Mathieu Coureuil

Type 4a pili (T4aP), the most widespread and functionally versatile subtype of the type 4 filaments (T4F) superfamily, are functionalised at their tip by the adhesin PilC/PilY1. Although AlphaFold models predict how this unusually large non-pilin protein is displayed at the pilus tip – by interacting through a short C-terminal stretch of residues with the PilHIJK complex of minor pilins – how PilC/PilY1 drives pilus biogenesis at a stage occurring after filament assembly remains unknown. Here, we answer this question in Neisseria meningitidis . After showing that the last 12 residues of PilC, which we name “plug”, are both necessary and sufficient for pilus biogenesis, we demonstrate that the plug interacts with PilK through β-strand augmentation by determining a crystal structure of a PilK-PLUG fusion protein. We establish that this interaction is strong and sequence-specific (by performing binding assays with purified PilK and synthetic peptides) and takes place at a late stage of pilus biogenesis occurring after filament assembly (by showing that extracellular addition of plug peptide to cultures of a ΔpilC mutant is able to restore pilus biogenesis by acting outside the cells). Finally, we demonstrate that the plug motif markedly stabilises PilK, providing an explanation for the requirement of PilC in pilus biogenesis and for the ability of a PilK-PLUG fusion protein to bypass this requirement. Together, these findings delineate the mechanism and chronology of events underlying PilC/PilY1 role in pilus biogenesis, reconcile all previous observations about this protein, and establish a broadly applicable model for T4aP biogenesis. SIGNIFICANCE STATEMENT: Type 4a pili (T4aP) are widespread and functionally versatile bacterial surface appendages, functionalised by the tip-located adhesin PilC/PilY1. While PilC/PilY1 is known to drive pilus biogenesis by acting after filament assembly, the underlying mechanism has remained elusive. We demonstrate in Neisseria meningitidis , that a C-terminal 12-residue motif within PilC/PilY1, designated the “plug,” is necessary and sufficient for pilus biogenesis. Crystal structure, binding and stability assays reveal that the plug interacts with the minor pilin PilK via β-strand augmentation, sequence-specifically, after filament assembly, and markedly stabilises it. This study delineates the mechanism and chronology of events underlying PilC/PilY1 role in T4aP biogenesis, establishing a broadly applicable model consistent with all previous observations.

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.

Once passed into the bloodstream, bacterial pathogens have a limited time to interact with permissive receptors at the surface of host cells. Neisseria meningitidis has developed an extremely effective strategy allowing it to find its receptors in a few seconds. Here, we report that N. meningitidis type IV pili exploit the physical properties of host cells' plasma membranes to promote the formation of early tubular membrane structures essential for initial bacterial adhesion. These tubular structures, which form before any signaling events in host cells, concentrate and trap multiple plasma membrane-associated proteins in the vicinity of bacteria, thereby facilitating the selection, interaction and activation of specific adhesion and signaling receptors by bacterial ligands present on type IV pili. Our results define an additional paradigm for the recruitment of specific receptors by pathogenic bacteria, which depends on the physical property of bacterial pili to induce the formation of tubular plasma membrane structures enriched in integral plasma membrane receptors.

Despite the well-described association of skin lesions with Staphylococcus aureus , the distinct ability of clinical isolates to influence the local and systemic inflammatory response in a patient-specific manner is insufficiently characterized. In this study, we analyzed clinical recessive dystrophic epidermolysis bullosa (RDEB), which is characterized by wounds chronically colonized with S. aureus , to explore the relationship between inflammatory immune response and strain diversity. Children with RDEB (moderate phenotype, n = 5; severe phenotype, n = 10) and controls ( n = 18) were enrolled in the study. Profiling of plasma proteins ( n = 800), immune cells ( n = 30 subsets and cytokine-producing cells), and cytokines ( n = 38) identified a specific inflammatory signature in severe disease. Furthermore, patients with severe RDEB presented a high frequency of interleukin-17A+ (IL-17A+) cells among CD4+ and mucosal-associated invariant T (MAIT) lymphocytes. Positive S. aureus cultures from the skin of patients with RDEB allowed whole-genome sequencing of patient strains and assessment of primary keratinocyte immune response upon bacterial challenge. S. aureus secretome and conditioned medium from keratinocytes challenged with S. aureus strains from patients with severe but not from those with moderate RDEB promoted strong activation and a pro–IL-17 response in both CD4+ and MAIT cells. Our findings show that S. aureus strains isolated from patients with severe RDEB induce an IL-17–skewed immune response and pave the way for precision microbiology to explain and predict the highly variable virulence potential of bacterial clinical isolates.

Neisseria meningitidis is a human commensal bacterium that can opportunistically invade the bloodstream and cross the blood–brain barrier, where it can cause septicemia and meningitis. These diseases, if left untreated, can be lethal within hours. Hyperinvasive N. meningitidis strains often express a genomically encoded filamentous bacteriophage called MDAΦ, which promotes colonization of mucosal host surfaces to facilitate bacterial invasion. How this phage is organized and how it promotes biofilm formation and infection at the molecular level is unclear. Here, we present an electron cryomicroscopy structure of the MDA phage, showing that MDAΦ is a class I filamentous inovirus, with the major capsid protein (MCP) arranged within the phage as a highly curved and densely packed α-helix. Comparison with other filamentous bacteriophages offers clues about inoviral genome encapsidation mechanisms, providing a framework for understanding the evolutionary diversity of inoviruses. A disordered, N-terminal segment in the MCP presents hydrophobic patches on the surface of assembled phage particles, which, together with electron cryotomography data of phage bundles, furnishes a structural rationale for phage–phage interactions that were seen previously in an epithelium adhesion infection model of N. meningitidis . Taken together, our results shed light on the structure, organization, and higher-order assembly of a biomedically relevant phage encoded in the genome of a human pathogen. Molecular insights gleaned from this study increase our understanding of phage evolution, phage-mediated bacterial adhesion, and pathogenicity.

Once passed into the bloodstream, bacterial pathogens have a limited time to interact with permissive receptors at the surface of host cells. Neisseria meningitidis has developed an extremely effective strategy allowing it to find its receptors in a few seconds. Here, we report that N. meningitidis type IV pili exploit the physical properties of host cells plasma membranes to promote the formation of early tubular membrane structures essential for initial bacterial adhesion. These tubular structures, which form before any signaling events in host cells, concentrate and trap multiple plasma membrane-associated proteins in the vicinity of bacteria, thereby facilitating the selection, interaction and activation of specific adhesion and signaling receptors by bacterial ligands bound to type IV pili. Our results define an additional paradigm for the recruitment of specific receptors by pathogenic bacteria, which depends on the physical property of bacterial pili to induce the formation of tubular plasma membrane structures enriched in integral membrane receptor.

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.

Neisseria meningitidis is a human commensal bacterium that can opportunistically invade the bloodstream and cross the blood-brain barrier, where it can cause septicaemia and meningitis. These diseases, if left untreated, can be lethal within hours. Hyperinvasive N. meningitidis strains often express a genomically encoded filamentous bacteriophage called MDAΦ, which promotes colonisation of mucosal host surfaces to facilitate invasion. How this phage is organised and how it promotes biofilm formation and infection at the molecular level is unclear. Here, we present an electron cryomicroscopy structure of the MDA phage, showing that MDAΦ is a class I filamentous inovirus, with the major capsid protein arranged within the phage as a highly curved and densely packed α-helix. Comparison with other filamentous bacteriophages offers clues about inoviral genome encapsidation mechanisms, providing a framework for understanding the evolutionary diversity of inoviruses. A disordered, N-terminal segment in the major capsid protein presents hydrophobic patches on the surface of assembled phage particles, which, together with electron cryotomography data of phage bundles, furnishes a structural rationale for phage-phage interactions that were seen previously in an epithelium adhesion infection model of N. meningitidis . Taken together, our results shed light on the structure, organisation, and higher order assembly of a biomedically relevant phage encoded in the genome of a human pathogen. Molecular insights gleaned from this study increase our understanding of phage evolution, phage-mediated bacterial adhesion and pathogenicity.