Timothy J. Mitchell

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.

American Power and Anti-Americanism in the Middle East was published in Anti-Americanism on page 87.

Pneumonia is the most common cause of the acute respiratory distress syndrome (ARDS). Here, we identified loss of endothelial cystic fibrosis transmembrane conductance regulator (CFTR) as an important pathomechanism leading to lung barrier failure in pneumonia-induced ARDS. CFTR was down-regulated after Streptococcus pneumoniae infection ex vivo or in vivo in human or murine lung tissue, respectively. Analysis of isolated perfused rat lungs revealed that CFTR inhibition increased endothelial permeability in parallel with intracellular chloride ion and calcium ion concentrations ([Cl ⁻ ] _i and [Ca ²⁺ ] _i ). Inhibition of the chloride ion–sensitive with-no-lysine kinase 1 (WNK1) protein with tyrphostin 47 or WNK463 replicated the effect of CFTR inhibition on endothelial permeability and endothelial [Ca ²⁺ ] _i , whereas WNK1 activation by temozolomide attenuated it. Endothelial [Ca ²⁺ ] _i transients and permeability in response to inhibition of either CFTR or WNK1 were prevented by inhibition of the cation channel transient receptor potential vanilloid 4 (TRPV4). Mice deficient in Trpv4 ( Trpv4 ^−/− ) developed less lung edema and protein leak than their wild-type littermates after infection with S. pneumoniae . The CFTR potentiator ivacaftor prevented lung CFTR loss, edema, and protein leak after S. pneumoniae infection in wild-type mice. In conclusion, lung infection caused loss of CFTR that promoted lung edema formation through intracellular chloride ion accumulation, inhibition of WNK1, and subsequent disinhibition of TRPV4, resulting in endothelial calcium ion influx and vascular barrier failure. Ivacaftor prevented CFTR loss in the lungs of mice with pneumonia and may, therefore, represent a possible therapeutic strategy in people suffering from ARDS due to severe pneumonia.

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 toStreptococcus pneumoniaelysates 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.

Pneumolysin (PLY) is a pore-forming toxin of Streptococcus pneumoniae that contributes substantially to the inflammatory processes underlying pneumococcal pneumonia and lung injury. Host responses against S. pneumoniae are regulated in part by neutrophils and platelets, both individually and in cooperative interaction. Previous studies have shown that PLY can target both neutrophils and platelets, however, the mechanisms by which PLY directly affects these cells and alters their interactions are not completely understood. In this study, we characterize the effects of PLY on neutrophils and platelets and explore the mechanisms by which PLY may induce neutrophil–platelet interactions. In vitro studies demonstrated that PLY causes the formation of neutrophil extracellular traps (NETs) and the release of extracellular vesicles (EVs) from both human and murine neutrophils. In vivo, neutrophil EV (nEV) levels were increased in mice infected with S. pneumoniae. In platelets, treatment with PLY induced the cell surface expression of P-selectin (CD62P) and binding to annexin V and caused a significant release of platelet EVs (pl-EVs). Moreover, PLY-induced nEVs but not NETs promoted platelet activation. The pretreatment of nEVs with proteinase K inhibited platelet activation, indicating that the surface proteins of nEVs play a role in this process. Our findings demonstrate that PLY activates neutrophils and platelets to release EVs and support an important role for neutrophil EVs in modulating platelet functions in pneumococcal infections.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

Epigenetic modifications regulate gene expression in the host response to a diverse range of pathogens. The extent and consequences of epigenetic modification during macrophage responses to Streptococcus pneumoniae , and the role of pneumolysin, a key Streptococcus pneumoniae virulence factor, in influencing these responses, are currently unknown. To investigate this, we infected human monocyte derived macrophages (MDMs) with Streptococcus pneumoniae and addressed whether pneumolysin altered the epigenetic landscape and the associated acute macrophage transcriptional response using a combined transcriptomic and proteomic approach. Transcriptomic analysis identified 503 genes that were differentially expressed in a pneumolysin-dependent manner in these samples. Pathway analysis highlighted the involvement of transcriptional responses to core innate responses to pneumococci including modules associated with metabolic pathways activated in response to infection, oxidative stress responses and NFκB, NOD-like receptor and TNF signalling pathways. Quantitative proteomic analysis confirmed pneumolysin-regulated protein expression, early after bacterial challenge, in representative transcriptional modules associated with innate immune responses. In parallel, quantitative mass spectrometry identified global changes in the relative abundance of histone post translational modifications (PTMs) upon pneumococcal challenge. We identified an increase in the relative abundance of H3K4me1, H4K16ac and a decrease in H3K9me2 and H3K79me2 in a PLY-dependent fashion. We confirmed that pneumolysin blunted early transcriptional responses involving TNF-α and IL-6 expression. Vorinostat, a histone deacetylase inhibitor, similarly downregulated TNF production, reprising the pattern observed with pneumolysin. In conclusion, widespread changes in the macrophage transcriptional response are regulated by pneumolysin and are associated with global changes in histone PTMs. Modulating histone PTMs can reverse pneumolysin-associated transcriptional changes influencing innate immune responses, suggesting that epigenetic modification by pneumolysin plays a role in dampening the innate responses to pneumococci. Author summary: Pneumolysin is a toxin that contributes to how Streptococcus pneumoniae , the leading cause of pneumonia, causes disease. In this study, the toxin alters gene expression in immune cells called macrophages, one of the first lines of defence against bacteria at sites of infection. Modulation involved multiple immune responses, including generation of chemical signals coordinating responses in immune cells termed cytokines. In addition, changes were observed in histone proteins that are involved in controlling gene expression in the cell. Pneumolysin reduced early production of the cytokine TNF-α and a medicine vorinostat that modifies these ‘epigenetic’ histone modifications had a similar affect, suggesting epigenetic mechanisms contribute to the ability of pneumolysin to reduce immune responses.

Antimicrobial therapy has provided the main component of chemotherapy against bacterial pathogens. The effectiveness of this strategy has, however, been increasingly challenged by the emergence of antimicrobial resistance which now threatens the sustained utility of this approach. Humans and animals are constantly exposed to bacteria and have developed effective strategies to control pathogens involving innate and adaptive immune responses. Impaired pathogen handling by the innate immune system is a key determinant of susceptibility to bacterial infection. However, the essential components of this response, specifically those which are amenable to re-calibration to improve host defense, remain elusive despite extensive research. We provide a mini-review focusing on therapeutic targeting of microbicidal responses in macrophages and neutrophils to de-stress reliance on antimicrobial therapy. We highlight pre-clinical and clinical data pointing toward potential targets and therapies. We suggest that developing focused host-directed therapeutic strategies to enhance “pauci-inflammatory” microbial killing in myeloid phagocytes that maximizes pathogen clearance while minimizing the harmful consequences of the inflammatory response merits particular attention. We also suggest the importance of One Health approaches in developing host-based approaches through model development and comparative medicine in informing our understanding of how to deliver this strategy.

This manuscript describes the use of a bacterial pore-forming toxin as a method to deliver gold nanoparticles into cells. Targeted drug delivery overcomes some of the issues of degradation of the delivery system in the endosomal network of the cell. Listeriolysin is a member of the large pore-forming family of toxins. Mutagenised Listeriolysin O (LLO) is coupled to nanoparticles via functionalization of the particles with nitrile acetic acid, which then allows coupling of the protein via a histidine tag on the protein. The nanoparticles are then taken up and the endosome acidifies, which releases the toxin from the nanoparticles and allows the formation of pores that perforate the endosome and allows the escape of the nanoparticles to deliver their cargo of drugs or other agents to the cytoplasm of the cell.