Jiangtao Ma

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.

Natural antibodies against several pneumococcal virulence proteins in children during the pre-pneumococcal-vaccine era: the generation R study.

ABSTRACT The currently available pneumococcal vaccines do not protect against all serotypes of Streptococcus pneumoniae. A shift toward nonvaccine serotypes causing colonization and invasive disease has occurred, and studies on protein-based vaccines have been undertaken. We assessed the association between specific antibodies against pneumococcal virulence proteins and colonization and respiratory tract infections (RTIs). Additionally, we assessed the extent to which colonization induces a humoral immune response. Nasopharyngeal swabs collected from children at 1.5, 6, 14, and 24 months of age were cultured for pneumococcus. Serum samples were obtained at birth and at 6, 14, and 24 months (n = 57 children providing 177 serum samples). Data were collected prior to the pneumococcal vaccine era. IgG, IgA, and IgM levels against 17 pneumococcal protein vaccine candidates were measured using a bead-based flow cytometry technique (xMAP; Luminex Corporation). Information regarding RTIs was questionnaire derived. Levels of IgG against all proteins were high in cord blood, decreased in the first 6 months and increased again thereafter, in contrast to the course of IgA and IgM levels. Specific antibodies were induced upon colonization. Increased levels of IgG against BVH-3, NanA, and SP1003 at 6 months, NanA, PpmA, PsaA, SlrA, SP0189, and SP1003 at 14 months, and SlrA at 24 months were associated with a decreased number of RTIs in the third year of life but not with colonization. Maternal antipneumococcal antibodies did not protect against pneumococcal colonization and infection. Certain antibodies against pneumococcal virulence proteins, some of which are induced by colonization, are associated with a decreased number of RTIs in children. This should be taken into account in future pneumococcal vaccine studies.

Novel mucosal vaccines generated by genetic conjugation of heterologous proteins to pneumolysin (PLY) from Streptococcus pneumoniae

Induction of immunity at mucosal surfaces is thought to be an essential feature in the protection of the host against the many pathogens that gain access through these surfaces. Here we describe how strong local and systemic immune responses can be generated when proteins are genetically conjugated to pneumolysin (PLY) from Streptococcus pneumoniae. Using green fluorescent protein (eGFP) and PsaA from S. pneumoniae, we have shown that genetic fusion (eGFPPLY and PsaAPLY) is essential to ensure high levels of antigen specific IgG and IgA in the serum and at mucosal surfaces. This form of vaccination is highly effective with antigen specific antibodies detected after a single dose of nanogram quantities of the conjugated proteins. In addition, generation of a non-toxic variant (eGFPDelta6PLY) indicated that while the toxic activity of PLY was not essential for adjuvanticity, it contributed to the magnitude of the response generated. Whilst vaccination with the PsaAPLY fusion proteins did not protect the animals from challenge, these studies confirm the utility of pneumolysin to act as a novel mucosal adjuvant to substantially increase the local and systemic humoral response to genetically fused protein antigens.

Bacterial Cytolysin during Meningitis Disrupts the Regulation of Glutamate in the Brain, Leading to Synaptic Damage

Streptococcus pneumoniae (pneumococcal) meningitis is a common bacterial infection of the brain. The cholesterol-dependent cytolysin pneumolysin represents a key factor, determining the neuropathogenic potential of the pneumococci. Here, we demonstrate selective synaptic loss within the superficial layers of the frontal neocortex of post-mortem brain samples from individuals with pneumococcal meningitis. A similar effect was observed in mice with pneumococcal meningitis only when the bacteria expressed the pore-forming cholesterol-dependent cytolysin pneumolysin. Exposure of acute mouse brain slices to only pore-competent pneumolysin at disease-relevant, non-lytic concentrations caused permanent dendritic swelling, dendritic spine elimination and synaptic loss. The NMDA glutamate receptor antagonists MK801 and D-AP5 reduced this pathology. Pneumolysin increased glutamate levels within the mouse brain slices. In mouse astrocytes, pneumolysin initiated the release of glutamate in a calcium-dependent manner. We propose that pneumolysin plays a significant synapto- and dendritotoxic role in pneumococcal meningitis by initiating glutamate release from astrocytes, leading to subsequent glutamate-dependent synaptic damage. We outline for the first time the occurrence of synaptic pathology in pneumococcal meningitis and demonstrate that a bacterial cytolysin can dysregulate the control of glutamate in the brain, inducing excitotoxic damage.

Changes in Astrocyte Shape Induced by Sublytic Concentrations of the Cholesterol-Dependent Cytolysin Pneumolysin Still Require Pore-Forming Capacity

Streptococcus pneumoniae is a common pathogen that causes various infections, such as sepsis and meningitis. A major pathogenic factor of S. pneumoniae is the cholesterol-dependent cytolysin, pneumolysin. It produces cell lysis at high concentrations and apoptosis at lower concentrations. We have shown that sublytic amounts of pneumolysin induce small GTPase-dependent actin cytoskeleton reorganization and microtubule stabilization in human neuroblastoma cells that are manifested by cell retraction and changes in cell shape. In this study, we utilized a live imaging approach to analyze the role of pneumolysin’s pore-forming capacity in the actin-dependent cell shape changes in primary astrocytes. After the initial challenge with the wild-type toxin, a permeabilized cell population was rapidly established within 20-40 minutes. After the initial rapid permeabilization, the size of the permeabilized population remained unchanged and reached a plateau. Thus, we analyzed the non-permeabilized (non-lytic) population, which demonstrated retraction and shape changes that were inhibited by actin depolymerization. Despite the non-lytic nature of pneumolysin treatment, the toxin’s lytic capacity remained critical for the initiation of cell shape changes. The non-lytic pneumolysin mutants W433F-pneumolysin and delta6-pneumolysin, which bind the cell membrane with affinities similar to that of the wild-type toxin, were not able to induce shape changes. The initiation of cell shape changes and cell retraction by the wild-type toxin were independent of calcium and sodium influx and membrane depolarization, which are known to occur following cellular challenge and suggested to result from the ion channel-like properties of the pneumolysin pores. Excluding the major pore-related phenomena as the initiation mechanism of cell shape changes, the existence of a more complex relationship between the pore-forming capacity of pneumolysin and the actin cytoskeleton reorganization is suggested.

Direct transmembrane interaction between actin and the pore-competent, cholesterol-dependent cytolysin pneumolysin.

The eukaryotic actin cytoskeleton is an evolutionarily well-established pathogen target, as a large number of bacterial factors disturb its dynamics to alter the function of the host cells. These pathogenic factors modulate or mimic actin effector proteins or they modify actin directly, leading to an imbalance of the precisely regulated actin turnover. Here, we show that the pore-forming, cholesterol-dependent cytolysin pneumolysin (PLY), a major neurotoxin of Streptococcus pneumoniae, has the capacity to bind actin directly and to enhance actin polymerisation in vitro. In cells, the toxin co-localised with F-actin shortly after exposure, and this direct interaction was verified by Förster resonance energy transfer. PLY was capable of exerting its effect on actin through the lipid bilayer of giant unilamellar vesicles, but only when its pore competence was preserved. The dissociation constant of G-actin binding to PLY in a biochemical environment was 170–190 nM, which is indicative of a high-affinity interaction, comparable to the affinity of other intracellular actin-binding factors. Our results demonstrate the first example of a direct interaction of a pore-forming toxin with cytoskeletal components, suggesting that the cross talk between pore-forming cytolysins and cells is more complex than previously thought.

Extracellular Calcium Reduction Strongly Increases the Lytic Capacity of Pneumolysin From Streptococcus Pneumoniae in Brain Tissue

Background. Streptococcus pneumoniae causes serious diseases such as pneumonia and meningitis. Its major pathogenic factor is the cholesterol-dependent cytolysin pneumolysin, which produces lytic pores at high concentrations. At low concentrations, it has other effects, including induction of apoptosis. Many cellular effects of pneumolysin appear to be calcium dependent. Methods. Live imaging of primary mouse astroglia exposed to sublytic amounts of pneumolysin at various concentrations of extracellular calcium was used to measure changes in cellular permeability (as judged by lactate dehydrogenase release and propidium iodide chromatin staining). Individual pore properties were analyzed by conductance across artificial lipid bilayer. Tissue toxicity was studied in continuously oxygenated acute brain slices. Results. The reduction of extracellular calcium increased the lytic capacity of the toxin due to increased membrane binding. Reduction of calcium did not influence the conductance properties of individual toxin pores. In acute cortical brain slices, the reduction of extracellular calcium from 2 to 1 mM conferred lytic activity to pathophysiologically relevant nonlytic concentrations of pneumolysin. Conclusions. Reduction of extracellular calcium strongly enhanced the lytic capacity of pneumolysin due to increased membrane binding. Thus, extracellular calcium concentration should be considered as a factor of primary importance for the course of pneumococcal meningitis.

Astrocytic tissue remodeling by the meningitis neurotoxin pneumolysin facilitates pathogen tissue penetration and produces interstitial brain edema

Astrocytes represent a major component of brain tissue and play a critical role in the proper functioning and protection of the brain. Streptococcus pneumoniae, the most common cause of bacterial meningitis, has a high lethality and causes serious disabilities in survivors. Pneumolysin (PLY), a member of the cholesterol‐dependent cytolysin group and a major S. pneumoniae neurotoxin, causes deterioration over the course of experimental S. pneumoniae meningitis. At disease‐relevant sub‐lytic concentrations, PLY produces actin and tubulin reorganization and astrocyte cell shape changes in vitro. In this article, we show that sub‐lytic amounts of PLY remodel brain tissue and produce astrocytic process retraction, cortical astroglial reorganization and increased interstitial fluid retention, which is manifested as tissue edema. These changes caused increased tissue permeability to macromolecules and bacteria. The pore‐forming capacity of PLY remained necessary for these changes because none of the nonpore‐forming mutants were capable of producing similar effects. We suggest that PLY can increase the permeability of brain tissue toward pathogenic factors and bacteria in the course of meningitis, thus contributing to the deterioration caused by the disease.

Genetic conjugation of components in two pneumococcal fusion protein vaccines enhances paediatric mucosal immune responses.

Streptococcus pneumoniae colonises the upper respiratory tract and can cause pneumonia, meningitis and otitis media. Existing pneumococcal conjugate vaccines are expensive to produce and only protect against 13 of the 90+ pneumococcal serotypes; hence there is an urgent need for the development of new vaccines. We have shown previously in mice that pneumolysin (Ply) and a non-toxic variant (Δ6Ply) enhance antibody responses when genetically fused to pneumococcal surface adhesin A (PsaA), a potentially valuable effect for future vaccines. We investigated this adjuvanticity in human paediatric mucosal primary immune cell cultures. Adenoidal mononuclear cells (AMNC) from children aged 0-15 years (n=46) were stimulated with conjugated, admixed or individual proteins, cell viability and CD4+ T-cell proliferative responses were assessed using flow cytometry and cytokine secretion was measured using multiplex technology. Proliferation of CD4+ T-cells in response to PsaAPly, was significantly higher than responses to individual or admixed proteins (p=0.002). In contrast, an enhanced response to PsaAΔ6Ply compared to individual or admixed proteins only occurred at higher concentrations (p<0.01). Evaluation of cytotoxicity suggested that responses occurred when Ply-induced cytolysis was inhibited, either by fusion or mutation, but importantly an additional toxicity independent immune enhancing effect was also apparent as a result of fusion. Responses were MHC class II dependent and had a Th1/Th17 profile. Genetic fusion of Δ6Ply to PsaA significantly modulates and enhances pro-inflammatory CD4+ T-cell responses without the cytolytic effects of some other pneumolysoids. Membrane binding activity of such proteins may confer valuable adjuvant properties as fusion may assist Δ6Ply to deliver PsaA to the APC surface effectively, contributing to the initiation of anti-pneumococcal CD4+ T-cell immunity.

Expression of the lux genes in Streptococcus pneumoniae modulates pilus expression and virulence

Bioluminescence has been harnessed for use in bacterial reporter systems and for in vivo imaging of infection in animal models. Strain Xen35, a bioluminescent derivative of Streptococcus pneumoniae serotype 4 strain TIGR4 was previously constructed for use for in vivo imaging of infections in animal models. We have shown that strain Xen35 is less virulent than its parent TIGR4 and that this is associated with the expression of the genes for bioluminescence. The expression of the luxA-E genes in the pneumococcus reduces virulence and down regulates the expression of the pneumococcal pilus.