Strategies to Isolate Extracellular Vesicles from Gram-Negative and Gram-Positive Bacteria.

Membrane vesicles are produced by all Gram-negative and Gram-positive bacteria investigated so far. Membrane vesicles are spherical bilayers of phospholipids released by the bacteria to their surrounding environment and whose average size is comprised between 20 and 300 nm. The purification of these vesicles is often a challenge, as the yield and purity are often crucial for further analyses or use. In this chapter, we describe the most used method to isolate membrane vesicles from culture supernatant of Streptococcus pneumoniae and Klebsiella pneumoniae using ultracentrifugation followed by a density gradient method.

Enterobacteria impair host p53 tumor suppressor activity through mRNA destabilization.

Increasing evidence highlights the role of bacteria in the physiopathology of cancer. However, the underlying molecular mechanisms remains poorly understood. Several cancer-associated bacteria have been shown to produce toxins which interfere with the host defense against tumorigenesis. Here, we show that lipopolysaccharides from Klebsiella pneumoniae and other Enterobacteria strongly inhibit the host tumor suppressor p53 pathway through a novel mechanism of p53 regulation. We found that lipopolysaccharides destabilize TP53 mRNA through a TLR4-NF-κB-mediated inhibition of the RNA-binding factor Wig-1. Importantly, we show that K. pneumoniae disables two major tumor barriers, oncogene-induced DNA damage signaling and senescence, by impairing p53 transcriptional activity upon DNA damage and oncogenic stress. Furthermore, we found an inverse correlation between the levels of TLR4 and p53 mutation in colorectal tumors. Hence, our data suggest that the repression of p53 by Enterobacteria via TLR4 alleviates the selection pressure for p53 oncogenic mutations and shapes the genomic evolution of cancer.

Neisseria meningitidis IgA1-specific serine protease exhibits novel cleavage activity against IgG3.

Neisseria meningitidis (meningococcus) is a common bacterial colonizer of the human nasopharynx but can occasionally cause very severe systemic infections with rapid onset. Meningococci are able to degrade IgA encountered during colonization of mucosal membranes using their IgA1-specific serine protease. During systemic infection, specific IgG can induce complement-mediated lysis of the bacterium. However, meningococcal immune evasion mechanisms in thwarting IgG remain undescribed. In this study, we report for the first time that the meningococcal IgA1-specific serine protease is able to degrade IgG3 in addition to IgA. The IgG3 heavy chain is specifically cleaved in the lower hinge region thereby separating the antigen binding part from its effector binding part. Through molecular characterization, we demonstrate that meningococcal IgA1-specific serine protease of cleavage type 1 degrades both IgG3 and IgA, whereas cleavage type 2 only degrades IgA. Epidemiological analysis of 7581 clinical meningococcal isolates shows a significant higher proportion of cleavage type 1 among isolates from invasive cases compared to carrier cases, regardless of serogroup. Notably, serogroup W cc11 which is an increasing cause of invasive meningococcal disease globally harbors almost exclusively cleavage type 1 protease. Our study also shows an increasing prevalence of meningococcal isolates encoding IgA1P cleavage type 1 compared to cleavage type 2 during the observed decade (2010-2019). Altogether, our work describes a novel mechanism of IgG3 degradation by meningococci and its association to invasive meningococcal disease.

The Role of Minor Pilins in Assembly and Function of the Competence Pilus of Streptococcus pneumoniae.

The remarkable genomic plasticity of Streptococcus pneumoniae largely depends on its ability to undergo natural genetic transformation. To take up extracellular DNA, S. pneumoniae assembles competence pili composed of the major pilin ComGC. In addition to ComGC, four minor pilins ComGD, E, F, and G are expressed during bacterial competence, but their role in pilus biogenesis and transformation is unknown. Here, using a combination of protein-protein interaction assays we show that all four proteins can directly interact with each other. Pneumococcal ComGG stabilizes the minor pilin ComGD and ComGF and can interact with and stabilize the major pilin ComGC, thus, deletion of ComGG abolishes competence pilus assembly. We further demonstrate that minor pilins are present in sheared pili fractions and find ComGF to be incorporated along the competence pilus by immunofluorescence and electron microscopy. Finally, mutants of the invariant Glu5 residue (E5), ComGDE5A or ComGEE5A, but not ComGFE5A, were severely impaired in pilus formation and function. Together, our results suggest that ComGG, lacking E5, is essential for competence pilus assembly and function, and plays a central role in connecting the pneumococcal minor pilins to ComGC.

Biofilm interfacial acidity evaluation by pH-Responsive luminescent nanoparticle films.

Biofilms are dense bacterial colonies that may adhere to the surfaces of medical devices and are major contributors to infections. These colonies are characterized by a self-produced matrix of extracellular polymeric substances (EPS). Bacterial biofilms are difficult to treat with the commonly used antibiotics partially because of their poor diffusion through the EPS and therefore require new targeted strategies to effectively fight them. Biofilms may produce an acidic microenvironment which can be exploited to design such targeted treatment strategies. However, there is currently a lack of high-throughput ways to determine the acidity of biofilms at their interface with the medical device. Here, a novel all-inorganic pH responsive system is developed from luminescent carbonated hydroxyapatite nanoparticles doped with Eu3+ ions which can determine the biofilm acidity fluorometrically due to carbonate removal in acidic environments that directly affects the nanoparticle luminescence. The pH responsive nanoparticles are in-situ deposited during their production onto substrates on which a variety of clinically-relevant biofilms are grown. The acidity of their interfacial (micro)environment depends on the bacterial species and strain even when differences in biofilm biomass are considered.

Mannose receptor-derived peptides neutralize pore-forming toxins and reduce inflammation and development of pneumococcal disease.

Cholesterol-dependent cytolysins (CDCs) are essential virulence factors for many human pathogens like Streptococcus pneumoniae (pneumolysin, PLY), Streptococcus pyogenes (streptolysin O, SLO), and Listeria monocytogenes (Listeriolysin, LLO) and induce cytolysis and inflammation. Recently, we identified that pneumococcal PLY interacts with the mannose receptor (MRC-1) on specific immune cells thereby evoking an anti-inflammatory response at sublytic doses. Here, we identified the interaction sites between MRC-1 and CDCs using computational docking. We designed peptides from the CTLD4 domain of MRC-1 that binds to PLY, SLO, and LLO, respectively. In vitro, the peptides blocked CDC-induced cytolysis and inflammatory cytokine production by human macrophages. Also, they reduced PLY-induced damage of the epithelial barrier integrity as well as blocked bacterial invasion into the epithelium in a 3D lung tissue model. Pre-treatment of human DCs with peptides blocked bacterial uptake via MRC-1 and reduced intracellular bacterial survival by targeting bacteria to autophagosomes. In order to use the peptides for treatment in vivo, we developed calcium phosphate nanoparticles (CaP NPs) as peptide nanocarriers for intranasal delivery of peptides and enhanced bioactivity. Co-administration of peptide-loaded CaP NPs during infection improved survival and bacterial clearance in both zebrafish and mice models of pneumococcal infection. We suggest that MRC-1 peptides can be employed as adjunctive therapeutics with antibiotics to treat bacterial infections by countering the action of CDCs.

Sex Steroids Induce Membrane Stress Responses and Virulence Properties in Pseudomonas aeruginosa.

Estrogen, a major female sex steroid hormone, has been shown to promote the selection of mucoid Pseudomonas aeruginosa in the airways of patients with chronic respiratory diseases, including cystic fibrosis. This results in long-term persistence, poorer clinical outcomes, and limited therapeutic options. In this study, we demonstrate that at physiological concentrations, sex steroids, including testosterone and estriol, induce membrane stress responses in P. aeruginosa This is characterized by increased virulence and consequent inflammation and release of proinflammatory outer membrane vesicles promoting in vivo persistence of the bacteria. The steroid-induced P. aeruginosa response correlates with the molecular polarity of the hormones and membrane fluidic properties of the bacteria. This novel mechanism of interaction between sex steroids and P. aeruginosa explicates the reported increased disease severity observed in females with cystic fibrosis and provides evidence for the therapeutic potential of the modulation of sex steroids to achieve better clinical outcomes in patients with hormone-responsive strains.IMPORTANCE Molecular mechanisms by which sex steroids interact with P. aeruginosa to modulate its virulence have yet to be reported. Our work provides the first characterization of a steroid-induced membrane stress mechanism promoting P. aeruginosa virulence, which includes the release of proinflammatory outer membrane vesicles, resulting in inflammation, host tissue damage, and reduced bacterial clearance. We further demonstrate that at nanomolar (physiological) concentrations, male and female sex steroids promote virulence in clinical strains of P. aeruginosa based on their dynamic membrane fluidic properties. This work provides, for the first-time, mechanistic insight to better understand and predict the P. aeruginosa related response to sex steroids and explain the interindividual patient variability observed in respiratory diseases such as cystic fibrosis that are complicated by gender differences and chronic P. aeruginosa infection.

Tracking the cargo of extracellular symbionts into host tissues with correlated electron microscopy and nanoscale secondary ion mass spectrometry imaging.

Extracellular bacterial symbionts communicate biochemically with their hosts to establish niches that foster the partnership. Using quantitative ion microprobe isotopic imaging (nanoscale secondary ion mass spectrometry [NanoSIMS]), we surveyed localization of 15 N-labelled molecules produced by the bacterium Vibrio fischeri within the cells of the symbiotic organ of its host, the Hawaiian bobtail squid, and compared that with either labelled non-specific species or amino acids. In all cases, two areas of the organ's epithelia were significantly more 15 N enriched: (a) surface ciliated cells, where environmental symbionts are recruited, and (b) the organ's crypts, where the symbiont population resides in the host. Label enrichment in all cases was strongest inside host cell nuclei, preferentially in the euchromatin regions and the nucleoli. This permissiveness demonstrated that uptake of biomolecules is a general mechanism of the epithelia, but the specific responses to V. fischeri cells recruited to the organ's surface are due to some property exclusive to this species. Similarly, in the organ's deeper crypts, the host responds to common bacterial products that only the specific symbiont can present in that location. The application of NanoSIMS allows the discovery of such distinct modes of downstream signalling dependent on location within the host and provides a unique opportunity to study the microbiogeographical patterns of symbiotic dialogue.

Insights into flagellar function and mechanism from the squid-vibrio symbiosis.

Flagella are essential and multifunctional nanomachines that not only move symbionts towards their tissue colonization site, but also play multiple roles in communicating with the host. Thus, untangling the activities of flagella in reaching, interacting, and signaling the host, as well as in biofilm formation and the establishment of a persistent colonization, is a complex problem. The squid-vibrio system offers a unique model to study the many ways that bacterial flagella can influence a beneficial association and, generally, other bacteria-host interactions. Vibrio fischeri is a bioluminescent bacterium that colonizes the Hawaiian bobtail squid, Euprymna scolopes. Over the last 15 years, the structure, assembly, and functions of V. fischeri flagella, including not only motility and chemotaxis, but also biofilm formation and symbiotic signaling, have been revealed. Here we discuss these discoveries in the perspective of other host-bacteria interactions.

Role and Recruitment of the TagL Peptidoglycan-Binding Protein during Type VI Secretion System Biogenesis.

The type VI secretion system (T6SS) is an injection apparatus that uses a springlike mechanism for effector delivery. The contractile tail is composed of a needle tipped by a sharpened spike and wrapped by the sheath that polymerizes in an extended conformation on the assembly platform, or baseplate. Contraction of the sheath propels the needle and effectors associated with it into target cells. The passage of the needle through the cell envelope of the attacker is ensured by a dedicated trans-envelope channel complex. This membrane complex (MC) comprises the TssJ lipoprotein and the TssL and TssM inner membrane proteins. MC assembly is a hierarchized mechanism in which the different subunits are recruited in a specific order: TssJ, TssM, and then TssL. Once assembled, the MC serves as a docking station for the baseplate. In enteroaggregative Escherichia coli, the MC is accessorized by TagL, a peptidoglycan-binding (PGB) inner membrane-anchored protein. Here, we show that the PGB domain is the only functional domain of TagL and that the N-terminal transmembrane region mediates contact with the TssL transmembrane helix. Finally, we conduct fluorescence microscopy experiments to position TagL in the T6SS biogenesis pathway, demonstrating that TagL is recruited to the membrane complex downstream of TssL and is not required for baseplate docking.IMPORTANCE Bacteria use weapons to deliver effectors into target cells. One of these weapons, called the type VI secretion system (T6SS), could be compared to a nano-spear gun using a springlike mechanism for effector injection. By targeting bacteria and eukaryotic cells, the T6SS reshapes bacterial communities and hijacks host cell defenses. In enteroaggregative Escherichia coli, the T6SS is a multiprotein machine that comprises a cytoplasmic tail and a peptidoglycan-anchored trans-envelope channel. In this work, we show that TagL comprises an N-terminal domain that mediates contact with the channel and a peptidoglycan-binding domain that binds the cell wall. We then determine at which stage of T6SS biogenesis TagL is recruited and how TagL absence impacts the assembly pathway.

Gram-Positive Type IV Pili and Competence.

Type IV pili (T4P) are remarkable bacterial surface appendages that carry out a range of functions. Various types of T4P have been identified in bacteria and archaea, making them almost universal structures in prokaryotes. T4P are best characterized in Gram-negative bacteria, in which pilus biogenesis and T4P-mediated functions have been studied for decades. Recent advances in microbial whole-genome sequencing have provided ample evidence for the existence of T4P also in many Gram-positive species. However, comparatively little is known, and T4P in Gram-positive bacteria are just beginning to be dissected. So far, they have mainly been studied in Clostridium and Streptococcus spp. and are involved in diverse cellular processes such as adhesion, motility, and horizontal gene transfer. Here we summarize the current understanding of T4P in Gram-positive species and their functions, with particular focus on the type IV competence pilus produced by the human pathogen Streptococcus pneumoniae and its role in natural transformation.

Factor H binding proteins protect division septa on encapsulated Streptococcus pneumoniae against complement C3b deposition and amplification.

Streptococcus pneumoniae evades C3-mediated opsonization and effector functions by expressing an immuno-protective polysaccharide capsule and Factor H (FH)-binding proteins. Here we use super-resolution microscopy, mutants and functional analysis to show how these two defense mechanisms are functionally and spatially coordinated on the bacterial cell surface. We show that the pneumococcal capsule is less abundant at the cell wall septum, providing C3/C3b entry to underlying nucleophilic targets. Evasion of C3b deposition at division septa and lateral amplification underneath the capsule requires localization of the FH-binding protein PspC at division sites. Most pneumococcal strains have one PspC protein, but successful lineages in colonization and disease may have two, PspC1 and PspC2, that we show affect virulence differently. We find that spatial localization of these FH-recruiting proteins relative to division septa and capsular layer is instrumental for pneumococci to resist complement-mediated opsonophagocytosis, formation of membrane-attack complexes, and for the function as adhesins.

Structure of the competence pilus major pilin ComGC in Streptococcus pneumoniae.

Type IV pili are important virulence factors on the surface of many pathogenic bacteria and have been implicated in a wide range of diverse functions, including attachment, twitching motility, biofilm formation, and horizontal gene transfer. The respiratory pathogen Streptococcus pneumoniae deploys type IV pili to take up DNA during transformation. These "competence pili" are composed of the major pilin protein ComGC and exclusively assembled during bacterial competence, but their biogenesis remains unclear. Here, we report the high resolution NMR structure of N-terminal truncated ComGC revealing a highly flexible and structurally divergent type IV pilin. It consists of only three α-helical segments forming a well-defined electronegative cavity and confined electronegative and hydrophobic patches. The structure is particularly flexible between the first and second α-helix with the first helical part exhibiting slightly slower dynamics than the rest of the pilin, suggesting that the first helix is involved in forming the pilus structure core and that parts of helices two and three are primarily surface-exposed. Taken together, our results provide the first structure of a type IV pilin protein involved in the formation of competence-induced pili in Gram-positive bacteria and corroborate the remarkable structural diversity among type IV pilin proteins.

Molecular Dissection of the Interface between the Type VI Secretion TssM Cytoplasmic Domain and the TssG Baseplate Component.

The type VI secretion system (T6SS) is a multiprotein complex that catalyses toxin secretion through the bacterial cell envelope of various Gram-negative bacteria including important human pathogens. This machine uses a bacteriophage-like contractile tail to puncture the prey cell and inject harmful toxins. The T6SS tail comprises an inner tube capped by the cell-puncturing spike and wrapped by the contractile sheath. This structure is built on an assembly platform, the baseplate, which is anchored to the bacterial cell envelope by the TssJLM membrane complex (MC). This MC serves as both a tail docking station and a channel for the passage of the inner tube. The TssM transmembrane protein is a key component of the MC as it connects the inner and outer membranes. In this study, we define the TssM topology, highlighting a large but poorly studied 35-kDa cytoplasmic domain, TssMCyto, located between two transmembrane segments. Protein-protein interaction assays further show that TssMCyto oligomerises and makes contacts with several baseplate components. Using computer predictions, we delineate two subdomains in TssMCyto, including a nucleotide triphosphatase (NTPase) domain, followed by a 110-aa extension. Finally, site-directed mutagenesis coupled to functional assays reveals the contribution of these subdomains and conserved motifs to the interaction with T6SS partners and to the function of the secretion apparatus.

Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development.

UNLABELLED: Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis; however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. IMPORTANCE: Determining the function(s) of sheathed flagella in bacteria has been challenging, because no known mutation results only in the loss of this outer membrane-derived casing. Nevertheless, the presence of a sheathed flagellum in such host-associated genera as Vibrio, Helicobacter, and Brucella has led to several proposed functions, including physical protection of the flagella and masking of their immunogenic flagellins. Using the squid-vibrio light organ symbiosis, we demonstrate another role, that of V. fischeri cells require rotating flagella to induce apoptotic cell death within surface epithelium, which is a normal step in the organ's development. Further, we present evidence that this rotation releases apoptosis-triggering lipopolysaccharide in the form of outer membrane vesicles. Such release may also occur by pathogens but with different outcomes for the host.

Vibrio fischeri-derived outer membrane vesicles trigger host development.

Outer membrane vesicles (OMV) are critical elements in many host-cell/microbe interactions. Previous studies of the symbiotic association between Euprymna scolopes and Vibrio fischeri had shown that within 12 h of colonizing crypts deep within the squid's light organ, the symbionts trigger an irreversible programme of tissue development in the host. Here, we report that OMV produced by V. fischeri are powerful contributors to this process. The first detectable host response to the OMV is an increased trafficking of macrophage-like cells called haemocytes into surface epithelial tissues. We showed that exposing the squid to other Vibrio species fails to induce this trafficking; however, addition of a high concentration of their OMV, which can diffuse into the crypts, does. We also provide evidence that tracheal cytotoxin released by the symbionts, which can induce haemocyte trafficking, is not part of the OMV cargo, suggesting two distinct mechanisms to induce the same morphogenesis event. By manipulating the timing and localization of OMV signal delivery, we showed that haemocyte trafficking is fully induced only when V. fischeri, the sole species able to reach and grow in the crypts, succeeds in establishing a sustained colonization. Further, our data suggest that the host's detection of OMV serves as a symbiotic checkpoint prior to inducing irreversible morphogenesis.

A phospholipase A1 antibacterial Type VI secretion effector interacts directly with the C-terminal domain of the VgrG spike protein for delivery.

The Type VI secretion system (T6SS) is a multiprotein machine that delivers protein effectors in both prokaryotic and eukaryotic cells, allowing interbacterial competition and virulence. The mechanism of action of the T6SS requires the contraction of a sheath-like structure that propels a needle towards target cells, allowing the delivery of protein effectors. Here, we provide evidence that the entero-aggregative Escherichia coli Sci-1 T6SS is required to eliminate competitor bacteria. We further identify Tle1, a toxin effector encoded by this cluster and showed that Tle1 possesses phospholipase A1 and A2 activities required for the interbacterial competition. Self-protection of the attacker cell is secured by an outer membrane lipoprotein, Tli1, which binds Tle1 in a 1:1 stoichiometric ratio with nanomolar affinity, and inhibits its phospholipase activity. Tle1 is delivered into the periplasm of the prey cells using the VgrG1 needle spike protein as carrier. Further analyses demonstrate that the C-terminal extension domain of VgrG1, including a transthyretin-like domain, is responsible for the interaction with Tle1 and its subsequent delivery into target cells. Based on these results, we propose an additional mechanism of transport of T6SS effectors in which cognate effectors are selected by specific motifs located at the C-terminus of VgrG proteins.

Biogenesis and structure of a type VI secretion membrane core complex.

Bacteria share their ecological niches with other microbes. The bacterial type VI secretion system is one of the key players in microbial competition, as well as being an important virulence determinant during bacterial infections. It assembles a nano-crossbow-like structure in the cytoplasm of the attacker cell that propels an arrow made of a haemolysin co-regulated protein (Hcp) tube and a valine-glycine repeat protein G (VgrG) spike and punctures the prey's cell wall. The nano-crossbow is stably anchored to the cell envelope of the attacker by a membrane core complex. Here we show that this complex is assembled by the sequential addition of three type VI subunits (Tss)-TssJ, TssM and TssL-and present a structure of the fully assembled complex at 11.6 Å resolution, determined by negative-stain electron microscopy. With overall C5 symmetry, this 1.7-megadalton complex comprises a large base in the cytoplasm. It extends in the periplasm via ten arches to form a double-ring structure containing the carboxy-terminal domain of TssM (TssMct) and TssJ that is anchored in the outer membrane. The crystal structure of the TssMct-TssJ complex coupled to whole-cell accessibility studies suggest that large conformational changes induce transient pore formation in the outer membrane, allowing passage of the attacking Hcp tube/VgrG spike.