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In pneumococcal meningitis, bacterial growth in the cerebrospinal fluid results in lysis, the release of toxic factors, and subsequent neuroinflammation. Exposure of primary murine glia to Streptococcus pneumoniae lysates leads to strong proinflammatory cytokine and chemokine production, blocked by inhibition of the intracellular innate receptor Nod1. Lysates enhance dynamin-dependent endocytosis, and dynamin inhibition reduces neuroinflammation, blocking ligand internalization. Here we identify the cholesterol-dependent cytolysin pneumolysin as a pro-endocytotic factor in lysates, its elimination reduces their proinflammatory effect. Only pore-competent pneumolysin enhances endocytosis in a dynamin-, phosphatidylinositol-3-kinase- and potassium-dependent manner. Endocytic enhancement is limited to toxin-exposed parts of the membrane, the effect is rapid and pneumolysin permanently alters membrane dynamics. In a murine model of pneumococcal meningitis, mice treated with chlorpromazine, a neuroleptic with a complementary endocytosis inhibitory effect show reduced neuroinflammation. Thus, the dynamin-dependent endocytosis emerges as a factor in pneumococcal neuroinflammation, and its enhancement by a cytolysin represents a proinflammatory control mechanism. Pneumococcal infection of the cerebrospinal fluid results in bacterial lysis, the release of toxic factors and induction of neuroinflammation. Here, the authors show that the virulence factor pneumolysin enhances the neuroinflammatory response to Streptococcus pneumoniae by the enhancement of endocytosis.

Abstract Rationale Antimicrobial resistance challenges therapy of pneumonia. Enhancing macrophage microbicidal responses would combat this problem but is limited by our understanding of how alveolar macrophages (AMs) kill bacteria. Objectives To define the role and mechanism of AM apoptosis–associated bacterial killing in the lung. Methods We generated a unique CD68.hMcl-1 transgenic mouse with macrophage-specific overexpression of the human antiapoptotic Mcl-1 protein, a factor upregulated in AMs from patients at increased risk of community-acquired pneumonia, to address the requirement for apoptosis-associated killing. Measurements and Main Results Wild-type and transgenic macrophages demonstrated comparable ingestion and initial phagolysosomal killing of bacteria. Continued ingestion (for ≥12 h) overwhelmed initial killing, and a second, late-phase microbicidal response killed viable bacteria in wild-type macrophages, but this response was blunted in CD68.hMcl-1 transgenic macrophages. The late phase of bacterial killing required both caspase-induced generation of mitochondrial reactive oxygen species and nitric oxide, the peak generation of which coincided with the late phase of killing. The CD68.hMcl-1 transgene prevented mitochondrial reactive oxygen species but not nitric oxide generation. Apoptosis-associated killing enhanced pulmonary clearance of Streptococcus pneumoniae and Haemophilus influenzae in wild-type mice but not CD68.hMcl-1 transgenic mice. Bacterial clearance was enhanced in vivo in CD68.hMcl-1 transgenic mice by reconstitution of apoptosis with BH3 mimetics or clodronate-encapsulated liposomes. Apoptosis-associated killing was not activated during Staphylococcus aureus lung infection. Conclusions Mcl-1 upregulation prevents macrophage apoptosis–associated killing and establishes that apoptosis-associated killing is required to allow AMs to clear ingested bacteria. Engagement of macrophage apoptosis should be investigated as a novel, host-based antimicrobial strategy.

Key Points Streptococci are often carried on mucosal surfaces and skin without causing disease. Streptococci can cause diseases that range from dental caries to necrotizing fasciitis. The type of disease caused depends on the virulence factors produced by the bacterium, as well as host susceptibility. Streptococcus mutans causes dental caries; this requires the ability of this organism to form biofilms and to produce acid on the tooth surface. Streptococcus agalactiae — group B streptococcus (GBS) — can cause neonatal meningitis. The organism is usually acquired by the neonate during passage through the birth canal of a colonized mother. Key virulence factors are the capsule, surface proteins and a haemolysin. Streptococcus pyogenes — group A streptococcus (GAS) — can cause a wide range of diseases. Toxic-shock syndrome that is caused by this organism is mediated by the production of superantigens. The organism makes a capsule, a wide range of toxins and enzymes, and molecules that interfere with innate immunity. Streptococcus pneumoniae (the pneumococcus) can cause pneumonia and meningitis. The organism induces overactivation of the inflammatory response, and disease symptoms partly reflect inappropriate production of host immune mediators. Key virulence factors are the capsule, surface proteins and pneumolysin. Complete genome sequences are available for nine strains of these four species of streptococci (one for S. mutans , two for GBS, four for GAS and two for pneumococcus). Analysis of the genes present in all four species shows that the highly conserved genes are concerned with core function, such as nucleic-acid metabolism and protein synthesis. There is also significant variation in gene content, even between strains of the same species. Simultaneous analysis of expression of all genes in the genome is now possible using microarray technology. It is also possible to examine the expression of genes in organisms that cause infection in model systems or in human disease. Such studies are beginning to allow an understanding of the detailed interactions that occur between pathogens and their hosts. The development of bacterial disease has been likened to a 'molecular arms race', in which the host tries to eliminate the bacteria, while the bacteria try to survive in the host. Although most bacteria do not cause disease, some cause serious human infection in a large proportion of encounters. Between these two extremes are bacteria that can coexist with humans in a carriage state but, under appropriate circumstances, cause disease. The streptococci exemplify this group of organisms, and by studying them we can begin to address why bacteria cause such a wide spectrum of disease.

Abstract BACKGROUND: Recent findings associated with resting-state cortical networks have provided insight into the brain's organizational structure. In addition to their neuroscientific implications, the networks identified by resting-state functional magnetic resonance imaging (rs-fMRI) may prove useful for clinical brain mapping. OBJECTIVE: To demonstrate that a data-driven approach to analyze resting-state networks (RSNs) is useful in identifying regions classically understood to be eloquent cortex as well as other functional networks. METHODS: This study included 6 patients undergoing surgical treatment for intractable epilepsy and 7 patients undergoing tumor resection. rs-fMRI data were obtained before surgery and 7 canonical RSNs were identified by an artificial neural network algorithm. Of these 7, the motor and language networks were then compared with electrocortical stimulation (ECS) as the gold standard in the epilepsy patients. The sensitivity and specificity for identifying these eloquent sites were calculated at varying thresholds, which yielded receiver-operating characteristic (ROC) curves and their associated area under the curve (AUC). RSNs were plotted in the tumor patients to observe RSN distortions in altered anatomy. RESULTS: The algorithm robustly identified all networks in all patients, including those with distorted anatomy. When all ECS-positive sites were considered for motor and language, rs-fMRI had AUCs of 0.80 and 0.64, respectively. When the ECS-positive sites were analyzed pairwise, rs-fMRI had AUCs of 0.89 and 0.76 for motor and language, respectively. CONCLUSION: A data-driven approach to rs-fMRI may be a new and efficient method for preoperative localization of numerous functional brain regions.

The opportunistic pathogen Streptococcus pneumoniae has dual lifestyles: one of an asymptomatic colonizer in the human nasopharynx and the other of a deadly pathogen invading sterile host compartments. The latter triggers an overwhelming inflammatory response, partly driven via pore forming activity of the cholesterol dependent cytolysin (CDC), pneumolysin. Although pneumolysin-induced inflammation drives person-to-person transmission from nasopharynx, the primary reservoir for pneumococcus, it also contributes to high mortality rates, creating a bottleneck that hampers widespread bacterial dissemination, thus acting as a double-edged sword. Serotype 1 ST306, a widespread pneumococcal clone, harbours a non-hemolytic variant of pneumolysin (Ply-NH). Performing crystal structure analysis of Ply-NH, we identified Y150H and T172I as key substitutions responsible for loss of its pore forming activity. We uncovered a novel inter-molecular cation-π interaction, governing formation of the transmembrane β-hairpins (TMH) in the pore state of Ply, which can be extended to other CDCs. H150 in Ply-NH disrupts this interaction, while I172 provides structural rigidity to domain-3, through hydrophobic interactions, inhibiting TMH formation. Loss of pore forming activity enabled improved cellular invasion and autophagy evasion, promoting an atypical intracellular lifestyle for pneumococcus, a finding that was corroborated in in vivo infection models. Attenuation of inflammatory responses and tissue damage promoted tolerance of Ply-NH-expressing pneumococcus in the lower respiratory tract. Adoption of this altered lifestyle may be necessary for ST306 due to its limited nasopharyngeal carriage, with Ply-NH, aided partly by loss of its pore forming ability, facilitating a benign association of SPN in an alternative, intracellular host niche.

A major Streptococcus pneumoniae pathogenic factor is the cholesterol-dependent cytolysin pneumolysin, binding membrane cholesterol and producing permanent lytic or transient pores. During brain infections, vascular damage with variable ischemia occurs. The role of ischemia on pneumolysin’s pore-forming capacity remains unknown. In acute brain slice cultures and primary cultured glia, we studied acute toxin lysis (via propidium iodide staining and LDH release) and transient pore formation (by analyzing increases in the intracellular calcium). We analyzed normal peripheral tissue glucose conditions (80 mg%), normal brain glucose levels (20 mg%), and brain hypoglycemic conditions (3 mg%), in combinations either with normoxia (8% oxygen) or hypoxia (2% oxygen). At 80 mg% glucose, hypoxia enhanced cytolysis via pneumolysin. At 20 mg% glucose, hypoxia did not affect cell lysis, but impaired calcium restoration after non-lytic pore formation. Only at 3 mg% glucose, during normoxia, did pneumolysin produce stronger lysis. In hypoglycemic (3 mg% glucose) conditions, pneumolysin caused a milder calcium increase, but restoration was missing. Microglia bound more pneumolysin than astrocytes and demonstrated generally stronger calcium elevation. Thus, our work demonstrated that the toxin pore-forming capacity in cells continuously diminishes when oxygen is reduced, overlapping with a continuously reduced ability of cells to maintain homeostasis of the calcium influx once oxygen and glucose are reduced.

(pneumococcus) meningitis is a serious disease with substantial lethality and long-term disability in survivors. Loss of synaptic staining in the superficial layers of the neocortex in rodent models and in humans, and pneumolysin (a major pneumococcal toxin)-dependent dendritic spine collapse in brain slices have been described. It remains unclear how deep in the neocortex more discrete changes are present, how soon after disease onset these changes occur, and whether other properties of dendrites are also affected. Using a mouse model of pneumococcal meningitis, we studied changes in the neocortex shortly (3-6 h) after the onset of clinical symptoms modified Golgi-Cox silver staining. Dendritic changes were present in areas with otherwise unchanged cell numbers and no signs of necrosis or other apparent neuronal pathology. Mature dendritic spines were reduced in the pyramidal neurons running through layers 1-5. Additionally, spine morphology changes (swelling, spine neck distortion), were also observed in the deeper layers 4 and 5 of the neocortex. Immature spines (filopodia) remained unchanged between groups, as well as the dendritic arborization of the analyzed neurons. In a third of the animals with meningitis, massive mechanical distortion of the primary dendrites of most of the pyramidal neurons through layers 1-5 was observed. This distortion was reproduced in acute brain slices after exposure to pneumolysin-containing bacterial lysates ( D39 strain), but not to lysates of pneumolysin-deficient bacteria, which we explain by the tissue remodeling effect of the toxin. Experimental mechanical dendrite distortion in primary neural cultures demonstrated diminished FRAP diffusion of neuronally-expressed enhanced green fluorescent protein (eGFP), indicative of disturbed dendritic diffusion. Our work extends earlier knowledge of synaptic loss in the superficial cortical layers during meningitis to deeper layers. These changes occurred surprisingly early in the course of the disease, substantially limiting the effective therapeutic window. Methodologically, we demonstrate that the dendritic spine collapse readout is a highly reliable and early marker of neural damage in pneumococcal meningitis models, allowing for reduction of the total number of animals used per a group due to much lower variation among animals.

Background Community-acquired pneumonia (CAP) is a significant cause of morbidity and mortality worldwide. Despite effective antimicrobial therapy, CAP can induce pulmonary endothelial hyperpermeability resulting in life-threatening lung failure due to an exaggerated host-pathogen interaction. Treatment of acute lung injury is mainly supportive because key elements of inflammation-induced barrier disruption remain undetermined. Angiopoietin-1 (Ang-1)-mediated Tie2 activation reduces, and the Ang-1 antagonist Ang-2 increases, inflammation and endothelial permeability in sepsis. Vasculotide (VT) is a polyethylene glycol-clustered Tie2-binding peptide that mimics the actions of Ang-1. The aim of our study was to experimentally test whether VT is capable of diminishing pneumonia-induced lung injury. Methods VT binding and phosphorylation of Tie2 were analyzed using tryptophan fluorescence spectroscopy and phospho-Tie-2 enzyme-linked immunosorbent assay. Human and murine lung endothelial cells were investigated by immunofluorescence staining and electric cell-substrate impedance sensing. Pulmonary hyperpermeability was quantified in VT-pretreated, isolated, perfused, and ventilated mouse lungs stimulated with the pneumococcal exotoxin pneumolysin (PLY). Furthermore, Streptococcus pneumoniae -infected mice were therapeutically treated with VT. Results VT showed dose-dependent binding and phosphorylation of Tie2. Pretreatment with VT protected lung endothelial cell monolayers from PLY-induced disruption. In isolated mouse lungs, VT decreased PLY-induced pulmonary permeability. Likewise, therapeutic treatment with VT of S. pneumoniae -infected mice significantly reduced pneumonia-induced hyperpermeability. However, effects by VT on the pulmonary or systemic inflammatory response were not observed. Conclusions VT promoted pulmonary endothelial stability and reduced lung permeability in different models of pneumococcal pneumonia. Thus, VT may provide a novel therapeutic perspective for reduction of permeability in pneumococcal pneumonia-induced lung injury.

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.

Background Streptococcus pneumoniae is the cause of a highly lethal form of meningitis in humans. Microglial cells in the brain represent the first line of defense against pathogens, and they participate in the inflammatory response. The cholesterol-dependent cytolysin pneumolysin and the bacterial capsule are key pathogenic factors, known to exacerbate the course of pneumococcal meningitis. Methods We utilized live imaging and immunostaining of glial cells in dissociated and acute brain slice cultures to study the effect of pneumococcal factors, including the cholesterol-dependent cytolysin pneumolysin and the pneumococcal capsule, on microglial motility and taxis. Results In brain tissue, primary microglia cells showed an enhanced response towards lysates from bacteria lacking capsules and pneumolysin as they moved rapidly to areas with an abundance of bacterial factors. The presence of bacterial capsules and pneumolysin cumulatively inhibited microglial taxis. In mixed cultures of astrocytes and microglia, the motility of microglia was inhibited by capsular components within minutes after exposure. The reduced motility was partially reversed by mannan, a mannose receptor inhibitor. The effects on microglia were not mediated by astrocytes because pure microglial cells responded to various pneumococcal lysates similarly with distinct cell shape changes as seen in mixed cultures. Conclusions Our data indicate that microglia possess the capacity for a very agile response towards bacterial pathogens, but key pathogenic factors, such as pneumococcal capsules and pneumolysin, inhibited this response shortly after a bacterial challenge. Furthermore, we demonstrate for the first time that the bacterial capsule affects cellular behaviors such as motility and taxis.