Filamentous phages belong to Inoviruses, a family of non-lytic phages that are mutually beneficial to their bacterial hosts. These phages have a role in bacterial dissemination, biofilm formation or immune evasion. In Neisseria meningitidis, the strains harbouring the MDA filamentous phage are associated with invasive diseases through the MDAΦ key role in epithelial cell colonisation. Using MDAΦ and N. meningitidis as model organisms, we aimed to understand the relationship between filamentous phage infection, type IV pili antigenic variation and bacterial colonisation. The paradigm of filamentous phage infection has been defined for the phages Ff and CTX, where binding to the tips of bacterial type IV pili is a means of infecting their bacterial host. In contrast, we showed that MDAΦ binds N. meningitidis type IV pili along the length of the filament, rather than at the tip, with preferential binding to positively charged variants of PilE (the major fibre-forming pilin) demonstrating a role for antigenic variation in phage infection. Strikingly, bacteria expressing the more positively charged PilE were the most adhesive, meaning that MDAΦ primarily target the most adhesive bacteria. Finally, we showed that adhesion to human cells is sufficient to amplify the phage-positive meningococcal population. Taken together, this study reveals how the propagation strategy of a filamentous phage, aimed at selecting the best coloniser as a host, can promote the selection of hyperadhesive bacterial variants, linking phage infection to bacterial virulence.

Filamentous phages are non-lytic phages mutually beneficial to their bacterial hosts. In Neisseria meningitidis, the filamentous phage MDA is associated with invasive diseases thanks to its key role in the formation of biofilm during epithelium colonisation. The infection model for filamentous phages has been defined for phages Ff and CTX. These phages bind to the tips of bacterial pili before being translocated into the periplasm of their hosts. The aim of this study is to investigate the relationship between filamentous phage infection and type IV pili, using the bacterium Neisseria meningitidis and the bacteriophage MDA as model organisms. We show that MDAΦ rather binds to type IV pili along their entire length with preferential binding to positively charged variants of the major fibre-forming pilin, demonstrating a role for antigenic variation in phage infection. Strikingly, bacteria expressing the more positively charged pilin are also the most adhesive, suggesting that MDAΦ primarily target the most adhesive bacteria. Finally, we show that adhesion to human cells is sufficient to amplify the phage-positive meningococcal population. Overall, this study reveals how a filamentous phage can target hyperadhesive bacterial variants and promote their selection, thereby establishing a link between phage infection and bacterial colonisation.

Dedicator of cytokinesis (DOCK) proteins play a central role in actin cytoskeleton regu-lation. This is highlighted by the DOCK2 and DOCK8 deficiencies leading to actinopathies and immune deficiencies. DOCK8 and DOCK11 activate CDC42, a Rho-guanosine triphosphate hydrolases involved in actin cytoskeleton dynamics, among many cellular functions. The role of DOCK11 in human immune disease has been long suspected but, to the best of our knowledge, has never been described to date. We studied 8 male patients, from 7 unrelated families, with hemizygous DOCK11 missense variants leading to reduced DOCK11 expression. The patients were presenting with early-onset autoim-munity, including cytopenia, systemic lupus erythematosus, skin, and digestive manifes-tations. Patients' platelets exhibited abnormal ultrastructural morphology and spreading as well as impaired CDC42 activity. In vitro activated T cells and B-lymphoblastoid cell lines from patients exhibited aberrant protrusions and abnormal migration speed in confined channels concomitant with altered actin polymeri-zation during migration. Knock down of DOCK11 recapitulated these abnormal cellular phenotypes in monocytes-derived dendritic cells and primary activated T cells from healthy controls. Lastly, in line with the patients' autoim-mune manifestations, we also observed abnormal regulatory T-cell (Treg) phenotype with profoundly reduced FOXP3 and IKZF2 expression. Moreover, we found reduced T-cell proliferation and impaired STAT5B phosphorylation upon interleukin-2 stimulation of the patients' lymphocytes. In conclusion, DOCK11 deficiency is a new X-linked immune -related actinopathy leading to impaired CDC42 activity and STAT5 activation, and is associated with abnormal actin cytoskeleton remodeling as well as Treg phenotype, culminating in immune dysregulation and severe early-onset autoimmunity.

Variants in the UNC45A cochaperone have been recently associated with a syndrome combining diarrhea, cholestasis, deafness, and bone fragility. Yet the mechanism underlying intestinal failure in UNC45A deficiency remains unclear. Here, biallelic variants in UNC45A were identified by next-generation sequencing in 6 patients with congenital diarrhea. Corroborating in silico prediction, variants either abolished UNC45A expression or altered protein conformation. Myosin VB was identified by mass spectrometry as client of the UNC45A chaperone and was found misfolded in UNC45AKO Caco-2 cells. In keeping with impaired myosin VB function, UNC45AKO Caco-2 cells showed abnormal epithelial morphogenesis that was restored by full-length UNC45A, but not by mutant alleles. Patients and UNC45AKO 3D organoids displayed altered luminal development and microvillus inclusions, while 2D cultures revealed Rab11 and apical transporter mislocalization as well as sparse and disorganized microvilli. All those features resembled the subcellular abnormalities observed in duodenal biopsies from patients with microvillus inclusion disease. Finally, microvillus inclusions and shortened microvilli were evidenced in enterocytes from unc45a-deficient zebrafish. Taken together, our results provide evidence that UNC45A plays an essential role in epithelial morphogenesis through its cochaperone function of myosin VB and that UNC45A loss causes a variant of microvillus inclusion disease.

Mitochondria are dynamic organelles playing essential metabolic and signaling functions in cells. Their ultrastructure has largely been investigated with electron microscopy (EM) techniques, which provided a wide range of information on how mitochondria acquire a tissue-specific shape, how they change during development, and how they are altered in disease conditions. However, quantifying protein-protein proximities using EM is extremely challenging. Super-resolution microscopy techniques as direct stochastic optical reconstruction microscopy (dSTORM) now provide a fluorescent-based alternative to EM with a higher quantitative throughput. Recently, super-resolution microscopy approaches including dSTORM led to valuable advances in our knowledge of mitochondrial ultrastructure, and in linking it with new insights in organelle functions. Nevertheless, dSTORM is currently used to image integral mitochondrial proteins only, and there is little or no information on proteins transiently present at this compartment. The cancer-related Aurora kinase A/AURKA is a protein localized at various subcellular locations, including mitochondria. After performing dSTORM, we here use the Geo-coPositioning System (GcoPS) image analysis method to quantify the degree of colocalization of AURKA with compartment-specific mitochondrial markers. We show that two-color dSTORM provides sufficient spatial resolution to visualize AURKA in the mitochondrial matrix. We conclude by demonstrating that optimizing fixation procedures is a key step to follow AURKA in the matrix. In this light, we show that a methanol-based fixation leads to a better detection of the matrix pool of AURKA than an aldehyde-based fixation. Our results indicate that dSTORM coupled to GcoPS colocalization analysis is a suitable approach to explore the compartmentalization of non-integral mitochondrial proteins as AURKA, in a qualitative and quantitative manner. This method also opens up the possibility of analyzing the proximity between AURKA and its multiple mitochondrial partners with exquisite spatial resolution, thereby allowing novel insights into the mitochondrial functions controlled by AURKA.

Bacterial virulence factors are attractive targets for the development of therapeutics. Type IV pili, which are associated with a remarkable array of properties including motility, the interaction between bacteria and attachment to biotic and abiotic surfaces, represent particularly appealing virulence factor targets. Type IV pili are present in numerous bacterial species and are critical for their pathogenesis. In this study, we report that trifluoperazine and related phenothiazines block functions associated with Type IV pili in different bacterial pathogens, by affecting piliation within minutes. Using Neisseria meningitidis as a paradigm of Gram-negative bacterial pathogens that require Type IV pili for pathogenesis, we show that piliation is sensitive to altered activity of the Na+ pumping NADH-ubiquinone oxidoreductase (Na+-NQR) complex and that these compounds probably altered the establishment of the sodium gradient. In vivo, these compounds exert a strong protective effect. They reduce meningococcal colonization of the human vessels and prevent subsequent vascular dysfunctions, intravascular coagulation and overwhelming inflammation, the hallmarks of invasive meningococcal infections. Finally, they reduce lethality. This work provides a proof of concept that compounds with activity against bacterial Type IV pili could beneficially participate in the treatment of infections caused by Type IV pilus-expressing bacteria.

Neisseria meningitidis (meningococcus) is an invasive bacterial pathogen that colonizes human vessels, causing thrombotic lesions and meningitis. Establishment of tight interactions with endothelial cells is crucial for meningococci to resist haemodynamic forces. Two endothelial receptors, CD147 and the β2-adrenergic receptor (β 2 AR), are sequentially engaged by meningococci to adhere and promote signalling events leading to vascular colonization, but their spatiotemporal coordination is unknown. Here we report that CD147 and β 2 AR form constitutive hetero-oligomeric complexes. The scaffolding protein α-actinin-4 directly binds to the cytosolic tail of CD147 and governs the assembly of CD147–β 2 AR complexes in highly ordered clusters at bacterial adhesion sites. This multimolecular assembly process increases the binding strength of meningococci to endothelial cells under shear stress, and creates molecular platforms for the elongation of membrane protrusions surrounding adherent bacteria. Thus, the specific organization of cellular receptors has major impacts on host–pathogen interaction.

Hepcidin, a circulating regulatory hormone peptide produced by hepatocytes, functions as the master regulator of cellular iron export by controlling the amount of ferroportin, an iron exporter present on the basolateral surface of intestinal enterocytes and macrophages. Hepcidin binding to ferroportin induces its internalization and degradation, resulting in cellular iron retention and decreased iron export. Whether hepatocytes express ferroportin that could be targeted by hepcidin has remained a subject of debate. Here, we describe a hepatocyte culture system expressing high levels of ferroportin, and demonstrate that both endogenously secreted and synthetic hepcidin are fully active in down-regulating membrane-associated ferroportin. In agreement with this result, ferroportin is stabilized in liver hepatocytes of hepcidin-deficient mice and accumulates in periportal areas, supporting the centrolobular iron deposition observed in these mice. In conclusion, we show that hepcidin can trigger ferroportin degradation in hepatocytes, which must be taken into account when considering hepcidin therapeutics.

Background/Aims: The iron-regulatory peptide hepcidin is synthesized in the liver as an 84-aa pre-pro-hormone maturated by proteolysis through a consensus furin cleavage site to generate the bioactive 25-aa peptide secreted in the circulation. This peptide regulates iron export from enterocytes and macrophages by binding the membrane iron exporter, ferroportin, leading to its degradation. Whether pro-hepcidin could be secreted and reflect hepcidin levels remains an open question. However, the activity of the pro-peptide on ferroportin degradation has never been addressed.Methods: To answer this question, we produced recombinant pro-hepcidin, both the wild-type form and a furin cleavage site mutant, and tested their activity on ferroportin levels in macrophagic J774 cells. Furin activity was also modulated using furin inhibitor or siRNA-mediated furin mRNA knockdown.Results: We found that pro-hepcidin could fully induce ferroportin degradation, but only when processed by furin to generate the mature hepcidin-25 form. Pro-hepcidin activity was abolished in the presence of furin inhibitor and diminished after siRNA-mediated knockdown of furin mRNA. Furthermore, the mutated version of pro-hepcidin was completely inefficient at degrading ferroportin in macrophages.Conclusions: Our results demonstrate that pro-hepcidin lacks biological activity, unless fully maturated by a furin-dependent process to yield the bioactive 25-aa peptide. (C) 2008 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

We have generated transgenic mouse lines expressing exclusively a human INS transgene on an Ins1/Ins2 double knockout (mIKO) background.The transgene expression was driven by either a 4000 bp or a 353 bp promoter.These transgenic lines, designated mIKO:INS 4000 and mIKO:INS 353 , were viable and fertile.Determination of the amounts of insulin transcripts and total pancreatic insulin content revealed relative insulin underproduction in both lines, from birth to adulthood.Total pancreatic insulin stores in mIKO:INS 4000 and mIKO:INS 353 mice represented only about 50% and 27%, respectively, as compared to wild-type mice.Morphometric analysis of pancreas did not show any compensatory betacell hyperplasia.The majority of animals in both lines remained normoglycemic throughout their lives.Nevertheless, glucose tolerance tests revealed glucose intolerance in nearly half of mIKO:INS 4000 male mice, likely due to impaired insulin secretion detected in those animals.In addition, a small fraction (2-4%) of male mice in both lines spontaneously developed diabetes with very distinct pathophysiological features.Diabetes was never seen in female animals.The diabetes developed by mIKO:INS 353 mice was rapidly lethal, accompanied by a dramatic depletion of pancreatic insulin stores whereas the mIKO:INS 4000 diabetic animals could live for several months.This suggests a possible link between the structure of the human INS gene promoter and the type of diabetes developed in these lines.

The Serum Response Factor (SRF) is widely expressed transcription factor acting at the confluence of multiple signaling pathways and has been implicated in the control of differentiation, growth, and cell death. In the present study, we found that SRF is expressed in the developing and adult pancreas. To explore the possible role of SRF in this organ, we have generated mutant mice with conditional disruption of the Srf gene. Such mutants presented normal development of both the exocrine and endocrine pancreas indicating that SRF is dispensable for pancreas ontogenesis. However, after weaning, these mice developed profound morphological alterations of the exocrine pancreas, which were reminiscent of severe pancreatitis. In these mice, massive acinar injury, Nuclear Factor Kappa B activation and proinflammatory cytokines release led to complete destruction of the exocrine pancreas and its replacement by adipose tissue. Despite these changes, the organization and function of the endocrine islets of Langerhans remained well-preserved. This new animal model of spontaneous pancreatitis could prove a valuable tool to gain further insight into the physiopathology of this disease.

The authors have derived a new beta-cell line (betaIns2(-/-lacZ)) from Ins2-/- mice that carry the lacZ reporter gene under control of the Ins2 promoter. betaIns2(-/-lacZ) cells stained positively using anti-insulin antibody, expressed beta-cell-specific genes encoding the transcription factor PDX-1, glucokinase, and Glut-2, retained glucose-responsiveness for insulin secretion, and expressed the lacZ gene. Analysis of Ins1 expression by reverse transcriptase-polymerase chain reaction (RT-PCR) showed that Ins1 transcripts were significantly raised to compensate for the lack of Ins2 transcripts in betaIns2(-/-lacZ) cells, as compared to those found in betaTC1 cells expressing both Ins1/Ins2. Thus, transcriptional up-regulation of the remaining functional insulin gene in Ins2-/- mice could potentially contribute to the beta-cell adaptation exhibited by these mutants, in addition to the increase in beta-cell mass that we previously reported. We have also shown that lacZ expression, as analyzed by determining beta-galactosidase activity, was up-regulated by incubating betaIns2(-/-lacZ) cells with GLP-1 and/or IBMX, 2 known stimulators of insulin gene expression. These cells thus represent a new tool for testing of molecules capable of stimulating Ins2 promoter activity.