Intermittent Pili-Mediated Forces Fluidize Neisseria meningitidis Aggregates Promoting Vascular Colonization.

Neisseria meningitidis, a bacterium responsible for meningitis and septicemia, proliferates and eventually fills the lumen of blood capillaries with multicellular aggregates. The impact of this aggregation process and its specific properties are unknown. We first show that aggregative properties are necessary for efficient infection and study their underlying physical mechanisms. Micropipette aspiration and single-cell tracking unravel unique features of an atypical fluidized phase, with single-cell diffusion exceeding that of isolated cells. A quantitative description of the bacterial pair interactions combined with active matter physics-based modeling show that this behavior relies on type IV pili active dynamics that mediate alternating phases of bacteria fast mutual approach, contact, and release. These peculiar fluid properties proved necessary to adjust to the geometry of capillaries upon bacterial proliferation. Intermittent attractive forces thus generate a fluidized phase that allows for efficient colonization of the blood capillary network during infection.

TrackMate 7: integrating state-of-the-art segmentation algorithms into tracking pipelines.

TrackMate is an automated tracking software used to analyze bioimages and is distributed as a Fiji plugin. Here, we introduce a new version of TrackMate. TrackMate 7 is built to address the broad spectrum of modern challenges researchers face by integrating state-of-the-art segmentation algorithms into tracking pipelines. We illustrate qualitatively and quantitatively that these new capabilities function effectively across a wide range of bio-imaging experiments.

Do Not Waste Time─Ensure Success in Your Cross-Linking Mass Spectrometry Experiments before You Begin.

Cross-linking mass spectrometry (XL-MS) has become a very useful tool for studying protein complexes and interactions in living systems. It enables the investigation of many large and dynamic assemblies in their native state, providing an unbiased view of their protein interactions and restraints for integrative modeling. More researchers are turning toward trying XL-MS to probe their complexes of interest, especially in their native environments. However, due to the presence of other potentially higher abundant proteins, sufficient cross-links on a system of interest may not be reached to achieve satisfactory structural and interaction information. There are currently no rules for predicting whether XL-MS experiments are likely to work or not; in other words, if a protein complex of interest will lead to useful XL-MS data. Here, we show that a simple iBAQ (intensity-based absolute quantification) analysis performed from trypsin digest data can provide a good understanding of whether proteins of interest are abundant enough to achieve successful cross-linking data. Comparing our findings to large-scale data on diverse systems from several other groups, we show that proteins of interest should be at least in the top 20% abundance range to expect more than one cross-link found per protein. We foresee that this guideline is a good starting point for researchers who would like to use XL-MS to study their protein of interest and help ensure a successful cross-linking experiment from the beginning. Data are available via ProteomeXchange with identifier PXD045792.

Deep mutational scanning of the Neisseria meningitidis major pilin reveals the importance of pilus tip-mediated adhesion.

Type IV pili (TFP) are multifunctional micrometer-long filaments expressed at the surface of many prokaryotes. In Neisseria meningitidis, TFP are crucial for virulence. Indeed, these homopolymers of the major pilin PilE mediate interbacterial aggregation and adhesion to host cells. However, the mechanisms behind these functions remain unclear. Here, we simultaneously determined regions of PilE involved in pilus display, auto-aggregation, and adhesion by using deep mutational scanning and started mining this extensive functional map. For auto-aggregation, pili must reach a minimum length to allow pilus-pilus interactions through an electropositive cluster of residues centered around Lys140. For adhesion, results point to a key role for the tip of the pilus. Accordingly, purified pili interacting with host cells initially bind via their tip-located major pilin and then along their length. Overall, these results identify functional domains of PilE and support a direct role of the major pilin in TFP-dependent aggregation and adhesion.

Inhibitors of the Neisseria meningitidis PilF ATPase provoke type IV pilus disassembly.

Despite the availability of antibiotics and vaccines, Neisseria meningitidis remains a major cause of meningitis and sepsis in humans. Due to its extracellular lifestyle, bacterial adhesion to host cells constitutes an attractive therapeutic target. Here, we present a high-throughput microscopy-based approach that allowed the identification of compounds able to decrease type IV pilus-mediated interaction of bacteria with endothelial cells in the absence of bacterial or host cell toxicity. Compounds specifically inhibit the PilF ATPase enzymatic activity that powers type IV pilus extension but remain inefficient on the ATPase that promotes pilus retraction, thus leading to rapid pilus disappearance from the bacterial surface and loss of pili-mediated functions. Structure activity relationship of the most active compound identifies specific moieties required for the activity of this compound and highlights its specificity. This study therefore provides compounds targeting pilus biogenesis, thereby inhibiting bacterial adhesion, and paves the way for a novel therapeutic option for meningococcal infections.

Advanced In Vivo Cross-Linking Mass Spectrometry Platform to Characterize Proteome-Wide Protein Interactions.

Chemical cross-linking (XL) coupled to mass spectrometry (MS) has become a powerful approach to probe the structure of protein assemblies. Although most of the applications concerned purified complexes, latest developments focus on large-scale in vivo studies. Pushing in this direction, we developed an advanced in vivo cross-linking mass spectrometry platform to study the cellular interactome of living bacterial cells. It is based on in vivo labeling and involves a one-step enrichment by click chemistry on a solid support. Our approach shows an impressive efficiency on Neisseria meningitidis, leading to the identification of about 3300 cross-links for the LC-MS/MS analysis of a biological triplicate using a benchtop high-resolution Orbitrap mass spectrometer. Highly dynamic multiprotein complexes were successfully captured and characterized in all bacterial compartments, showing the great potential and precision of our proteome-wide approach. Our workflow paves new avenues for the large-scale and nonbiased analysis of protein-protein interactions. All raw data, databases, and processing parameters are available on ProteomeXchange via PRIDE repository (data set identifier PXD021553).

The number of Neisseria meningitidis type IV pili determines host cell interaction.

As mediators of adhesion, autoaggregation and bacteria-induced plasma membrane reorganization, type IV pili are at the heart of Neisseria meningitidis infection. Previous studies have proposed that two minor pilins, PilV and PilX, are displayed along the pilus structure and play a direct role in mediating these effects. In contrast with this hypothesis, combining imaging and biochemical approaches we found that PilV and PilX are located in the bacterial periplasm rather than along pilus fibers. Furthermore, preventing exit of these proteins from the periplasm by fusing them to the mCherry protein did not alter their function. Deletion of the pilV and pilX genes led to a decrease in the number, but not length, of pili displayed on the bacterial surface indicating a role in the initiation of pilus biogenesis. By finely regulating the expression of a central component of the piliation machinery, we show that the modest reductions in the number of pili are sufficient to recapitulate the phenotypes of the pilV and pilX mutants. We further show that specific type IV pili-dependent functions require different ranges of pili numbers.

Neisseria meningitidis Type IV Pili Composed of Sequence Invariable Pilins Are Masked by Multisite Glycosylation.

The ability of pathogens to cause disease depends on their aptitude to escape the immune system. Type IV pili are extracellular filamentous virulence factors composed of pilin monomers and frequently expressed by bacterial pathogens. As such they are major targets for the host immune system. In the human pathogen Neisseria meningitidis, strains expressing class I pilins contain a genetic recombination system that promotes variation of the pilin sequence and is thought to aid immune escape. However, numerous hypervirulent clinical isolates express class II pilins that lack this property. This raises the question of how they evade immunity targeting type IV pili. As glycosylation is a possible source of antigenic variation it was investigated using top-down mass spectrometry to provide the highest molecular precision on the modified proteins. Unlike class I pilins that carry a single glycan, we found that class II pilins display up to 5 glycosylation sites per monomer on the pilus surface. Swapping of pilin class and genetic background shows that the pilin primary structure determines multisite glycosylation while the genetic background determines the nature of the glycans. Absence of glycosylation in class II pilins affects pilus biogenesis or enhances pilus-dependent aggregation in a strain specific fashion highlighting the extensive functional impact of multisite glycosylation. Finally, molecular modeling shows that glycans cover the surface of class II pilins and strongly decrease antibody access to the polypeptide chain. This strongly supports a model where strains expressing class II pilins evade the immune system by changing their sugar structure rather than pilin primary structure. Overall these results show that sequence invariable class II pilins are cloaked in glycans with extensive functional and immunological consequences.

Adhesion of Neisseria meningitidis to dermal vessels leads to local vascular damage and purpura in a humanized mouse model.

Septic shock caused by Neisseria meningitidis is typically rapidly evolving and often fatal despite antibiotic therapy. Further understanding of the mechanisms underlying the disease is necessary to reduce fatality rates. Postmortem samples from the characteristic purpuric rashes of the infection show bacterial aggregates in close association with microvessel endothelium but the species specificity of N. meningitidis has previously hindered the development of an in vivo model to study the role of adhesion on disease progression. Here we introduced human dermal microvessels into SCID/Beige mice by xenografting human skin. Bacteria injected intravenously exclusively associated with the human vessel endothelium in the skin graft. Infection was accompanied by a potent inflammatory response with the secretion of human inflammatory cytokines and recruitment of inflammatory cells. Importantly, infection also led to local vascular damage with hemostasis, thrombosis, vascular leakage and finally purpura in the grafted skin, replicating the clinical presentation for the first time in an animal model. The adhesive properties of the type IV pili of N. meningitidis were found to be the main mediator of association with the dermal microvessels in vivo. Bacterial mutants with altered type IV pili function also did not trigger inflammation or lead to vascular damage. This work demonstrates that local type IV pili mediated adhesion of N. meningitidis to the vascular wall, as opposed to circulating bacteria, determines vascular dysfunction in meningococcemia.

Early sequence of events triggered by the interaction of Neisseria meningitidis with endothelial cells.

Neisseria meningitidis is a bacterium responsible for severe sepsis and meningitis. Following type IV pilus-mediated adhesion to endothelial cells, bacteria proliferating on the cellular surface trigger a potent cellular response that enhances the ability of adhering bacteria to resist the mechanical forces generated by the blood flow. This response is characterized by the formation of numerous 100 nm wide membrane protrusions morphologically related to filopodia. Here, a high-resolution quantitative live-cell fluorescence microscopy procedure was designed and used to study this process. A farnesylated plasma membrane marker was first detected only a few seconds after bacterial contact, rapidly followed by actin cytoskeleton reorganization and bulk cytoplasm accumulation. The bacterial type IV pili-associated minor pilin PilV is necessary for the initiation of this cascade. Plasma membrane composition is a key factor as cholesterol depletion with methyl-ß-cyclodextrin completely blocks the initiation of the cellular response. In contrast membrane deformation does not require the actin cytoskeleton. Strikingly, plasma membrane remodelling undermicrocolonies is also independent of common intracellular signalling pathways as cellular ATP depletion is not inhibitory. This study shows that bacteria-induced plasma membrane reorganization is a rapid event driven by a direct cross-talk between type IV pili and the plasma membrane rather than by the activation of an intracellular signalling pathway that would lead to actin remodelling.

Complete posttranslational modification mapping of pathogenic Neisseria meningitidis pilins requires top-down mass spectrometry.

In pathogenic bacteria, posttranslationally modified proteins have been found to promote bacterial survival, replication, and evasion from the host immune system. In the human pathogen Neisseria meningitidis, the protein PilE (15-18 kDa) is the major building block of type IV pili, extracellular filamentous organelles that play a major role in mediating pathogenesis. Previous reports have shown that PilE can be expressed as a number of different proteoforms, each harboring its own set of PTMs and that specific proteoforms are key in promoting bacterial virulence. Efficient tools that allow complete PTM mapping of proteins involved in bacterial infection are therefore strongly needed. As we show in this study, a simple combination of mass profiling and bottom-up proteomics is fundamentally unable to achieve this goal when more than two proteoforms are present simultaneously. In a N. meningitidis strain isolated from a patient with meningitis, mass profiling revealed the presence of four major proteoforms of PilE, in a 1:1:1:1 ratio. Due to the complexity of the sample, a top-down approach was required to achieve complete PTM mapping for all four proteoforms, highlighting an unprecedented extent of glycosylation. Top-down MS therefore appears to be a promising tool for the analysis of highly posttranslationally modified proteins involved in bacterial virulence.

Epithelial control of the human pDC response to extracellular bacteria.

Plasmacytoid pre-dendritic cells (pDCs) are specialized in responding to nucleic acids, and link innate with adaptive immunity. Although the response of pDCs to viruses is well established, whether pDCs can respond to extracellular bacteria remains controversial. Here, we demonstrate that extracellular bacteria such as Neisseria meningitidis, Haemophilus influenzae, and Staphylococcus aureus activate pDCs to produce IFN-α, TNF-α, IL-6, and to upregulate CD86 expression. We observed that pDCs were present within tonsillar crypts and oro-nasopharyngeal epithelium, where they may contact extracellular bacteria, in situ. Tonsil epithelium-conditioned supernatants inhibited IFN-α, TNF-α, and IL-6 triggered by the direct contact of N. meningitidis or S. aureus with pDCs. However, pDC priming of naive T cells was not affected, suggesting that tonsil epithelium micro-environment limits local inflammation while preserving adaptive immunity in response to extracellular bacteria. Our results reveal an important and novel function of pDCs in the initiation of the mucosal innate and adaptive immunity to extracellular bacteria.

Assessment of the interplay between blood and skin vascular abnormalities in adult purpura fulminans.

RATIONALE: Purpura fulminans in adults is a rare but devastating disease. Its pathophysiology is not well known. OBJECTIVES: To understand the pathophysiology of skin lesions in purpura fulminans, the interplay between circulating blood and vascular alterations was assessed. METHODS: Prospective multicenter study in four intensive care units. Patients with severe sepsis without skin lesions were recruited as control subjects. MEASUREMENTS AND MAIN RESULTS: Twenty patients with severe sepsis and purpura fulminans were recruited for blood sampling, and skin biopsy was performed in deceased patients. High severity of disease and mortality rates (80%) was observed. Skin biopsies in purpura fulminans lesions revealed thrombosis and extensive vascular damage: vascular congestion and dilation, endothelial necrosis, alteration of markers of endothelial integrity (CD31) and of the protein C pathway receptors (endothelial protein C receptor, thrombomodulin). Elevated plasminogen activating inhibitor-1 mRNA was also observed. Comparison with control patients showed that these lesions were specific to purpura fulminans. By contrast, no difference was observed for blood hemostasis parameters, including soluble thrombomodulin, activated protein C, and disseminated intravascular coagulation markers. Bacterial presence at the vascular wall was observed specifically in areas of vascular damage in eight of nine patients tested (including patients with Streptococcus pneumoniae, Neisseria meningitidis, Escherichia coli, and Pseudomonas aeruginosa infection). CONCLUSIONS: Thrombi and extensive vascular damage with multifaceted prothrombotic local imbalance are characteristics of purpura fulminans. A "vascular wall infection" hypothesis, responsible for endothelial damage and subsequent skin lesions, can be put forward.

Cryo-EM structures of type IV pili complexed with nanobodies reveal immune escape mechanisms.

Type IV pili (T4P) are prevalent, polymeric surface structures in pathogenic bacteria, making them ideal targets for effective vaccines. However, bacteria have evolved efficient strategies to evade type IV pili-directed antibody responses. Neisseria meningitidis are prototypical type IV pili-expressing Gram-negative bacteria responsible for life threatening sepsis and meningitis. This species has evolved several genetic strategies to modify the surface of its type IV pili, changing pilin subunit amino acid sequence, nature of glycosylation and phosphoforms, but how these modifications affect antibody binding at the structural level is still unknown. Here, to explore this question, we determine cryo-electron microscopy (cryo-EM) structures of pili of different sequence types with sufficiently high resolution to visualize posttranslational modifications. We then generate nanobodies directed against type IV pili which alter pilus function in vitro and in vivo. Cyro-EM in combination with molecular dynamics simulation of the nanobody-pilus complexes reveals how the different types of pili surface modifications alter nanobody binding. Our findings shed light on the impressive complementarity between the different strategies used by bacteria to avoid antibody binding. Importantly, we also show that structural information can be used to make informed modifications in nanobodies as countermeasures to these immune evasion mechanisms.

The meningococcal minor pilin PilX is responsible for type IV pilus conformational changes associated with signaling to endothelial cells.

Neisseria meningitidis crosses the blood-brain barrier (BBB) following the activation of the ß2-adrenergic receptor by the type IV pili (TFP). Two components of the type IV pili recruit the ß2-adrenergic receptor, the major pilin PilE and the minor pilin PilV. Here, we report that a strain deleted of PilX, one of the three minor pilins, is defective in endothelial cell signaling. The signaling role of PilX was abolished when pili were not retractable. Purified PilX was unable to recruit the ß2-adrenergic receptor, thus suggesting that PilX was playing an indirect role in endothelial cell signaling. Considering the recent finding that type IV pili can transition into a new conformation (N. Biais, D. L. Higashi, J. Brujic, M. So, and M. P. Sheetz, Proc. Natl. Acad. Sci. U. S. A. 107:11358-11363, 2010), we hypothesized that PilX was responsible for a structural modification of the fiber and allowed hidden epitopes to be exposed. To confirm this hypothesis, we showed that a monoclonal antibody which recognizes a linear epitope of PilE bound fibers only when bacteria adhered to endothelial cells. On the other hand, this effect was not observed in PilX-deleted pili. A deletion of a region of PilX exposed on the surface of the fiber had phenotypical consequences identical to those of a PilX deletion. These data support a model in which surface-exposed motifs of PilX use forces generated by pilus retraction to promote conformational changes required for TFP-mediated signaling.

Cerebral microcirculation shear stress levels determine Neisseria meningitidis attachment sites along the blood-brain barrier.

Neisseria meningitidis is a commensal bacterium of the human nasopharynx. Occasionally, this bacterium reaches the bloodstream and causes meningitis after crossing the blood-brain barrier by an unknown mechanism. An immunohistological study of a meningococcal sepsis case revealed that neisserial adhesion was restricted to capillaries located in low blood flow regions in the infected organs. This study led to the hypothesis that drag forces encountered by the meningococcus in the bloodstream determine its attachment site in vessels. We therefore investigated the ability of N. meningitidis to bind to endothelial cells in the presence of liquid flow mimicking the bloodstream with a laminar flow chamber. Strikingly, average blood flows reported for various organs strongly inhibited initial adhesion. As cerebral microcirculation is known to be highly heterogeneous, cerebral blood velocity was investigated at the level of individual vessels using intravital imaging of rat brain. In agreement with the histological study, shear stress levels compatible with meningococcal adhesion were only observed in capillaries, which exhibited transient reductions in flow. The flow chamber assay revealed that, after initial attachment, bacteria resisted high blood velocities and even multiplied, forming microcolonies resembling those observed in the septicemia case. These results argue that the combined mechanical properties of neisserial adhesion and blood microcirculation target meningococci to transiently underperfused cerebral capillaries and thus determine disease development.

Revisiting the extracellular lifestyle.

In microbiology textbooks infectious agents are traditionally classified as intracellular or extracellular pathogens depending on whether they multiply inside or outside host cells. In recent literature an increasing number of extracellular pathogens are described in close apposition with the host cell surface embedded in plasma membrane folds, making it difficult to classify them as strictly extracellular pathogens. This review further explores this emerging new lifestyle category tentatively named 'epicellular' in reference to earlier work describing the location of the parasite Cryptosporidium parvum. The lifestyles of three diverse such pathogens were examined: the parasite Cryptosporidium parvum, the Gram-negative bacterium Neisseria meningitidis and the human T cell leukaemia virus type 1 (HTLV-1). The specific cellular location, the mechanisms of adhesion, the induction of plasma membrane folds and the subsequent functional consequences will be compared. Although current knowledge suggests different underlying mechanisms, a concept that emerges is that the particular location of these pathogens has important functional consequences for the pathogens in terms of nutrient acquisition, immune escape, resistance to mechanical stress and transmission. Re-examining the lifestyle of other classically extracellular pathogens might thus shed a new light on the way pathogens interact with cells and point to new areas of investigation.

Dual role for pilus in adherence to epithelial cells and biofilm formation in Streptococcus agalactiae.

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.

Extracellular bacterial pathogen induces host cell surface reorganization to resist shear stress.

Bacterial infections targeting the bloodstream lead to a wide array of devastating diseases such as septic shock and meningitis. To study this crucial type of infection, its specific environment needs to be taken into account, in particular the mechanical forces generated by the blood flow. In a previous study using Neisseria meningitidis as a model, we observed that bacterial microcolonies forming on the endothelial cell surface in the vessel lumen are remarkably resistant to mechanical stress. The present study aims to identify the molecular basis of this resistance. N. meningitidis forms aggregates independently of host cells, yet we demonstrate here that cohesive forces involved in these bacterial aggregates are not sufficient to explain the stability of colonies on cell surfaces. Results imply that host cell attributes enhance microcolony cohesion. Microcolonies on the cell surface induce a cellular response consisting of numerous cellular protrusions similar to filopodia that come in close contact with all the bacteria in the microcolony. Consistent with a role of this cellular response, host cell lipid microdomain disruption simultaneously inhibited this response and rendered microcolonies sensitive to blood flow-generated drag forces. We then identified, by a genetic approach, the type IV pili component PilV as a triggering factor of plasma membrane reorganization, and consistently found that microcolonies formed by a pilV mutant are highly sensitive to shear stress. Our study shows that bacteria manipulate host cell functions to reorganize the host cell surface to form filopodia-like structures that enhance the cohesion of the microcolonies and therefore blood vessel colonization under the harsh conditions of the bloodstream.

Structure and function of interacting IcmR-IcmQ domains from a type IVb secretion system in Legionella pneumophila.

During infection, Legionella pneumophila creates a replication vacuole within eukaryotic cells and this requires a Type IVb secretion system (T4bSS). IcmQ plays a critical role in the translocase and associates with IcmR. In this paper, we show that the N-terminal domain of IcmQ (Qn) mediates self-dimerization, whereas the C-terminal domain with a basic linker promotes membrane association. In addition, the binding of IcmR to IcmQ prevents self-dimerization and also blocks membrane permeabilization. However, IcmR does not completely block membrane binding by IcmQ. We then determined crystal structures of Qn with the interacting region of IcmR. In this complex, each protein forms an alpha-helical hairpin within a parallel four-helix bundle. The amphipathic nature of helices in Qn suggests two possible models for membrane permeabilization by IcmQ. The Rm-Qn structure also suggests how IcmR-like proteins in other L. pneumophila species may interact with their IcmQ partners.