Epithelial cells are sensitive detectors of bacterial pore-forming toxins

Epithelial cells act as an interface between human mucosal surfaces and the surrounding environment. As a result, they are responsible for the initiation of local immune responses, which may be crucial for prevention of invasive infection. Here we show that epithelial cells detect the presence of bacterial pore-forming toxins (including pneumolysin from Streptococcus pneumoniae, alpha-hemolysin from Staphylococcus aureus, streptolysin O from Streptococcus pyogenes, and anthrolysin O from Bacillus anthracis) at nanomolar concentrations, far below those required to cause cytolysis. Phosphorylation of p38 MAPK appears to be a conserved response of epithelial cells to subcytolytic concentrations of bacterial poreforming toxins, and this activity is inhibited by the addition of high molecular weight osmolytes to the extracellular medium. By sensing osmotic stress caused by the insertion of a sublethal number of pores into their membranes, epithelial cells may act as an early warning system to commence an immune response, while the local density of toxin-producing bacteria remains low. Osmosensing may thus represent a novel innate immune response to a common bacterial virulence strategy.     

The NLRP3 inflammasome is differentially activated by pneumolysin variants and contributes to host defense in pneumococcal pneumonia

Streptococcus pneumoniae is a leading cause of pneumonia, meningitis, and sepsis. Pneumococci can be divided into >90 serotypes that show differences in the pathogenicity and invasiveness. We tested the hypotheses that the innate immune inflammasome pathway is involved in fighting pneumococcal pneumonia and that some invasive pneumococcal types are not recognized by this pathway. We show that human and murine mononuclear cells responded to S. pneumoniae expressing hemolytic pneumolysin by producing IL-1β. This IL-1β production depended on the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. Some serotype 1, serotype 8, and serotype 7F bacteria, which have previously been associated with increased invasiveness and with production of toxins with reduced hemolytic activity, or bacterial mutants lacking pneumolysin did not stimulate notable IL-1β production. We further found that NLRP3 was beneficial for mice during pneumonia caused by pneumococci expressing hemolytic pneumolysin and was involved in cytokine production and maintenance of the pulmonary microvascular barrier. Overall, the inflammasome pathway is protective in pneumonia caused by pneumococci expressing hemolytic toxin but is not activated by clinically important pneumococcal sequence types causing invasive disease. The study indicates that a virulence factor polymorphism may substantially affect the recognition of bacteria by the innate immune system.     

The role of innate immune responses in the outcome of interspecies competition for colonization of mucosal surfaces

Since mucosal surfaces may be simultaneously colonized by multiple species, the success of an organism may be determined by its ability to compete with co-inhabitants of its niche. To explore the contribution of host factors to polymicrobial competition, a murine model was used to study the initiation of colonization by Haemophilus influenzae and Streptococcus pneumoniae. Both bacterial species, which occupy a similar microenvironment within the nasopharynx, persisted during colonization when given individually. Co-colonization, however, resulted in rapid clearance of S. pneumoniae from the upper respiratory tract, associated with increased recruitment of neutrophils into paranasal spaces. Systemic depletion of either neutrophil-like cells or complement was sufficient to eliminate this competitive effect, indicating that clearance was likely due to enhanced opsonophagocytic killing. The hypothesis that modulation of opsonophagocytic activity was responsible for host-mediated competition was tested using in vitro killing assays with elicited neutrophil-like cells. Components of H. influenzae (but not S. pneumoniae) stimulated complement-dependent phagocytic killing of S. pneumoniae. Thus, the recruitment and activation of neutrophils through selective microbial pattern recognition may underlie the H. influenzae-induced clearance of S. pneumoniae. This study demonstrates how innate immune responses may mediate competitive interactions between species and dictate the composition of the colonizing flora.     

PKCθ synergizes with TLR-dependent TRAF6 signaling pathway to upregulate MUC5AC mucin via CARMA1

CARD-containing MAGUK protein 1 (CARMA1) plays a crucial role in regulating adaptive immune responses upon T-cell receptor (TCR) activation in T cells. Its role in regulating host mucosal innate immune response such as upregulation of mucin remains unknown. Here we show that CARMA1 acts as a key signaling mediator for synergistic upregulation of MUC5AC mucin by bacterium nontypeable Haemophilus influenzae (NTHi) and phorbol ester PMA in respiratory epithelial cells. NTHi-induced TLR-dependent TRAF6-MKK3-p38 MAPK signaling pathway synergizes with PKCθ-MEK-ERK signaling pathway. CARMA1 plays a crucial role in mediating this synergistic effect via TRAF6, thereby resulting in synergistic upregulation of MUC5AC mucin. Thus our study unveils a novel role for CARMA1 in mediating host mucosal innate immune response.     

Serotype 1 and 8 Pneumococci Evade Sensing by Inflammasomes in Human Lung Tissue

Streptococcus pneumoniae is a major cause of pneumonia, sepsis and meningitis. The pore-forming toxin pneumolysin is a key virulence factor of S. pneumoniae, which can be sensed by the NLRP3 inflammasome. Among the over 90 serotypes, serotype 1 pneumococci (particularly MLST306) have emerged across the globe as a major cause of invasive disease. The cause for its particularity is, however, incompletely understood. We therefore examined pneumococcal infection in human cells and a human lung organ culture system mimicking infection of the lower respiratory tract. We demonstrate that different pneumococcal serotypes differentially activate inflammasome-dependent IL-1β production in human lung tissue and cells. Whereas serotype 2, 3, 6B, 9N pneumococci expressing fully haemolytic pneumolysins activate NLRP3 inflammasome-dependent responses, serotype 1 and 8 strains expressing non-haemolytic toxins are poor activators of IL-1β production. Accordingly, purified haemolytic pneumolysin but not serotype 1-associated non-haemolytic toxin activates strong IL-1β production in human lungs. Our data suggest that the evasion of inflammasome-dependent innate immune responses by serotype 1 pneumococci might contribute to their ability to cause invasive diseases in humans.     

Pneumolysin: a double-edged sword during the host-pathogen interaction

The cholesterol-dependent cytolysins are pore-forming toxins. Pneumolysin is the cytolysin produced by Streptococcus pneumoniae and is a key virulence factor. The protein contains 471 amino acids and four structural domains. Binding to cholesterol is followed by oligomerization and membrane pore formation. Pneumolysin also activates the classical pathway of complement. Mutational analysis of the toxin and knowledge of sequence variation in outbreak strains suggests that additional activities of biologic importance exist. Pneumolysin activates a large number of genes, some by epigenetic modification, in eukaryotic cells and multiple signal transduction pathways. Cytolytic effects contribute to lung injury and neuronal damage while pro-inflammatory effects compound tissue damage. Nevertheless pneumolysin is a focal point of the immune response to pneumococci. Toll-like receptor 4-mediated recognition, osmosensing and T-cell responses to pneumolysin have been identified. In some animal models mutants that lack pneumolysin are associated with impaired bacterial clearance. Pneumolysin, which itself may induce apoptosis in neurones and other cells can activate host-mediated apoptosis in macrophages enhancing clearance. Disease pathogenesis, which has traditionally focused on the harmful effects of the toxin, increasingly recognises that a precarious balance between limited host responses to pneumolysin and either excessive immune responses or toxin-mediated subversion of host immunity exists.     

Studies on the structure and mechanism of a bacterial protein toxin by analytical ultracentrifugation and small-angle neutron scattering.

Pneumolysin, an important virulence factor of the human pathogen Streptococcus pneumoniae, is a pore-forming toxin which also possesses the ability to activate the complement system directly. Pneumolysin binds to cholesterol in cell membrane surfaces as a prelude to pore formation, which involves the oligomerization of the protein. Two important aspects of the pore-forming activity of pneumolysin are therefore the effect of the toxin on bilayer membrane structure and the nature of the self-association into oligomers undergone by it. We have used analytical ultracentrifugation (AUC) to investigate oligomerization and small-angle neutron scattering (SANS) to investigate the changes in membrane structure accompanying pore formation.Pneumolysin self-associates in solution to form oligomeric structures apparently similar to those which appear on the membrane coincident with pore formation. It has previously been demonstrated by us using site-specific chemical derivatization of the protein that the self-interaction preceding oligomerization involves its C-terminal domain. The AUC experiments described here involved pneumolysin toxoids harbouring mutations in different domains, and support our previous conclusions that self-interaction via the C-terminal domain leads to oligomerization and that this may be related to the mechanism by which pneumolysin activates the complement system.SANS data at a variety of neutron contrasts were obtained from liposomes used as model cell membranes in the absence of pneumolysin, and following the addition of toxin at a number of concentrations. These experiments were designed to allow visualization of the effect that pneumolysin has on bilayer membrane structure resulting from oligomerization into a pore-forming complex. The structure of the liposomal membrane alone and following addition of pneumolysin was calculated by the fitting of scattering equations directly to the scattering curves. The fitting equations describe scattering from simple three-dimensional scattering volume models for the structures present in the sample, whose dimensions were varied iteratively within the fitting program. The overall trend was a thinning of the liposome surface on toxin attack, which was countered by the formation of localized structures thicker than the liposome bilayer itself, in a manner dependent on pneumolysin concentration. At the neutron contrast match point of the liposomes, pneumolysin oligomers were observed. Inactive toxin appeared to bind to the liposome but not to cause membrane alteration; subsequent activation of pneumolysin in situ brought about changes in liposome structure similar to those seen in the presence of active toxin. We propose that the changes in membrane structure on toxin attack which we have observed are related to the mechanism by which pneumolysin forms pores and provide an important perspective on protein/membrane interactions in general. We discuss these results in the light of published data concerning the interaction of gramicidin with bilayers and the hydrophobic mismatch effect.     

Additive inhibition of complement deposition by pneumolysin and PspA facilitates Streptococcus pneumoniae septicemia

Streptococcus pneumoniae is a common cause of septicemia in the immunocompetent host. To establish infection, S. pneumoniae has to overcome host innate immune responses, one component of which is the complement system. Using isogenic bacterial mutant strains and complement-deficient immune naive mice, we show that the S. pneumoniae virulence factor pneumolysin prevents complement deposition on S. pneumoniae, mainly through effects on the classical pathway. In addition, using a double pspA-/ply- mutant strain we demonstrate that pneumolysin and the S. pneumoniae surface protein PspA act in concert to affect both classical and alternative complement pathway activity. As a result, the virulence of the pspA-/ply- strain in models of both systemic and pulmonary infection is greatly attenuated in wild-type mice but not complement deficient mice. The sensitivity of the pspA-/ply- strain to complement was exploited to demonstrate that although early innate immunity to S. pneumoniae during pulmonary infection is partially complement-dependent, the main effect of complement is to prevent spread of S. pneumoniae from the lungs to the blood. These data suggest that inhibition of complement deposition on S. pneumoniae by pneumolysin and PspA is essential for S. pneumoniae to successfully cause septicemia. Targeting mechanisms of complement inhibition could be an effective therapeutic strategy for patients with septicemia due to S. pneumoniae or other bacterial pathogens.     

Phospholipid scramblase 1 mediates type i interferon-induced protection against staphylococcal α-toxin

The opportunistic gram-positive pathogen Staphylococcus aureus is a leading cause of pneumonia and?sepsis. Staphylococcal α-toxin, a prototypical pore-forming toxin, is a major virulence factor of S.?aureus clinical isolates, and lung epithelial cells are highly sensitive to α-toxin's cytolytic activity. Type I interferon (IFN) signaling activated in response to S.?aureus increases pulmonary cell resistance to α-toxin, but the underlying mechanisms are uncharacterized. We show that IFNα protects human lung epithelial cells from α-toxin-induced intracellular ATP depletion and cell death by reducing extracellular ATP leakage. This effect depends on protein palmitoylation and induction of phospholipid scramblase 1 (PLSCR1). IFNα-induced PLSCR1 associates with the cytoskeleton after exposure to α-toxin, and cellular depletion of PLSCR1 negates IFN-induced protection from α-toxin. PLSCR1-deficient mice display enhanced sensitivity to inhaled α-toxin and an α-toxin-producing S.?aureus strain. These results uncover PLSCR1 activity as part of an innate protective mechanism to a bacterial pore-forming toxin.     

MKP1 regulates the induction of MCP1 by Streptococcus pneumoniae pneumolysin in human epithelial cells

Epithelial cells act as the first line of host defense against microbes by producing a range of different molecules for clearance. Chemokines facilitate the clearance of invaders through the recruitment of leukocytes. Thus, upregulation of chemokine expression represents an important innate host defense response against invading microbes such as Streptococcus pneumoniae. In this study, we report that the expression of Monocyte Chemotactic Protein 1 (MCP1) was highly induced in response to S. pneumoniae in vitro and in vivo. Among numerous virulence factors, pneumococcal pneumolysin was found to be the major factor responsible for this induction. Furthermore, MCP1 induction was mediated by the p38 mitogen-activated protein kinase (MAPK) whose activation was controlled by MAPK phosphatase 1 (MKP1). Therefore, this study reveals novel roles of pneumolysin in mediating MKP1 expression for the regulation of MCP1 expression in human epithelial cells.     

Bacterial membrane vesicles deliver peptidoglycan to NOD1 in epithelial cells

Gram‐negative bacterial peptidoglycan is specifically recognized by the host intracellular sensor NOD1, resulting in the generation of innate immune responses. Although epithelial cells are normally refractory to external stimulation with peptidoglycan, these cells have been shown to respond in a NOD1‐dependent manner to Gram‐negative pathogens that can either invade or secrete factors into host cells. In the present work, we report that Gram‐negative bacteria can deliver peptidoglycan to cytosolic NOD1 in host cells via a novel mechanism involving outer membrane vesicles (OMVs). We purified OMVs from the Gram‐negative mucosal pathogens: Helicobacter pylori, Pseudomonas aeruginosa and Neisseria gonorrhoea and demonstrated that these peptidoglycan containing OMVs upregulated NF‐κB and NOD1‐dependent responses in vitro. These OMVs entered epithelial cells through lipid rafts thereby inducing NOD1‐dependent responses in vitro. Moreover, OMVs delivered intragastrically to mice‐induced innate and adaptive immune responses via a NOD1‐dependent but TLR‐independent mechanism. Collectively, our findings identify OMVs as a generalized mechanism whereby Gram‐negative bacteria deliver peptidoglycan to cytosolic NOD1. We propose that OMVs released by bacteria in vivo may promote inflammation and pathology in infected hosts.     

Synergistic activation of NF-kappaB by nontypeable H. influenzae and S. pneumoniae is mediated by CK2, IKKbeta-IkappaBalpha, and p38 MAPK

In review of the past studies on NF-kappaB regulation, most of them have focused on investigating how NF-kappaB is activated by a single inducer at a time. Given the fact that, in mixed bacterial infections in vivo, multiple inflammation inducers, including both nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae, are present simultaneously, a key issue that has yet to be addressed is whether NTHi and S. pneumoniae simultaneously activate NF-kappaB and the subsequent inflammatory response in a synergistic manner. Here, we show that NTHi and S. pneumoniae synergistically induce NF-kappaB-dependent inflammatory response via activation of multiple signaling pathways in vitro and in vivo. The classical IKKbeta-IkappaBalpha and p38 MAPK pathways are involved in synergistic activation of NF-kappaB via two distinct mechanisms, p65 nuclear translocation-dependent and -independent mechanisms. Moreover, casein kinase 2 (CK2) is involved in synergistic induction of NF-kappaB via a mechanism dependent on phosphorylation of p65 at both Ser536 and Ser276 sites. These studies bring new insights into the molecular mechanisms underlying the NF-kappaB-dependent inflammatory response in polymicrobial infections and may lead to development of novel therapeutic strategies for modulating inflammation in mixed infections for patients with otitis media and chronic obstructive pulmonary diseases.     

Role of p38 MAP kinase and transforming growth factor-beta signaling in transepithelial migration of invasive bacterial pathogens

Streptococcus pneumoniae and Haemophilus influenzae are human pathogens that often asymptomatically colonize the mucosal surface of the upper respiratory tract, but also occasionally cause invasive disease. The ability of these species to traverse the epithelium of the airway mucosa was modeled in vitro using polarized respiratory epithelial cells in culture. Migration across the epithelial barrier was preceded by loss of transepithelial resistance. Membrane products of S. pneumoniae that included lipoteichoic acid induced disruption of the epithelial barrier in a Toll-like receptor 2-dependent manner. This result correlates with a recent genetic study that associates increased TLR2 signaling with increased rates of invasive pneumococcal disease in humans. Loss of transepithelial resistance by the TLR2 ligand correlated with activation of p38 MAP kinase and transforming growth factor (TGF)-beta signaling. Activation of p38 MAPK and TGF-beta signaling in epithelial cells upon nasal infection with S. pneumoniae was also demonstrated in vivo. Inhibition of either p38 MAPK or TGF-beta signaling was sufficient to inhibit the migration of S. pneumoniae or H. influenzae. Our data shows that diverse bacteria utilize common mechanisms, including MAPK and TGF-beta signaling pathways to disrupt epithelial barriers and promote invasion.     

Identification of a secreted cholesterol-dependent cytolysin (mitilysin) from Streptococcus mitis

We have detected a cholesterol-dependent cytolysin, which we have named mitilysin, in a small number of Streptococcus mitis isolates. We have sequenced the mitilysin gene from seven isolates of S. mitis. Comparisons with the pneumococcal pneumolysin gene show 15 amino acid substitutions. S. mitis appear to release mitilysin extracellularly. Certain alleles of mitilysin are not recognized by a monoclonal antibody raised to the related toxin pneumolysin. Based on enzyme-linked immunosorbent assay and neutralization assay results, one isolate of S. mitis may produce a further hemolytic toxin in addition to mitilysin. As genetic exchange is known to occur between S. mitis and Streptococcus pneumoniae, this finding may have implications for the development of vaccines or therapies for pneumococcal disease that are based on pneumolysin.     

MyD88 signaling is not essential for induction of antigen-specific B cell responses but is indispensable for protection against Streptococcus pneumoniae infection following oral vaccination with attenuated Salmonella expressing PspA antigen

TLRs directly induce innate host defense responses, but the mechanisms of TLR-mediated adaptive immunity remain subject to debate. In this study, we clarified a role of TLR-mediated innate immunity for induction of adaptive immunity by oral vaccination with a live recombinant attenuated Salmonella enteric serovar Typhimurium vaccine (RASV) strain expressing Streptococcus pneumoniae surface protein A (PspA) Ag. Of note, oral or intranasal vaccination with RASV expressing PspA resulted in identical or even significantly higher levels of PspA-specific IgG and IgA responses in the systemic and mucosal compartments of MyD88(-/-) mice of either BALB/c or C57BL/6 background when compared with those of wild-type mice. Although PspA-specific CD4(+) T cell proliferation in the MyD88(-/-) mice was minimal, depletion of CD4(+) T cells abolished PspA-specific IgG and IgA responses in the MyD88(-/-) mice of BALB/c background. Of the greatest interest, MyD88(-/-) mice that possessed high levels of PspA-specific IgG and IgA responses but minimal levels of CD4(+) T cell responses died earlier than nonvaccinated and vaccinated wild-type mice following i.v. or intranasal challenge with virulent S. pneumoniae. Taken together, these results suggest that innate immunity activated by MyD88 signals might not be necessary for Ag-specific Ab induction in both systemic and mucosal sites but is critical for protection following oral vaccination with attenuated Salmonella expressing PspA.     

Nod1 participates in the innate immune response to Pseudomonas aeruginosa

The mammalian innate immune system recognizes pathogen-associated molecular patterns through pathogen recognition receptors. Nod1 has been described recently as a cytosolic receptor that detects specifically diaminopimelate-containing muropeptides from Gram-negative bacteria peptidoglycan. In the present study we investigated the potential role of Nod1 in the innate immune response against the opportunistic pathogen Pseudomonas aeruginosa. We demonstrate that Nod1 detects the P. aeruginosa peptidoglycan leading to NF-kappaB activation and that this activity is diminished in epithelial cells expressing a dominant-negative Nod1 construct or in mouse embryonic fibroblasts from Nod1 knock-out mice infected with P. aeruginosa. Finally, we demonstrate that the cytokine secretion kinetics and bacterial killing are altered in Nod1-deficient cells infected with P. aeruginosa in the early stages of infection.