlighting the molecular role of ExsB as a pilotin. This involvement in the regulation of the T3S apparatus assembly may explain the localization of the ExsB-encoding gene within the regulatory gene operon.

Pharmacological activation of Rap1 antagonizes the endothelial barrier disruption induced by exotoxins ExoS and ExoT of Pseudomonas aeruginosa.

Most clinical strains of Pseudomonas aeruginosa, a leading agent of nosocomial infections, are multiresistant to antibiotherapy. Because of the paucity of new available antibiotics, the investigation of strategies aimed at limiting the action of its major virulence factors has gained much interest. The type 3 secretion system of P. aeruginosa and its effectors are known to be major determinants of toxicity and are required for bacterial dissemination in the host. Bacterial transmigration across the vascular wall is considered to be an important step in the infectious process. Using human endothelial primary cells, we demonstrate that forskolin (FSK), a drug inducing cyclic AMP (cAMP) elevation in eukaryotic cells, strikingly reduced the cell retraction provoked by two type 3 toxins, ExoS and ExoT, found in the majority of clinical strains. Conversely, cytotoxicity of a strain carrying the type 3 effector ExoU was unaffected by FSK. In addition, FSK altered the capacity of two ExoS/ExoT strains to transmigrate across cell monolayers. In agreement with these findings, other drugs and a cytokine inducing the increase of cAMP intracellular levels have also protected cells from retraction. cAMP is an activator of both protein kinase A and EPAC, a GTPase exchange factor of Rap1. Using activators or inhibitors of either pathway, we show that the beneficial effect of FSK is exerted by the activation of the EPAC/Rap1 axis, suggesting that its protective effect is mediated by reinforcing cell-cell and cell-substrate adhesion.

VE-cadherin cleavage by LasB protease from Pseudomonas aeruginosa facilitates type III secretion system toxicity in endothelial cells.

Infection of the vascular system by Pseudomonas aeruginosa (Pa) occurs during bacterial dissemination in the body or in blood-borne infections. Type 3 secretion system (T3SS) toxins from Pa induce a massive retraction when injected into endothelial cells. Here, we addressed the role of type 2 secretion system (T2SS) effectors in this process. Mutants with an inactive T2SS were much less effective than wild-type strains at inducing cell retraction. Furthermore, secretomes from wild-types were sufficient to trigger cell-cell junction opening when applied to cells, while T2SS-inactivated mutants had minimal activity. Intoxication was associated with decreased levels of vascular endothelial (VE)-cadherin, a homophilic adhesive protein located at endothelial cell-cell junctions. During the process, the protein was cleaved in the middle of its extracellular domain (positions 335 and 349). VE-cadherin attrition was T3SS-independent but T2SS-dependent. Interestingly, the epithelial (E)-cadherin was unaffected by T2SS effectors, indicating that this mechanism is specific to endothelial cells. We showed that one of the T2SS effectors, the protease LasB, directly affected VE-cadherin proteolysis, hence promoting cell-cell junction disruption. Furthermore, mouse infection with Pa to induce acute pneumonia lead to significant decreases in lung VE-cadherin levels, whereas the decrease was minimal with T2SS-inactivated or LasB-deleted mutant strains. We conclude that the T2SS plays a pivotal role during Pa infection of the vascular system by breaching the endothelial barrier, and propose a model in which the T2SS and the T3SS cooperate to intoxicate endothelial cells.

A type III secretion negative clinical strain of Pseudomonas aeruginosa employs a two-partner secreted exolysin to induce hemorrhagic pneumonia.

Virulence of Pseudomonas aeruginosa is typically attributed to its type III secretion system (T3SS). A taxonomic outlier, the P. aeruginosa PA7 strain, lacks a T3SS locus, and no virulence phenotype is attributed to PA7. We characterized a PA7-related, T3SS-negative P. aeruginosa strain, CLJ1, isolated from a patient with fatal hemorrhagic pneumonia. CLJ1 is highly virulent in mice, leading to lung hemorrhage and septicemia. CLJ1-infected primary endothelial cells display characteristics of membrane damage and permeabilization. Proteomic analysis of CLJ1 culture supernatants identified a hemolysin/hemagglutinin family pore-forming toxin, Exolysin (ExlA), that is exported via ExlB, representing a putative two-partner secretion system. A recombinant P. aeruginosa PAO1ΔpscD::exlBA strain, deficient for T3SS but engineered to express ExlA, gained lytic capacity on endothelial cells and full virulence in mice, demonstrating that ExlA is necessary and sufficient for pathogenicity. This highlights clinically relevant T3SS-independent hypervirulence, isolates, and points to a broader P. aeruginosa pathogenic repertoire.

Structural basis of cytotoxicity mediated by the type III secretion toxin ExoU from Pseudomonas aeruginosa.

The type III secretion system (T3SS) is a complex macromolecular machinery employed by a number of Gram-negative pathogens to inject effectors directly into the cytoplasm of eukaryotic cells. ExoU from the opportunistic pathogen Pseudomonas aeruginosa is one of the most aggressive toxins injected by a T3SS, leading to rapid cell necrosis. Here we report the crystal structure of ExoU in complex with its chaperone, SpcU. ExoU folds into membrane-binding, bridging, and phospholipase domains. SpcU maintains the N-terminus of ExoU in an unfolded state, required for secretion. The phospholipase domain carries an embedded catalytic site whose position within ExoU does not permit direct interaction with the bilayer, which suggests that ExoU must undergo a conformational rearrangement in order to access lipids within the target membrane. The bridging domain connects catalytic domain and membrane-binding domains, the latter of which displays specificity to PI(4,5)P2. Both transfection experiments and infection of eukaryotic cells with ExoU-secreting bacteria show that ExoU ubiquitination results in its co-localization with endosomal markers. This could reflect an attempt of the infected cell to target ExoU for degradation in order to protect itself from its aggressive cytotoxic action.

Fibrillin-1 genetic deficiency leads to pathological ageing of arteries in mice.

Fibrillin-1, the major component of extracellular microfibrils that associate with insoluble elastin in elastic fibres, is mainly synthesized during development and postnatal growth and is believed to guide elastogenesis. Mutations in the fibrillin-1 gene cause Marfan syndrome, a multisystem disorder characterized by aortic aneurysms and dissections. The recent finding that early deficiency of elastin modifies vascular ageing has raised the possibility that fibrillin-1 deficiency could also contribute to late-onset pathology of vascular remodelling. To address this question, we examined cardiovascular function in 3-week-old, 6-month-old, and 24-month-old mice that are heterozygous for a hypomorphic structural mutation of fibrillin-1 (Fbn1{+/mgΔ} mice). Our results indicate that Fbn1{+/mgΔ} mice, particularly those that are 24 months old, are slightly more hypotensive than wild-type littermates. Additionally, aneurysm and aortic insufficiency were more frequently observed in ageing Fbn1{+/mgΔ}$ mice than in the wild-type counterparts. We also noted substantial fragmentation and decreased number of elastic lamellae in the aortic wall of Fbn1{+/mgΔ} mice, which were correlated with an increase in aortic stiffness, a decrease in vasoreactivity, altered expression of elastic fibre-related genes, including fibrillin-1 and elastin, and a decrease in the relative ratio between tissue elastin and collagen. Collectively, our findings suggest that the heterozygous mgΔ mutation accelerates some aspects of vascular ageing and eventually leads to aortic manifestations resembling those of Marfan syndrome. Importantly, our data also indicate that vascular abnormalities in Fbn1{+/mgΔ} mice are opposite to those induced by elastin haploinsufficiency during ageing that affect blood pressure, vascular dimensions, and number of elastic lamellae.

Protocadherin-12 cleavage is a regulated process mediated by ADAM10 protein: evidence of shedding up-regulation in pre-eclampsia.

Protocadherins are a group of transmembrane proteins with homophilic binding activity, members of the cadherin superfamily. Apart from their role in adhesion, the cellular functions of protocadherins are essentially unknown. Protocadherin (PCDH)12 was previously identified in invasive trophoblasts and endothelial and mesangial cells in the mouse. Invalidation studies revealed that the protein was required for optimal placental development. In this article, we show that its human homolog is abundantly expressed in various trophoblast subtypes of the human placenta and at lower levels in endothelial cells. We demonstrate that PCDH12 is shed at high rates in vitro. The shedding mechanism depends on ADAM10 and results in reduced cellular adhesion in a cell migration assay. PCDH12 is subsequently cleaved by the γ-secretase complex, and its cytoplasmic domain is rapidly degraded by the proteasome. PCDH12 shedding is regulated by interlinked intracellular pathways, including those involving protein kinase C, PI3K, and cAMP, that either increase or inhibit cleavage. In endothelial cells, VEGF, prostaglandin E(2), or histamine regulates PCDH12 shedding. The extracellular domain of PCDH12 was also detected in human serum and urine, thus providing evidence of PCDH12 shedding in vivo. Importantly, we observed an increase in circulating PCDH12 in pregnant women who later developed a pre-eclampsia, a frequent pregnancy syndrome and a major cause of maternal and fetal morbidity and mortality. In conclusion, we speculate that, like in mice, PCDH12 may play an important role in human placental development and that proteolytic cleavage in response to external factors, such as cytokines and pathological settings, regulates its activity.