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Supplementary Figure from Escherichia coli–Specific CXCL13-Producing TFH Are Associated with Clinical Efficacy of Neoadjuvant PD-1 Blockade against Muscle-Invasive Bladder Cancer

Biomarkers guiding the neoadjuvant use of immune-checkpoint blockers (ICB) are needed for patients with localized muscle-invasive bladder cancers (MIBC). Profiling tumor and blood samples, we found that follicular helper CD4+ T cells (TFH) are among the best therapeutic targets of pembrolizumab correlating with progression-free survival. TFH were associated with tumoral CD8 and PD-L1 expression at baseline and the induction of tertiary lymphoid structures after pembrolizumab. Blood central memory TFH accumulated in tumors where they produce CXCL13, a chemokine found in the plasma of responders only. IgG4+CD38+ TFH residing in bladder tissues correlated with clinical benefit. Finally, TFH and IgG directed against urothelium-invasive Escherichia coli dictated clinical responses to pembrolizumab in three independent cohorts. The links between tumor infection and success of ICB immunomodulation should be prospectively assessed at a larger scale.Significance:In patients with bladder cancer treated with neoadjuvant pembrolizumab, E. coli–specific CXCL13 producing TFH and IgG constitute biomarkers that predict clinical benefit. Beyond its role as a biomarker, such immune responses against E. coli might be harnessed for future therapeutic strategies.This article is highlighted in the In This Issue feature, p. 2221

BackgroundDrug-induced QT prolongation (diLQT) is a feared side-effect as exposing susceptible individuals to fatal arrhythmias. The occurrence of diLQT is primarily attributed to unintended drug interactions with cardiac ion channels, notably the hERG channels that generate the repolarizing current (IKr) and thereby regulate the late repolarization phase. There is an important inter-individual susceptibility to develop diLQT which is of unknown origin but can be reproduced in patient-specific iPSC-derived cardiomyocytes (iPS-CMs).ObjectiveWe aimed to investigate the dynamics of hERG channels in response to sotalol and to identify regulators of the susceptibility to developing diLQT.MethodsWe measured electrophysiological activity and cellular distribution of hERG channels after hERG blocker treatment in iPS-CMs derived from patients with highest or lowest sensitivity (HS or LS) to sotalol administrationin vivo(i.e., based on the measure of the maximal change in QT interval 3 hours after administration). Specific small-interfering RNAs (siRNA) and CAVIN1-T2A-GFP adenovirus were used to manipulateCAVIN1expression.ResultsWhile HS and LS iPS-CMs showed similar electrophysiological characteristics at the baseline, the late repolarization phase was prolonged, and IKrsignificantly decreased after exposure of HS iPS-CMs to low sotalol concentrations. IKrreduction was caused by a rapid translocation of hERG channel from the plasma membrane to the cytoskeleton upon sotalol application. This phenomenon was suppressed by blocking active endocytosis using dynasore.CAVIN1, essential for caveolae biogenesis, was two-times more expressed in HS iPS-CMs and its knockdown using siRNA decreased their sensitivity to sotalol.CAVIN1overexpression in LS iPS-CMs using adenovirus showed reciprocal effects. Mechanistically, we found that treatment with sotalol promoted trafficking of the hERG channel from the plasma membrane to the cytoskeleton through caveolae and in a manner dependent on CAVIN1 expression.CAVIN1silencing reduced the number of caveolae at the membrane and abrogated the internalization of hERG channel in sotalol-treated HS iPS-CMs. CAVIN1 also controlled cardiomyocyte responses to other hERG blockers such as E4031, vandetanib, and clarithromycin.ConclusionsOur study identifies unbridled turnover of the potassium channel hERG as a mechanism supporting the inter-individual susceptibility underlying diLQT development and demonstrates how this phenomenon is finely tuned by CAVIN1.CLINICAL PERSPECTIVEWhat is new?-The inter-individual susceptibility underlying diLQT development involves unbridled turnover of cardiac ion channels from the plasma membrane.-This phenomenon is finely tuned by CAVIN1, a protein that is essential for essential for caveolae biogenesis.-Treatment with hERG blocker promoted trafficking of the hERG channel from the plasma membrane to the cytoskeleton through caveolae and in a manner dependent on CAVIN1 expression.What are the clinical implications?-While congenital long QT is primarily from a genetic origin, the development of drug-induced long QT involves differences in the trafficking machinery of cardiac ion channels.-The prediction of CAVIN1 expression levels could help preventing drug-induced cardiotoxicity.

While establishing an invasive infection, the dormant conidia of Aspergillus fumigatus transit through swollen and germinating stages, to form hyphae. During this morphotype transition, the conidial cell wall undergoes dynamic remodeling, which poses challenges to the host immune system and antifungal drugs. However, such cell wall reorganization during conidial germination has not been studied so far. Here, we explored the molecular rearrangement of Aspergillus fumigatus cell wall polysaccharides during different stages of germination. We took advantage of magic-angle spinning NMR to investigate the cell wall polysaccharides, without employing any destructive method for sample preparation. The breaking of dormancy was associated with a significant change in the molar ratio between the major polysaccharides β-1,3-glucan and α-1,3-glucan, while chitin remained equally abundant. The use of various polarization transfers allowed the detection of rigid and mobile polysaccharides; the appearance of mobile galactosaminogalactan was a molecular hallmark of germinating conidia. We also report for the first time highly abundant triglyceride lipids in the mobile matrix of conidial cell walls. Water to polysaccharides polarization transfers revealed an increased surface exposure of glucans during germination, while chitin remained embedded deeper in the cell wall, suggesting a molecular compensation mechanism to keep the cell wall rigidity. We complement the NMR analysis with confocal and atomic force microscopies to explore the role of melanin and RodA hydrophobin on the dormant conidial surface. Exemplified here using Aspergillus fumigatus as a model, our approach provides a powerful tool to decipher the molecular remodeling of fungal cell walls during their morphotype switching.

International audience; Cilia are unique eukaryotic organelles and exhibit remarkable conservation across evolution. Nevertheless, very different types of configurations are encountered, raising the question of their evolution. Cilia are constructed by intraflagellar transport (IFT), the movement of large protein complexes or trains that deliver cilia components to the distal tip for assembly. Recent data revealed that IFT trains are restricted to some but not all nine doublet microtubules in the protist Trypanosoma brucei. Here, we propose that restricted positioning of IFT trains could offer potent options for cilia to evolve towards more complex (addition of new structural elements like in spermatozoa) or simpler configuration (loss of some elements like in primary cilia), and therefore be a driver of cilia diversification. We present two hypotheses to explain how IFT trains could be restricted to some doublets, either by a triage process taking place at the basal body level or by the development of molecular differences between ciliary microtubules

Biomarkers guiding the neoadjuvant use of immune-checkpoint blockers (ICB) are needed for patients with localized muscle-invasive bladder cancers (MIBC). Profiling tumor and blood samples, we found that follicular helper CD4+ T cells (TFH) are among the best therapeutic targets of pembrolizumab correlating with progression-free survival. TFH were associated with tumoral CD8 and PD-L1 expression at baseline and the induction of tertiary lymphoid structures after pembrolizumab. Blood central memory TFH accumulated in tumors where they produce CXCL13, a chemokine found in the plasma of responders only. IgG4+CD38+ TFH residing in bladder tissues correlated with clinical benefit. Finally, TFH and IgG directed against urothelium-invasive Escherichia coli dictated clinical responses to pembrolizumab in three independent cohorts. The links between tumor infection and success of ICB immunomodulation should be prospectively assessed at a larger scale. Significance: In patients with bladder cancer treated with neoadjuvant pembrolizumab, E. coli–specific CXCL13 producing TFH and IgG constitute biomarkers that predict clinical benefit. Beyond its role as a biomarker, such immune responses against E. coli might be harnessed for future therapeutic strategies. This article is highlighted in the In This Issue feature, p. 2221

Collective transmission via structures containing several virions has recently emerged as a highly efficient mode of viral spread. Here, we demonstrate that HIV-1 spreads between T lymphocytes in the form of viral particles colonies that are concentrated and sheltered in an extracellular matrix (ECM) lattice enabling their collective transmission upon cell contacts. Intrinsically, ECM-clustered viruses infect T lymphocytes more efficiently than individual viral particles. They preserve HIV-1 transmission from antiretroviral treatment (ArT) and potent broadly neutralizing antibodies. We also show that collagen induced by HIV-1 infection controls the clustering of virions and their collective spread, thereby enhancing infectivity. CD4+ T cells from HIV-1-infected patients produce and transmit ECM-virus clusters, supporting that they could be involved in vivo. This study provides new insights into modes of HIV-1 transmission and identifies a novel fundamental role for collagen in this process. HIV-1 spread via ECM-virus clusters may have important implications for viral dissemination and persistence, including during therapy.

Understanding how SARS-CoV-2 spreads within the respiratory tract is important to define the parameters controlling the severity of COVID-19. We examined the functional and structural consequences of SARS-CoV-2 infection in a reconstituted human bronchial epithelium model. SARS-CoV-2 replication caused a transient decrease in epithelial barrier function and disruption of tight junctions, though viral particle crossing remained limited. Rather, SARS-CoV-2 replication led to a rapid loss of the ciliary layer, characterized at the ultrastructural level by axoneme loss and misorientation of remaining basal bodies. The motile cilia function was compromised, as measured in a mucociliary clearance assay. Epithelial defense mechanisms, including basal cell mobilization and interferon-lambda induction, ramped up only after the initiation of cilia damage. Analysis of SARS-CoV-2 infection in Syrian hamsters further demonstrated the loss of motile ciliain vivo. This study identifies cilia damage as a pathogenic mechanism that could facilitate SARS-CoV-2 spread to the deeper lung parenchyma.

Salmonella Typhimurium (S. Typhimurium) is an enteric bacterium capable of invading a wide range of hosts, including rodents and humans. It targets different host cell types showing different intracellular lifestyles. S. Typhimurium colonizes different intracellular niches and is able to either actively divide at various rates or remain dormant to persist. A comprehensive tool to determine these distinct S. Typhimurium lifestyles remains lacking. Here we developed a novel fluorescent reporter, Salmonella INtracellular Analyzer (SINA), compatible for fluorescence microscopy and flow cytometry in single-bacterium level quantification. This identified a S. Typhimurium subpopulation in infected epithelial cells that exhibits a unique phenotype in comparison to the previously documented vacuolar or cytosolic S. Typhimurium. This subpopulation entered a dormant state in a vesicular compartment distinct from the conventional Salmonella-containing vacuoles (SCV) as well as the previously reported niche of dormant S. Typhimurium in macrophages. The dormant S. Typhimurium inside enterocytes were viable and expressed Salmonella Pathogenicity Island 2 (SPI-2) virulence factors at later time points. We found that the formation of these dormant S. Typhimurium is not triggered by the loss of SPI-2 effector secretion but it is regulated by (p)ppGpp-mediated stringent response through RelA and SpoT. We predict that intraepithelial dormant S. Typhimurium represents an important pathogen niche and provides an alternative strategy for S. Typhimurium pathogenicity and its persistence., Author summary Salmonella Typhimurium is a clinically relevant bacterial pathogen that causes Salmonellosis. It can actively or passively invade various host cell types and reside in a Salmonella-containing vacuole (SCV) within host cells. The SCV can be remodeled into a replicative niche with the aid of Salmonella Type III Secretion System 2 (T3SS2) effectors or else, the SCV is ruptured for the access of the nutrient-rich host cytosol. Depending on the infected host cell type, S. Typhimurium undertake different lifestyles that are distinct by their subcellular localization, replication rate and metabolic rate. We present here a novel fluorescent reporter system that rapidly detects S. Typhimurium lifestyles using fluorescence microscopy and flow cytometry. We identified a dormant S. Typhimurium population within enterocyte that displays capacities in host cell persistence, dormancy exit and antibiotic tolerance. We deciphered the (p)ppGpp stringent response pathway that suppresses S. Typhimurium dormancy in enterocytes while promoting dormancy in macrophages, pinpointing a divergent physiological consequence regulated by the same set of S. Typhimurium molecular mediators. Altogether, our work demonstrated the potential of fluorescent reporters in facile bacterial characterization, and revealed a dormant S. Typhimurium population in human enterocytes that are phenotypically distinct from that observed in macrophages and fibroblasts.

The mitochondrion is crucial for ATP generation by oxidative phosphorylation, among other processes. Cristae are invaginations of the mitochondrial inner membrane that house nearly all the macromolecular complexes that perform oxidative phosphorylation. The unicellular parasite Trypanosoma brucei undergoes during its life cycle extensive remodeling of its single mitochondrion, which reflects major changes in its energy metabolism. While the bloodstream form (BSF) generates ATP exclusively by substrate-level phosphorylation and has a morphologically highly reduced mitochondrion, the insect-dwelling procyclic form (PCF) performs oxidative phosphorylation and has an expanded and reticulated organelle. Here, we have performed high-resolution 3D reconstruction of BSF and PCF mitochondria, with a particular focus on their cristae. By measuring the volumes and surface areas of these structures in complete or nearly complete cells, we have found that mitochondrial cristae are more prominent in BSF than previously thought and their biogenesis seems to be maintained during the cell cycle. Furthermore, PCF cristae exhibit a surprising range of volumes in situ, implying that each crista is acting as an independent bioenergetic unit. Cristae appear to be particularly enriched in the region of the organelle between the nucleus and kinetoplast, the mitochondrial genome, suggesting this part has distinctive properties., SCOPUS: ar.j, info:eu-repo/semantics/published

International audience; Serum osteocalcin was measured in patients with idiopathic hypoparathyroidism or pseudohypoparathyroidism, before or during the treatment with active vitamin D3 (1,25(OH)2D3 or 1 alpha OHD3). Serum osteocalcin and plasma 1,25(OH)2D were decreased in 11 patients with idiopathic hypoparathyroidism before treatment (2.8 +/- 1.27 ng/ml, P less than 0.001 and 14.3 +/- 4.27 pg/ml, P less than 0.001, respectively). In 24 patients with idiopathic hypoparathyroidism during the treatment, serum osteocalcin and plasma 1,25(OH)2D were within the normal range (4.5 +/- 0.74 ng/ml and 25.7 +/- 5.69 pg/ml, respectively). In five patients with pseudohypoparathyroidism before treatment, plasma 1,25(OH)2D was decreased (15.6 +/- 10.6 pg/ml, P less than 0.001) but serum osteocalcin was normal (7.8 +/- 1.66 ng/ml). In nine patients with pseudohypoparathyroidism during the treatment with active vitamin D3, serum osteocalcin and plasma 1,25(OH)2D were normal (6.8 +/- 1.47 ng/ml and 27.2 +/- 6.0 pg/ml, respectively). Serum PTH in pseudohypoparathyroidism was increased before treatment (0.70 +/- 0.34 ng/ml, P less than 0.05) and was normal during the treatment (0.50 +/- 0.13 ng/ml). In idiopathic hypoparathyroidism, the active vitamin D3 increased serum osteocalcin without PTH. In pseudohypoparathyroidism, PTH may increase serum osteocalcin or modulate the effect of active vitamin D3 on serum osteocalcin.

Biochemical processes often require spatial regulation and specific microenvironments. The general lack of organelles in bacteria limits the potential of bioengineering complex intracellular reactions. Here, we demonstrate synthetic membraneless organelles in Escherichia coli termed transcriptionally engineered addressable RNA solvent droplets (TEARS). TEARS are assembled from RNA-binding protein recruiting domains fused to poly-CAG repeats that spontaneously drive liquid-liquid phase separation from the bulk cytoplasm. Targeting TEARS with fluorescent proteins revealed multilayered structures with composition and reaction robustness governed by non-equilibrium dynamics. We show that TEARS provide organelle-like bioprocess isolation for sequestering biochemical pathways, controlling metabolic branch points, buffering mRNA translation rates, and scaffolding protein-protein interactions. We anticipate TEARS to be a simple and versatile tool for spatially controlling E. coli biochemistry. Particularly, the modular design of TEARS enables applications without expression fine-tuning, simplifying the design-build-test cycle of bioengineering.

Biochemical processes often require spatial regulation and specific microenvironments. The general lack of organelles in bacteria limits the potential of bioengineering complex intracellular reactions. Here we demonstrate Transcriptionally Engineered Addressable RNA Solvent droplets (TEARS) as synthetic microdomains within theEscherichia coli.TEARS are assembled from RNA-binding protein recruitment domains fused to poly-CAG repeats that spontaneously drive liquid-liquid phase separation from the bulk cytoplasm. Targeting TEARS with fluorescent proteins revealed multilayered structures and a non-equilibrium mechanism controlling their composition and reaction robustness. We show that TEARS provide organelle-like bioprocess isolation for sequestering biochemical pathways, controlling metabolic branch points, buffering mRNA translation rates and scaffolding protein-protein interactions. TEARS are a simple and versatile tool for spatially controllingE. colibiochemistry.

Multicellular organisms assemble cilia and flagella of precise lengths differing from one cell to another, yet little is known about the mechanisms governing these differences. Similarly, protists assemble flagella of different lengths according to the stage of their life cycle. This is the case ofTrypanosoma bruceithat assembles flagella of 3 to 30 µm during its development in the tsetse fly. It provides an opportunity to examine how cells naturally modulate organelle length. Flagella are constructed by addition of new blocks at their distal end via intraflagellar transport (IFT). Immunofluorescence assays, 3-D electron microscopy and live cell imaging revealed that IFT was present in all life cycle stages. IFT proteins are concentrated at the base, IFT trains are located along doublets 3-4 & 7-8 and travel bidirectionally in the flagellum. Quantitative analysis demonstrated that the total amount of IFT proteins correlates with the length of the flagellum. Surprisingly, the shortest flagellum exhibited a supplementary large amount of dynamic IFT material at its distal end. The contribution of IFT and other factors to the regulation of flagellum length is discussed.Summary statementThis work investigated the assembly of flagella of different length during the development ofTrypanosoma bruceiin the tsetse fly, revealing a direct correlation between the amount of intraflagellar transport proteins and flagellum length.

Multicellular organisms assemble cilia and flagella of precise lengths differing from one cell to another, yet little is known about the mechanisms governing these differences. Similarly, protists assemble flagella of different lengths according to the stage of their life cycle.

Trypanosoma bruceiexhibits a complex life cycle alternating between tsetse flies and mammalian hosts. When parasites infect the fly, cells differentiate to adapt to life in various tissues, which is accompanied by drastic morphological and biochemical modifications especially in the proventriculus. This key step represents a bottleneck for salivary gland infection. Here we monitored flagellum assembly in trypanosomes during differentiation from the trypomastigote to the epimastigote stage, i.e. when the nucleus migrates to the posterior end of the cell. Three-dimensional electron microscopy (Focused Ion Bean Scanning Electron Microscopy, FIB-SEM) and immunofluorescence assays provided structural and molecular evidence that the new flagellum is assembled while the nucleus migrates towards the posterior region of the body. Two major differences with well known procyclic cells are reported. First, growth of the new flagellum begins when the associated basal body is found in a posterior position relative to the mature one. Second, the new flagellum acquires its own flagellar pocket before rotating on the left side of the anterior-posterior axis. FIB-SEM revealed the presence of a structure connecting the new and mature flagellum and serial sectioning confirmed morphological similarities with the flagella connector of procyclic cells. We discuss potential function of the flagella connector in trypanosomes from the proventriculus. These findings show thatT. bruceifinely modulates its cytoskeletal components to generate highly variable morphologies.Author SummaryTrypanosoma bruceiis a flagellated parasitic protist that causes human African trypanosomiasis, or sleeping sickness and that is transmitted by the bite of tsetse flies. The complex life cycle ofT. bruceiinside the tsetse digestive tract requires adaptation to specific organs and follow a strictly defined order. It is marked by morphological modifications in cell shape and size, as well organelle positioning. In the proventriculus of tsetse flies,T. bruceiundergoes a unique asymmetric division leading to two very different daughter cells: one with a short and one with a long flagellum. This organelle is crucial for the trypanosome life cycle as it is involved in motility, adhesion and morphogenesis. Here we investigated flagellum assembly using molecular and 3D Electron Microscopy approaches revealing that flagellum construction in proventricular trypanosomes is concomitant with parasite differentiation. During flagellum growth, the new flagellum is connected to the mature one and rotates around the mature one after its emergence at the cell surface. The sequence of events is different from what is observed in the well-studied procyclic stage in culture revealing different processes governing morphological development. These results highlight the importance to study pathogen development in their natural environment.

Aspergillus fumigatus, a ubiquitous human fungal pathogen, produces asexual spores (conidia), which are the main mode of propagation, survival and infection of this human pathogen. In this study, we present the molecular characterization of a novel regulator of conidiogenesis and conidial survival called MybA because the predicted protein contains a Myb DNA binding motif. Cellular localization of the MybA::Gfp fusion and immunoprecipitation of the MybA::Gfp or MybA::3xHa protein showed that MybA is localized to the nucleus. RNA sequencing data and a uidA reporter assay indicated that the MybA protein functions upstream of wetA, vosA and velB, the key regulators involved in conidial maturation. The deletion of mybA resulted in a very significant reduction in the number and viability of conidia. As a consequence, the ΔmybA strain has a reduced virulence in an experimental murine model of aspergillosis. RNA-sequencing and biochemical studies of the ΔmybA strain suggested that MybA protein controls the expression of enzymes involved in trehalose biosynthesis as well as other cell wall and membrane-associated proteins and ROS scavenging enzymes. In summary, MybA protein is a new key regulator of conidiogenesis and conidial maturation and survival, and plays a crucial role in propagation and virulence of A. fumigatus.

International audience; No abstract available

Initial adhesion of bacterial cells to surfaces or host tissues is a key step in colonisation and biofilm formation processes, and is mediated by cell surface appendages. It was previously demonstrated that Escherichia coli K-12 possesses an arsenal of silenced chaperone-usher fimbriae that were functional when constitutively expressed. Among them, production of prevalent Yad fimbriae induces adhesion to abiotic surfaces. Functional characterisation of Yad fimbriae were undertook, and YadN was identified as the most abundant and potential major pilin, and YadC as the potential tip-protein of Yad fimbriae. It was showed that Yad production participates to binding of E. coli K-12 to human eukaryotic cells (Caco-2) and inhibits macrophage phagocytosis, but also enhances E. coli K-12 binding to xylose, a major component of the plant cell wall, through its tip-lectin YadC. Consistently, it was demonstrated that Yad production provides E. coli with a competitive advantage in colonising corn seed rhizospheres. The latter phenotype is correlated with induction of Yad expression at temperatures below 37°C, and under anaerobic conditions, through a complex regulatory network. Taken together, these results suggest that Yad fimbriae are versatile adhesins that beyond potential capacities to modulate host-pathogen interactions might contribute to E. coli environmental persistence.

The shape of cellular membranes is highly regulated by a set of conserved mechanisms. These mechanisms can be manipulated by bacterial pathogens to infect cells. Human endothelial cell plasma membrane remodeling by the bacteriumNeisseria meningitidisis thought to be essential during the blood phase of meningococcal infection, but the underlying mechanisms are unknown. Here we show that plasma membrane remodeling occurs independently of F-actin, along meningococcal type IV pili fibers, by a novel physical mechanism we term “ onedimensional” membrane wetting. We provide a theoretical model that gives the physical basis of 1D wetting and show that this mechanism occurs in model membranes interacting with model nanofibers, and in human cells interacting with model extracellular matrices. It is thus a new general principle driving the interaction of cells with their environment at the nanoscale that is diverted by meningococcus during infection.

Intraflagellar transport (IFT) is the movement of large protein complexes responsible for the construction of cilia and flagella. Using a combination of three-dimensional electron microscopy and high-resolution live imaging, Bertiaux et al. show that IFT takes place on only four microtubule doublets out of the nine available in the trypanosome flagellum., Intraflagellar transport (IFT) is the rapid bidirectional movement of large protein complexes driven by kinesin and dynein motors along microtubule doublets of cilia and flagella. In this study, we used a combination of high-resolution electron and light microscopy to investigate how and where these IFT trains move within the flagellum of the protist Trypanosoma brucei. Focused ion beam scanning electron microscopy (FIB-SEM) analysis of trypanosomes showed that trains are found almost exclusively along two sets of doublets (3–4 and 7–8) and distribute in two categories according to their length. High-resolution live imaging of cells expressing mNeonGreen::IFT81 or GFP::IFT52 revealed for the first time IFT trafficking on two parallel lines within the flagellum. Anterograde and retrograde IFT occurs on each of these lines. At the distal end, a large individual anterograde IFT train is converted in several smaller retrograde trains in the space of 3–4 s while remaining on the same side of the axoneme.

Summary Salmonella invades epithelial cells and survives within a membrane-bound compartment, the Salmonella-containing vacuole (SCV). We isolated and determined the host protein composition of the SCV at 30 min and 3 h of infection to identify and characterize novel regulators of intracellular bacterial localization and growth. Quantitation of the SCV protein content revealed 392 host proteins specifically enriched at SCVs, out of which 173 associated exclusively with early SCVs, 124 with maturing SCV and 95 proteins during both time-points. Vacuole interactions with endoplasmic reticulum-derived coat protein complex II vesicles modulate early steps of SCV maturation, promoting SCV rupture and bacterial hyper-replication within the host cytosol. On the other hand, SCV interactions with VAMP7-positive lysosome-like vesicles promote Salmonella-induced filament formation and bacterial growth within the late SCV. Our results reveal that the dynamic communication between the SCV and distinct host organelles affects both intracellular Salmonella localization and growth at successive steps of host cell invasion.

Invasive bacterial pathogens often target cellular proteins involved in adhesion as a first event during infection. For example, Listeria monocytogenes uses the bacterial protein InlA to interact with E-cadherin, hijack the host adherens junction (AJ) machinery and invade non-phagocytic cells by a clathrin-dependent mechanism. Here, we investigate a potential role for clathrin in cell–cell adhesion. We observed that the initial steps of AJ formation trigger the phosphorylation of clathrin, and its transient localization at forming cell–cell contacts. Furthermore, we show that clathrin serves as a hub for the recruitment of proteins that are necessary for the actin rearrangements that accompany the maturation of AJs. Using an InlA/E-cadherin chimera, we show that adherent cells expressing the chimera form AJs with cells expressing E-cadherin. We demonstrate that non-adherent cells expressing the InlA chimera, as bacteria, can be internalized by E-cadherin-expressing adherent cells. Together these results reveal that a common clathrin-mediated machinery may regulate internalization and cell adhesion and that the relative mobility of one of the interacting partners plays an important role in the commitment to either one of these processes.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma and HTLV-1–associated myelopathy/tropical spastic paraparesis. In addition to blood transfusion and sexual transmission, HTLV-1 is transmitted mainly through prolonged breastfeeding, and such infection represents a major risk for the development of adult T-cell leukemia/lymphoma. Although HTLV-1–infected lymphocytes can be retrieved from maternal milk, the mechanisms of HTLV-1 transmission through the digestive tract remain unknown. In the present study, we assessed HTLV-1 transport across the epithelial barrier using an in vitro model. Our results show that the integrity of the epithelial barrier was maintained during coculture with HTLV-1–infected lymphocytes, because neither morphological nor functional alterations of the cell monolayer were observed. Enterocytes were not susceptible to HTLV-1 infection, but free infectious HTLV-1 virions could cross the epithelial barrier via a transcytosis mechanism. Such virions were able to infect productively human dendritic cells located beneath the epithelial barrier. Our data indicate that HTLV-1 crosses the tight epithelial barrier without disruption or infection of the epithelium to further infect target cells such as dendritic cells. The present study provides the first data pertaining to the mode of HTLV-1 transport across a tight epithelial barrier, as can occur during mother-to-child HTLV-1 transmission during breastfeeding.

Early events leading to the establishment of hepatitis C virus (HCV) infection are not completely understood. We show that intact and dynamic microtubules play a key role in the initiation of productive HCV infection. Microtubules were required for virus entry into cells, as evidenced using virus pseudotypes presenting HCV envelope proteins on their surface. Studies carried out using the recent infectious HCV model revealed that microtubules also play an essential role in early, postfusion steps of the virus cycle. Moreover, low concentrations of vinblastin and nocodazol, microtubule-affecting drugs, and paclitaxel, which stabilizes microtubules, inhibited infection, suggesting that microtubule dynamic instability and/or treadmilling mechanisms are involved in HCV internalization and early transport. By protein chip and direct core-dependent pull-down assays, followed by mass spectrometry, we identified β- and α-tubulin as cellular partners of the HCV core protein. Surface plasmon resonance analyses confirmed that core directly binds to tubulin with high affinity via amino acids 2-117. The interaction of core with tubulin in vitro promoted its polymerization and enhanced the formation of microtubules. Immune electron microscopy showed that HCV core associates, at least temporarily, with microtubules polymerized in its presence. Studies by confocal microscopy showed a juxtaposition of core with microtubules in HCV-infected cells. In summary, we report that intact and dynamic microtubules are required for virus entry into cells and for early postfusion steps of infection. HCV may exploit a direct interaction of core with tubulin, enhancing microtubule polymerization, to establish efficient infection and promote virus transport and/or assembly in infected cells.

International audience; Streptococcus agalactiae (or Group B Streptococcus, GBS) is a commensal bacterium present in the intestinal and urinary tracts of approximately 30% of humans. We and others previously showed that the PI-2a pilus polymers, made of the backbone pilin PilB, the tip adhesin PilA and the cell wall anchor protein PilC, promote adhesion to host epithelia and biofilm formation. Affinity-purified PI-2a pili from GBS strain NEM316 were recognized by N-acetylneuraminic acid (NeuNAc, also known as sialic acid) specific lectins such as Elderberry Bark Lectin (EBL) suggesting that pili are sialylated. Glycan profiling with twenty different lectins combined with monosaccharide composition by HPLC suggested that affinity-purified PI-2a pili are modified by N-glycosylation and decorated with sialic acid attached to terminal galactose. Analysis of various relevant mutants in the PI-2a pilus operon by flow-cytometry and electron microscopy analyses pointed to PilA as the pilus subunit modified by glycosylation. Double labeling using PilB antibody and EBL lectin, which specifically recognizes N-acetylneuraminic acid attached to galactose in α-2, 6, revealed a characteristic binding of EBL at the tip of the pilus structures, highly reminiscent of PilA localization. Expression of a secreted form of PilA using an inducible promoter showed that this recombinant PilA binds specifically to EBL lectin when produced in the native GBS context. In silico search for potentially glycosylated asparagine residues in PilA sequence pointed to N427 and N597, which appear conserved and exposed in the close homolog RrgA from S. pneumoniae, as likely candidates. Conversion of these two asparagyl residues to glutamyl resulted in a higher instability of PilA. Our results provide the first evidence that the tip PilA adhesin can be glycosylated, and suggest that this modification is critical for PilA stability and may potentially influence interactions with the host.

International audience; Group B Streptococcus (GBS) is an asymptomatic colonizer of human mucosal surfaces that is responsible for sepsis and meningitis in neonates. Bacterial persistence and pathogenesis often involves biofilm formation. We previously showed that biofilm formation in medium supplemented with glucose is mediated by the PI-2a pilus. Here, biofilm formation was tested in cell culture medium supplemented with human plasma. GBS strains were able to form biofilms in these conditions unlike Group A Streptococcus (GAS) or Staphylococcus aureus. Analysis of mutants impaired for various surface components revealed that the GBS capsule is a key component in this process.

Summary The interferon-induced transmembrane (IFITM) proteins protect cells from diverse virus infections by inhibiting virus-cell fusion. IFITM proteins also inhibit HIV-1 replication through mechanisms only partially understood. We show that when expressed in uninfected lymphocytes, IFITM proteins exert protective effects during cell-free virus infection, but this restriction can be overcome upon HIV-1 cell-to-cell spread. However, when present in virus-producing lymphocytes, IFITM proteins colocalize with viral Env and Gag proteins and incorporate into nascent HIV-1 virions to limit entry into new target cells. IFITM in viral membranes is associated with impaired virion fusion, offering additional and more potent defense against virus spread. Thus, IFITM proteins act additively in both productively infected cells and uninfected target cells to inhibit HIV-1 spread, potentially conferring these proteins with greater breadth and potency against enveloped viruses., Graphical Abstract, Highlights • IFITM proteins inhibit HIV spread from infected cells to new target cells • IFITM colocalize with viral Env and Gag proteins and incorporate into virions • When incorporated into virions, IFITM proteins reduce virus particle infectivity • IFITM in viral membranes impair virion fusion more potently than IFITM in cells, Interferon-induced transmembrane (IFITM) proteins are membrane-resident antiviral effectors, protecting cells against infection by diverse viruses. Compton et al. describe a function for IFITM proteins in which incorporation into the membrane of HIV-1 virions and inhibition of fusion between virus and target cell lead to a potent block of HIV-1 spread.

International audience; BACKGROUND: Streptococcus gallolyticus is a causative agent of infective endocarditis associated with colon cancer. Genome sequence of strain UCN34 revealed the existence of 3 pilus loci (pil1, pil2, and pil3). Pili are long filamentous structures playing a key role as adhesive organelles in many pathogens. The pil1 locus encodes 2 LPXTG proteins (Gallo2178 and Gallo2179) and 1 sortase C (Gallo2177). Gallo2179 displaying a functional collagen-binding domain was referred to as the adhesin, whereas Gallo2178 was designated as the major pilin. METHODS: S. gallolyticus UCN34, Pil1(+) and Pil1(-), expressing various levels of pil1, and recombinant Lactococcus lactis strains, constitutively expressing pil1, were studied. Polyclonal antibodies raised against the putative pilin subunits Gallo2178 and Gallo2179 were used in immunoblotting and immunogold electron microscopy. The role of pil1 was tested in a rat model of endocarditis. RESULTS: We showed that the pil1 locus (gallo2179-78-77) forms an operon differentially expressed among S. gallolyticus strains. Short pilus appendages were identified both on the surface of S. gallolyticus UCN34 and recombinant L. lactis-expressing pil1. We demonstrated that Pil1 pilus is involved in binding to collagen, biofilm formation, and virulence in experimental endocarditis. CONCLUSIONS: This study identifies Pil1 as the first virulence factor characterized in S. gallolyticus.

Clathrin assembles at bacterial adhesion sites and its phosphorylation is required for actin recruitment during bacterial infection., Bacterial pathogens recruit clathrin upon interaction with host surface receptors during infection. Here, using three different infection models, we observed that host–pathogen interactions induce tyrosine phosphorylation of clathrin heavy chain. This modification was critical for recruitment of actin at bacteria–host adhesion sites during bacterial internalization or pedestal formation. At the bacterial interface, clathrin assembled to form coated pits of conventional size. Because such structures cannot internalize large particles such as bacteria, we propose that during infection, clathrin-coated pits serve as platforms to initiate actin rearrangements at bacteria–host adhesion sites. We then showed that the clathrin–actin interdependency is initiated by Dab2 and depends on the presence of clathrin light chain and its actin-binding partner Hip1R, and that the fully assembled machinery can recruit Myosin VI. Together, our study highlights a physiological role for clathrin heavy chain phosphorylation and reinforces the increasingly recognized function of clathrin in actin cytoskeletal organization in mammalian cells.

Background Besides horizontal transmission, HTLV-1 is transmitted vertically mainly through prolonged breastfeeding. Maternal transmission via breast milk is the dominant mode of HTLV-1 spread in high endemic areas, and is inked to the presence of HTLV-1 infected cells (lymphocytes, epithelial cells..) in the milk. Infection in young childhood by such mean appears to be a major risk factor for development of ATL in adults. Materials and methods We developed an in vitro model of epithelial barrier (Caco-2 human enterocytic cell line) to assess the mode of passage of HTLV-1 through the digestive tract. Integrity of the barrier was checked by ultrastructural approach, measurement of the trans-epithelial resistance (TER), and diffusion of fluorescently labeled molecules. Results When enterocytes were co-cultured with HTLV-1infected lymphocytes, no structural modifications were detected in intercellular tight junctions. Moreover, the functional integrity of the epithelial barrier was maintained since no TER change was detected in the presence of infected lymphocytes, and passage of small fluorescent markers was unaffected. Although enterocytes were not found to be susceptible to HTLV-1 infection, free infectious HTLV-1 virions were detected in the basal compartment, and such a passage was temperaturedependent, suggesting a transcytotic mechanism of passage. When human dendritic cells were added to the basal compartment, they were found to be productively infected by HTLV-1 that had crossed the epithelial barrier.

International audience; The WalKR two-component system is essential for viability of Staphylococcus aureus, a major pathogen. We have shown that WalKR acts as the master controller of peptidoglycan metabolism, yet none of the identified regulon genes explain its requirement for cell viability. Transmission electron micrographs revealed cell wall thickening and aberrant division septa in the absence of WalKR, suggesting its requirement may be linked to its role in coordinating cell wall metabolism and cell division. We therefore tested whether uncoupling autolysin gene expression from WalKR-dependent regulation could compensate for its essential nature. Uncoupled expression of genes encoding lytic transglycosylases or amidases did not restore growth to a WalKR-depleted strain. We identified only two WalKR-regulon genes whose expression restored cell viability in the absence of WalKR: lytM and ssaA. Neither of these two genes are essential under our conditions and a ΔlytM ΔssaA mutant does not present any growth defect. LytM is a glycyl-glycyl endopeptidase, hydrolyzing the pentaglycine interpeptide crossbridge, and SsaA belongs to the CHAP amidase family, members of which such as LysK and LytA have been shown to have D-alanyl-glycyl endopeptidase activity, cleaving between the crossbridge and the stem peptide. Taken together, our results strongly suggest that peptidoglycan crosslinking relaxation through crossbridge hydrolysis plays a crucial role in the essential requirement of the WalKR system for cell viability.

International audience; Actin-based motility is used by various pathogens for dissemination within and between cells. Yet host factors restricting this process have not been identified. Septins are GTP-binding proteins that assemble as filaments and are essential for cell division. However, their role during interphase has remained elusive. Here, we report that septin assemblies are recruited to different bacteria that polymerize actin. We observed that intracytosolic Shigella either become compartmentalized in septin cage-like structures or form actin tails. Inactivation of septin caging increases the number of Shigella with actin tails and enhances cell-to-cell spread. TNF-α, a host cytokine produced upon Shigella infection, stimulates septin caging and restricts actin tail formation and cell-to-cell spread. Finally, we show that septin cages entrap bacteria targeted to autophagy. Together, these results reveal an unsuspected mechanism of host defense that restricts dissemination of invasive pathogens.

Streptococcus agalactiae is a common human commensal and a major life-threatening pathogen in neonates. Adherence to host epithelial cells is the first critical step of the infectious process. Pili have been observed on the surface of several gram-positive bacteria including S. agalactiae. We previously characterized the pilus-encoding operon gbs1479-1474 in strain NEM316. This pilus is composed of three structural subunit proteins: Gbs1478 (PilA), Gbs1477 (PilB), and Gbs1474 (PilC), and its assembly involves two class C sortases (SrtC3 and SrtC4). PilB, the bona fide pilin, is the major component; PilA, the pilus associated adhesin, and PilC, are both accessory proteins incorporated into the pilus backbone. We first addressed the role of the housekeeping sortase A in pilus biogenesis and showed that it is essential for the covalent anchoring of the pilus fiber to the peptidoglycan. We next aimed at understanding the role of the pilus fiber in bacterial adherence and at resolving the paradox of an adhesive but dispensable pilus. Combining immunoblotting and electron microscopy analyses, we showed that the PilB fiber is essential for efficient PilA display on the surface of the capsulated strain NEM316. We then demonstrated that pilus integrity becomes critical for adherence to respiratory epithelial cells under flow-conditions mimicking an in vivo situation and revealing the limitations of the commonly used static adherence model. Interestingly, PilA exhibits a von Willebrand adhesion domain (VWA) found in many extracellular eucaryotic proteins. We show here that the VWA domain of PilA is essential for its adhesive function, demonstrating for the first time the functionality of a prokaryotic VWA homolog. Furthermore, the auto aggregative phenotype of NEM316 observed in standing liquid culture was strongly reduced in all three individual pilus mutants. S. agalactiae strain NEM316 was able to form biofilm in microtiter plate and, strikingly, the PilA and PilB mutants were strongly impaired in biofilm formation. Surprisingly, the VWA domain involved in adherence to epithelial cells was not required for biofilm formation., Author Summary Streptococcus agalactiae (Group B Streptococcus) is a leading cause of sepsis (blood infection) and meningitis (brain infection) in newborns. Most bacterial pathogens have long filamentous structures known as pili or fimbriae, which are often involved in the initial adhesion of bacteria to host tissues but also in bacteria–bacteria interactions, resulting in biofilm formation. Our previous functional characterization of the pilus locus in S. agalactiae showed that it encodes a major pilin and two minor pilin subunits that are covalently polymerized by the action of two enzymes belonging to the sortase C family. One of the accessory pilins is responsible for the adhesive property of the pilus. However, this initial study raised two major questions that were addressed in the present work: i) what anchors the pilus to the cell wall and ii) what is the function of the pilus fiber itself. We showed that the pilus is essential for optimal display of the pilus-associated adhesin and overcomes the masking effect of the capsule. Pilus integrity was shown to be critical in adherence assays under flow conditions. We also report that GBS can form biofilms and that pili play an important role in this process.

Early events leading to the establishment of hepatitis C virus (HCV) infection are not completely understood. We show that intact and dynamic microtubules play a key role in the initiation of productive HCV infection. Microtubules were required for virus entry into cells, as evidenced using virus pseudotypes presenting HCV envelope proteins on their surface. Studies carried out using the recent infectious HCV model revealed that microtubules also play an essential role in early, postfusion steps of the virus cycle. Moreover, low concentrations of vinblastin and nocodazol, microtubule-affecting drugs, and paclitaxel, which stabilizes microtubules, inhibited infection, suggesting that microtubule dynamic instability and/or treadmilling mechanisms are involved in HCV internalization and early transport. By protein chip and direct core-dependent pull-down assays, followed by mass spectrometry, we identified β- and α-tubulin as cellular partners of the HCV core protein. Surface plasmon resonance analyses confirmed that core directly binds to tubulin with high affinity via amino acids 2-117. The interaction of core with tubulin in vitro promoted its polymerization and enhanced the formation of microtubules. Immune electron microscopy showed that HCV core associates, at least temporarily, with microtubules polymerized in its presence. Studies by confocal microscopy showed a juxtaposition of core with microtubules in HCV-infected cells. In summary, we report that intact and dynamic microtubules are required for virus entry into cells and for early postfusion steps of infection. HCV may exploit a direct interaction of core with tubulin, enhancing microtubule polymerization, to establish efficient infection and promote virus transport and/or assembly in infected cells.

We and others have uncovered the existence of human T-cell lymphotropic virus type 3 (HTLV-3). We have now generated an HTLV-3 proviral clone. We established that gag, env, pol, pro , and tax/rex as well as minus-strand mRNAs are present in cells transfected with the HTLV-3 clone. HTLV-3 p24 gag protein is detected in the cell culture supernatant. Transfection of 293T-long terminal repeat (LTR)-green fluorescent protein (GFP) cells with the HTLV-3 clone promotes formation of syncytia, a hallmark of Env expression, together with the appearance of fluorescent cells, demonstrating that Tax is expressed. Viral particles are visible by electron microscopy. These particles are infectious, as demonstrated by infection experiments with purified virions.

Members of the dual-specificity tyrosine-phosphorylated and regulated kinase (DYRK) family perform a variety of functions in eukaryotes. We used gene disruption, targeted pharmacologic inhibition, and genome-wide transcriptional profiling to dissect the function of the Yak1 DYRK in the human fungal pathogen Candida albicans. C. albicans strains with mutant yak1 alleles showed defects in the yeast-to-hypha transition and in maintaining hyphal growth. They also could not form biofilms. Despite their in vitro filamentation defect, C. albicans yak1Delta/yak1Delta mutants remained virulent in animal models of systemic and oropharyngeal candidiasis. Transcriptional profiling showed that Yak1 was necessary for the up-regulation of only a subset of hypha-induced genes. Although downstream targets of the Tec1 and Bcr1 transcription factors were down-regulated in the yak1Delta/yak1Delta mutant, TEC1 and BCR1 were not. Furthermore, 63% of Yak1-dependent, hypha-specific genes have been reported to be negatively regulated by the transcriptional repressor Tup1 and inactivation of TUP1 in the yak1Delta/yak1Delta mutant restored filamentation, suggesting that Yak1 may function upstream of Tup1 in governing hyphal emergence and maintenance.