Reconstitution of a minimal machinery capable of assembling periplasmic type IV pili.

Type IV pili (Tfp), which are key virulence factors in many bacterial pathogens, define a large group of multipurpose filamentous nanomachines widespread in Bacteria and Archaea. Tfp biogenesis is a complex multistep process, which relies on macromolecular assemblies composed of 15 conserved proteins in model gram-negative species. To improve our limited understanding of the molecular mechanisms of filament assembly, we have used a synthetic biology approach to reconstitute, in a nonnative heterologous host, a minimal machinery capable of building Tfp. Here we show that eight synthetic genes are sufficient to promote filament assembly and that the corresponding proteins form a macromolecular complex at the cytoplasmic membrane, which we have purified and characterized biochemically. Our results contribute to a better mechanistic understanding of the assembly of remarkable dynamic filaments nearly ubiquitous in prokaryotes.

Neisseria meningitis GNA1030 is a ubiquinone-8 binding protein.

Bexsero, a new vaccine against Neisseria meningitidis serogroup B (MenB), is composed of 3 main recombinant proteins and an outer membrane vesicle component. One of the main bactericidal antigens, neisseria heparin binding antigen (NHBA), is present as a fusion protein with the accessory protein genome-derived neisserial antigen (GNA) 1030 to further increase its immunogenicity. The gene encoding for GNA1030 is present and highly conserved in all Neisseria strains, and although orthologs are present in numerous species, its biologic function is unknown. Native mass spectrometry was used to demonstrate that GNA1030 forms a homodimer associated with 2 molecules of ubiquinone-8 (Ub8), a cofactor mainly involved in the electron transport chain and with antioxidant properties. Disc diffusion assays on the wild-type and knockout mutant of GNA1030, in the presence of various compounds, suggested that GNA1030 is not involved in oxidative stress or electron chain transport per se, although it contributes to constitutive refilling of the inner membrane with Ub8. These studies shed light on an accessory protein present in Bexsero and reveal functional insights into the family of related proteins. On the basis of our findings, we propose to name the protein neisseria ubiquinone binding protein (NUbp).

NAD-dependent ADP-ribosylation of the human antimicrobial and immune-modulatory peptide LL-37 by ADP-ribosyltransferase-1.

LL-37 is a cationic peptide belonging to the cathelicidin family that has antimicrobial and immune-modulatory properties. Here we show that the mammalian mono-ADP-ribosyltransferase-1 (ART1), which selectively transfers the ADP-ribose moiety from NAD to arginine residues, ADP-ribosylates LL-37 in vitro. The incorporation of ADP-ribose was first observed by Western blot analysis and then confirmed by MALDI-TOF. Mass-spectrometry showed that up to four of the five arginine residues present in LL-37 could be ADP-ribosylated on the same peptide when incubated at a high NAD concentration in the presence of ART1. The attachment of negatively charged ADP-ribose moieties considerably alters the positive charge of the arginine residues thus reducing the cationicity of LL-37. The cationic nature of LL-37 is key for its ability to interact with cell membranes or negatively charged biomolecules, such as DNA, RNA, F-actin and glycosaminoglycans. Thus, the ADP-ribosylation of LL-37 is expected to have the potential to modulate LL-37 biological activities in several physiological and pathological settings.

Arginine-specific mono ADP-ribosylation in vitro of antimicrobial peptides by ADP-ribosylating toxins.

Among the several toxins used by pathogenic bacteria to target eukaryotic host cells, proteins that exert ADP-ribosylation activity represent a large and studied family of dangerous and potentially lethal toxins. These proteins alter cell physiology catalyzing the transfer of the ADP-ribose unit from NAD to cellular proteins involved in key metabolic pathways. In the present study, we tested the capability of four of these toxins, to ADP-ribosylate α- and ß- defensins. Cholera toxin (CT) from Vibrio cholerae and heat labile enterotoxin (LT) from Escherichia coli both modified the human α-defensin (HNP-1) and ß- defensin-1 (HBD1), as efficiently as the mammalian mono-ADP-ribosyltransferase-1. Pseudomonas aeruginosa exoenzyme S was inactive on both HNP-1 and HBD1. Neisseria meningitidis NarE poorly recognized HNP-1 as a substrate but it was completely inactive on HBD1. On the other hand, HNP-1 strongly influenced NarE inhibiting its transferase activity while enhancing auto-ADP-ribosylation. We conclude that only some arginine-specific ADP-ribosylating toxins recognize defensins as substrates in vitro. Modifications that alter the biological activities of antimicrobial peptides may be relevant for the innate immune response. In particular, ADP-ribosylation of antimicrobial peptides may represent a novel escape mechanism adopted by pathogens to facilitate colonization of host tissues.

Large-scale study of the interactions between proteins involved in type IV pilus biology in Neisseria meningitidis: characterization of a subcomplex involved in pilus assembly.

The functionally versatile type IV pili (Tfp) are one of the most widespread virulence factors in bacteria. However, despite generating much research interest for decades, the molecular mechanisms underpinning the various aspects of Tfp biology remain poorly understood, mainly because of the complexity of the system. In the human pathogen Neisseria meningitidis for example, 23 proteins are dedicated to Tfp biology, 15 of which are essential for pilus biogenesis. One of the important gaps in our knowledge concerns the topology of this multiprotein machinery. Here we have used a bacterial two-hybrid system to identify and quantify the interactions between 11 Pil proteins from N. meningitidis. We identified 20 different binary interactions, many of which are novel. This represents the most complex interaction network between Pil proteins reported to date and indicates, among other things, that PilE, PilM, PilN and PilO, which are involved in pilus assembly, indeed interact. We focused our efforts on this subset of proteins and used a battery of assays to determine the membrane topology of PilN and PilO, map the interaction domains between PilE, PilM, PilN and PilO, and show that a widely conserved N-terminal motif in PilN is essential for both PilM-PilN interactions and pilus assembly. Finally, we show that PilP (another protein involved in pilus assembly) forms a complex with PilM, PilN and PilO. Taken together, these findings have numerous implications for understanding Tfp biology and provide a useful blueprint for future studies.

Fatal disseminated toxoplasmosis in a cardiac transplantation with seropositive match for Toxoplasma: should prophylaxis be extended?

In cardiac transplant, toxoplasmosis in the immunocompromised recipient can result either from the transmission of the parasite from a seropositive donor (D+) to a seronegative recipient (R-) with the transplanted organ (more common) or from the reactivation of a pre-transplant latent infection (D-/R+ or D+/R+). In the immunocompromised patient, toxoplasmosis is a life-threatening disease. We report a case of disseminated toxoplasmosis following heart transplantation in a Toxoplasma seropositive recipient before transplantation (R+) (IgG 1:160, IgM negative) who received an organ from a Toxoplasma seropositive donor (D+) (IgG 1:640, IgM negative). No anti-Toxoplasma prophylactic treatment was administered. A number of complications arose in the postoperative period, as well as Enterobacter cloacae and Cytomegalovirus (CMV) (reactivation) infections, but neither serological nor histological toxoplasma recrudescence was evidenced. The patient died on post transplant day 41. Post-autopsy histological examinations revealed an unexpected diffuse toxoplasmosis (lungs, brain, heart).

Biological features (inflammation and neoangiogenesis) and atherosclerotic risk factors in carotid plaques and calcified aortic valve stenosis: two different sites of the same disease?

Neoangiogenesis and inflammation have a pivotal role in atherosclerosis. Observations support the hypothesis that calcified aortic valve stenosis is an inflammatory process, similar to atherosclerosis in tissue features and risk factors. We studied 2 groups of cases: 47 were affected by hemodynamic atherosclerotic carotid plaque (group 1) and 35 by severe calcified aortic valve stenosis (group 2). We compared the groups for atherosclerosis risk factors, morphologic features, and immunohistochemical phenotypes. In both groups, men, smokers, and hypertensive subjects prevailed, and histologic analysis showed an elevated score for T-lymphocyte infiltrates, neoangiogenesis, calcium, and sclerosis. Adhesion molecule expression was present in both lesions. Expression of intercellular adhesion molecule 1 correlated with inflammatory infiltrates (group 1, P = .0007; group 2, P = .06). Neoangiogenesis also correlated with inflammatory infiltrates (group 1, P = .035; group 2, P = .045). In valves, neoangiogenesis correlated with calcium (P = .048). Carotid plaque and calcified valve stenosis showed common risk factors and biologic hallmarks of a chronic inflammatory process. Inflammation and neoangiogenesis have a crucial role in plaque evolution and in the progression of aortic valve stenosis.

Expression of matrix metalloproteinases, their tissue inhibitors, and osteopontin in the wall of thoracic and abdominal aortas with dilatative pathology.

Matrix metalloproteinases (MMPs) degrade extracellular matrix and may play a central role in the pathogenesis of aortic aneurysms. We studied 2 groups of patients: 15 with dilatative pathology of the ascending thoracic aorta and 17 with aneurysm of the abdominal aortic wall (AAA). We compared the expression of MMPs, tissue inhibitors of matrix metalloproteinases (TIMPs), and osteopontin in the wall of thoracic and abdominal aneurysms. In AAA, MMP-9 and TIMP-1 expression in inflammatory cells was higher than in smooth muscle cells (SMCs) (median score: 3.5 versus 1, P < .0001; 2 versus 1, P < .04, respectively), whereas MMP-2 demonstrated higher expression in SMCs than in inflammatory cells (median score: 0 versus 4, P < .0001). In ATA, MMP-2, MMP-9, TIMP-1, TIMP-2, TIMP-3, and osteopontin expression in SMCs was higher than in inflammatory cells (median score: 3 versus 0, P < .0001; 4 versus 1, P < .0005; 2 versus 0, P < .001; 5 versus 2, P < .0001; 2 versus 0, P < .005; and 5 versus 1.5, P < .0001, respectively), when both inflammatory cells of the media and the adventitia were considered together. The cellular expression of MMP-9 and their tissue inhibitors TIMP-1, TIMP-2, and TIMP-3 differs in the dilatative pathology of abdominal and thoracic aortas, so the hypothetical model of morphogenesis of AAA cannot completely explain the formation of dilatative pathology of the ascending thoracic aorta.