Impact of Endogenous Pneumococcal Hydrogen Peroxide on the Activity and Release of Pneumolysin.

Streptococcus pneumoniae is the leading cause of community-acquired pneumonia. The pore-forming cholesterol-dependent cytolysin (CDC) pneumolysin (PLY) and the physiological metabolite hydrogen peroxide (H2O2) can greatly increase the virulence of pneumococci. Although most studies have focused on the contribution of both virulence factors to the course of pneumococcal infection, it is unknown whether or how H2O2 can affect PLY activity. Of note, S. pneumoniae exploits endogenous H2O2 as an intracellular signalling molecule to modulate the activity of several proteins. Here, we demonstrate that H2O2 negatively affects the haemolytic activity of PLY in a concentration-dependent manner. Prevention of cysteine-dependent sulfenylation upon substitution of the unique and highly conserved cysteine residue to serine in PLY significantly reduces the toxin's susceptibility to H2O2 treatment and completely abolishes the ability of DTT to activate PLY. We also detect a clear gradual correlation between endogenous H2O2 generation and PLY release, with decreased H2O2 production causing a decline in the release of PLY. Comparative transcriptome sequencing analysis of the wild-type S. pneumoniae strain and three mutants impaired in H2O2 production indicates enhanced expression of several genes involved in peptidoglycan (PG) synthesis and in the production of choline-binding proteins (CPBs). One explanation for the impact of H2O2 on PLY release is the observed upregulation of the PG bridge formation alanyltransferases MurM and MurN, which evidentially negatively affect the PLY release. Our findings shed light on the significance of endogenous pneumococcal H2O2 in controlling PLY activity and release.

A Single Residue within the MCR-1 Protein Confers Anticipatory Resilience.

The envelope stress response (ESR) of Gram-negative enteric bacteria senses fluctuations in nutrient availability and environmental changes to avert damage and promote survival. It has a protective role toward antimicrobials, but direct interactions between ESR components and antibiotic resistance genes have not been demonstrated. Here, we report interactions between a central regulator of ESR viz., the two-component signal transduction system CpxRA (conjugative pilus expression), and the recently described mobile colistin resistance protein (MCR-1). Purified MCR-1 is specifically cleaved within its highly conserved periplasmic bridge element, which links its N-terminal transmembrane domain with the C-terminal active-site periplasmic domain, by the CpxRA-regulated serine endoprotease DegP. Recombinant strains harboring cleavage site mutations in MCR-1 are either protease resistant or degradation susceptible, with widely differing consequences for colistin resistance. Transfer of the gene encoding a degradation-susceptible mutant to strains that lack either DegP or its regulator CpxRA restores expression and colistin resistance. MCR-1 production in Escherichia coli imposes growth restriction in strains lacking either DegP or CpxRA, effects that are reversed by transactive expression of DegP. Excipient allosteric activation of the DegP protease specifically inhibits growth of isolates carrying mcr-1 plasmids. As CpxRA directly senses acidification, growth of strains at moderately low pH dramatically increases both MCR-1-dependent phosphoethanolamine (PEA) modification of lipid A and colistin resistance levels. Strains expressing MCR-1 are also more resistant to antimicrobial peptides and bile acids. Thus, a single residue external to its active site induces ESR activity to confer resilience in MCR-1-expressing strains to commonly encountered environmental stimuli, such as changes in acidity and antimicrobial peptides. Targeted activation of the nonessential protease DegP can lead to the elimination of transferable colistin resistance in Gram-negative bacteria. IMPORTANCE The global presence of transferable mcr genes in a wide range of Gram-negative bacteria from clinical, veterinary, food, and aquaculture environments is disconcerting. Its success as a transmissible resistance factor remains enigmatic, because its expression imposes fitness costs and imparts only moderate levels of colistin resistance. Here, we show that MCR-1 triggers regulatory components of the envelope stress response, a system that senses fluctuations in nutrient availability and environmental changes, to promote bacterial survival in low pH environments. We identify a single residue within a highly conserved structural element of mcr-1 distal to its catalytic site that modulates resistance activity and triggers the ESR. Using mutational analysis, quantitative lipid A profiling and biochemical assays, we determined that growth in low pH environments dramatically increases colistin resistance levels and promotes resistance to bile acids and antimicrobial peptides. We exploited these findings to develop a targeted approach that eliminates mcr-1 and its plasmid carriers.

SecA2 Associates with Translating Ribosomes and Contributes to the Secretion of Potent IFN-ß Inducing RNAs.

Protein secretion plays a central role in modulating interactions of the human pathogen Listeria monocytogenes with its environment. Recently, secretion of RNA has emerged as an important strategy used by the pathogen to manipulate the host cell response to its advantage. In general, the Sec-dependent translocation pathway is a major route for protein secretion in L. monocytogenes, but mechanistic insights into the secretion of RNA by these pathways are lacking. Apart from the classical SecA1 secretion pathway, L. monocytogenes also encodes for a SecA paralogue (SecA2) which targets the export of a specific subset of proteins, some of which are involved in virulence. Here, we demonstrated that SecA2 co-sediments with translating ribosomes and provided evidence that it associates with a subset of secreted small non-coding RNAs (sRNAs) that induce high levels of IFN-ß response in host cells. We found that enolase, which is translocated by a SecA2-dependent mechanism, binds to several sRNAs, suggesting a pathway by which sRNAs are targeted to the supernatant of L. monocytogenes.

Recombinant Porphyromonas gingivalis W83 FimA alters immune response and metabolic gene expression in oral squamous carcinoma cells.

OBJECTIVES: The Gram-negative anaerobic rod Porphyromonas gingivalis (P. gingivalis) is regarded as a keystone pathogen in periodontitis and expresses a multitude of virulence factors iincluding fimbriae that are enabling adherence to and invasion in cells and tissues. The progression of periodontitis is a consequence of the interaction between the host immune response and periodontal pathogens. The aim of this study was to investigate the genome-wide impact of recombinant fimbrial protein FimA from P. gingivalis W83 on the gene expression of oral squamous carcinoma cells by transcriptome analysis. MATERIALS AND METHODS: Human squamous cell carcinoma cells (SCC-25) were stimulated for 4 and 24 h with recombinant FimA. RNA sequencing was performed and differential gene expression and enrichment were analyzed using gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and REACTOME. The results of transcriptome analysis were validated using quantitative real-time polymerase chain reaction (PCR) with selected genes. RESULTS: Differential gene expression after 4 and 24 h revealed upregulation of 464 (4 h) and 179 genes (24 h) and downregulation of 69 (4 h) and 312 (24 h) genes. GO, KEGG, and REACTONE enrichment analysis identified a strong immunologic transcriptomic response signature after 4 h. After 24 h, mainly those genes were regulated, which belonged to cell metabolic pathways and replication. Real-time PCR of selected genes belonging to immune response and signaling demonstrated strong upregulation of CCL20, TNFAIP6, CXCL8, TNFAIP3, and NFkBIA after both stimulation times. CONCLUSIONS: These data shed light on the RNA transcriptome of human oral squamous carcinoma epithelial cells following stimulation with P. gingivalis FimA and identify a strong immunological gene expression response to this virulence factor. The data provide a base for future studies of molecular and cellular interactions between P. gingivalis and oral epithelium to elucidate basic mechanisms that may provide new prospects for periodontitis therapy and give new insights into the development and possible treatments of cancer.

Dichotomous Role of Tumor Necrosis Factor in Pulmonary Barrier Function and Alveolar Fluid Clearance.

Alveolar-capillary leak is a hallmark of the acute respiratory distress syndrome (ARDS), a potentially lethal complication of severe sepsis, trauma and pneumonia, including COVID-19. Apart from barrier dysfunction, ARDS is characterized by hyper-inflammation and impaired alveolar fluid clearance (AFC), which foster the development of pulmonary permeability edema and hamper gas exchange. Tumor Necrosis Factor (TNF) is an evolutionarily conserved pleiotropic cytokine, involved in host immune defense against pathogens and cancer. TNF exists in both membrane-bound and soluble form and its mainly -but not exclusively- pro-inflammatory and cytolytic actions are mediated by partially overlapping TNFR1 and TNFR2 binding sites situated at the interface between neighboring subunits in the homo-trimer. Whereas TNFR1 signaling can mediate hyper-inflammation and impaired barrier function and AFC in the lungs, ligand stimulation of TNFR2 can protect from ventilation-induced lung injury. Spatially distinct from the TNFR binding sites, TNF harbors within its structure a lectin-like domain that rather protects lung function in ARDS. The lectin-like domain of TNF -mimicked by the 17 residue TIP peptide- represents a physiological mediator of alveolar-capillary barrier protection. and increases AFC in both hydrostatic and permeability pulmonary edema animal models. The TIP peptide directly activates the epithelial sodium channel (ENaC) -a key mediator of fluid and blood pressure control- upon binding to its α subunit, which is also a part of the non-selective cation channel (NSC). Activity of the lectin-like domain of TNF is preserved in complexes between TNF and its soluble TNFRs and can be physiologically relevant in pneumonia. Antibody- and soluble TNFR-based therapeutic strategies show considerable success in diseases such as rheumatoid arthritis, psoriasis and inflammatory bowel disease, but their chronic use can increase susceptibility to infection. Since the lectin-like domain of TNF does not interfere with TNF's anti-bacterial actions, while exerting protective actions in the alveolar-capillary compartments, it is currently evaluated in clinical trials in ARDS and COVID-19. A more comprehensive knowledge of the precise role of the TNFR binding sites versus the lectin-like domain of TNF in lung injury, tissue hypoxia, repair and remodeling may foster the development of novel therapeutics for ARDS.

Data for microbe resistant engineered recombinant spider silk protein based 2D and 3D materials.

Data presented in this article describe bacterial and fungal repellent properties of 2D-films and 3D-hydrogels made of different recombinantly produced spider silk proteins based on consensus sequences of Araneus diadematus dragline silk proteins (fibroin 3 and 4). Here, the attachment, growth, and microbial colonization of Streptococcus mutans (S. mutans) as well as Candida albicans (C. albicans) on plane and micro-patterned films were visualized by scanning electron microscopy (SEM). Also, microbial viability data are provided of Escherichia coli (E. coli) and Pichia pastoris (P. pastoris) on hydrogels made of eADF4(C16) and eADF4(C16)-RGD, quantified using the Alamar blue assay. Experimental results, design of a post-operative contamination model of microbes with mammalian cells, and methods in the data article refer to the research paper "Engineered spider silk-based 2D and 3D materials prevent microbial infestation" published recently [1].

Impact of Bacterial Toxins in the Lungs.

Bacterial toxins play a key role in the pathogenesis of lung disease. Based on their structural and functional properties, they employ various strategies to modulate lung barrier function and to impair host defense in order to promote infection. Although in general, these toxins target common cellular signaling pathways and host compartments, toxin- and cell-specific effects have also been reported. Toxins can affect resident pulmonary cells involved in alveolar fluid clearance (AFC) and barrier function through impairing vectorial Na+ transport and through cytoskeletal collapse, as such, destroying cell-cell adhesions. The resulting loss of alveolar-capillary barrier integrity and fluid clearance capacity will induce capillary leak and foster edema formation, which will in turn impair gas exchange and endanger the survival of the host. Toxins modulate or neutralize protective host cell mechanisms of both the innate and adaptive immunity response during chronic infection. In particular, toxins can either recruit or kill central players of the lung's innate immune responses to pathogenic attacks, i.e., alveolar macrophages (AMs) and neutrophils. Pulmonary disorders resulting from these toxin actions include, e.g., acute lung injury (ALI), the acute respiratory syndrome (ARDS), and severe pneumonia. When acute infection converts to persistence, i.e., colonization and chronic infection, lung diseases, such as bronchitis, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) can arise. The aim of this review is to discuss the impact of bacterial toxins in the lungs and the resulting outcomes for pathogenesis, their roles in promoting bacterial dissemination, and bacterial survival in disease progression.

Phosphocholine Antagonizes Listeriolysin O-Induced Host Cell Responses of Listeria monocytogenes.

BACKGROUND: Bacterial toxins disrupt plasma membrane integrity with multitudinous effects on host cells. The secreted pore-forming toxin listeriolysin O (LLO) of the intracellular pathogen Listeria monocytogenes promotes egress of the bacteria from vacuolar compartments into the host cytosol often without overt destruction of the infected cell. Intracellular LLO activity is tightly controlled by host factors including compartmental pH, redox, proteolytic, and proteostatic factors, and inhibited by cholesterol. METHODS: Combining infection studies of L. monocytogenes wild type and isogenic mutants together with biochemical studies with purified phospholipases, we investigate the effect of their enzymatic activities on LLO. RESULTS: Here, we show that phosphocholine (ChoP), a reaction product of the phosphatidylcholine-specific phospholipase C (PC-PLC) of L. monocytogenes, is a potent inhibitor of intra- and extracellular LLO activities. Binding of ChoP to LLO is redox-independent and leads to the inhibition of LLO-dependent induction of calcium flux, mitochondrial damage, and apoptosis. ChoP also inhibits the hemolytic activities of the related cholesterol-dependent cytolysins (CDC), pneumolysin and streptolysin. CONCLUSIONS: Our study uncovers a strategy used by L. monocytogenes to modulate cytotoxic LLO activity through the enzymatic activity of its PC-PLC. This mechanism appears to be widespread and also used by other CDC pore-forming toxin-producing bacteria.

Listeria goaensis sp. nov.

Two Listeria-like isolates obtained from mangrove swamps in Goa, India were characterized using polyphasic combinations of phenotypic, chemotaxonomic and whole-genome sequence (WGS)-based approaches. The isolates presented as short, non-spore-forming, Gram-positive rods, that were non-motile, oxidase-negative, catalase-positive and exhibited α-haemolysis on 5 % sheep- and horse-blood agar plates. The 16S rRNA gene sequences exhibited 93.7-99.7 % nucleotide identity to other Listeria species and had less than 92 % nucleotide identity to species of closely related genera, indicating that the isolates are de facto members of the genus Listeria. Their overall fatty acid composition resembled that of other Listeria species, with quantitative differences in iso C15 : 0, anteiso C15 : 0, iso C16 : 0, C16 : 0, iso C17 : 0 and anteiso C17 : 0 fatty acid profiles. Phylogeny based on 406 core coding DNA sequences grouped these two isolates in a monophyletic clade within the genus Listeria. WGS-based average nucleotide identity and in silico DNA-DNA hybridization values were lower than the recommended cut-off values of 95 and 70 %, respectively, to the other Listeria species, indicating that they are founding members of a novel Listeria species. We suggest the name Listeriagoaensis sp. nov. be created and the type strain is ILCC801T (=KCTC 33909;=DSM 29886;=MCC 3285).

Listeriolysin O Causes ENaC Dysfunction in Human Airway Epithelial Cells.

Pulmonary permeability edema is characterized by reduced alveolar Na⁺ uptake capacity and capillary barrier dysfunction and is a potentially lethal complication of listeriosis. Apical Na⁺ uptake is mainly mediated by the epithelial sodium channel (ENaC) and initiates alveolar liquid clearance. Here we examine how listeriolysin O (LLO), the pore-forming toxin of Listeria monocytogenes, impairs the expression and activity of ENaC. To that purpose, we studied how sub-lytic concentrations of LLO affect negative and positive regulators of ENaC expression in the H441 airway epithelial cell line. LLO reduced expression of the crucial ENaC-α subunit in H441 cells within 2 h and this was preceded by activation of PKC-α, a negative regulator of the channel's expression. At later time points, LLO caused a significant reduction in the phosphorylation of Sgk-1 at residue T256 and of Akt-1 at residue S473, both of which are required for full activation of ENaC. The TNF-derived TIP peptide prevented LLO-mediated PKC-α activation and restored phospho-Sgk-1-T256. The TIP peptide also counteracted the observed LLO-induced decrease in amiloride-sensitive Na⁺ current and ENaC-α expression in H441 cells. Intratracheally instilled LLO caused profound pulmonary edema formation in mice, an effect that was prevented by the TIP peptide; thus indicating the therapeutic potential of the peptide for the treatment of pore-forming toxin-associated permeability edema.

Epithelial Sodium Channel-α Mediates the Protective Effect of the TNF-Derived TIP Peptide in Pneumolysin-Induced Endothelial Barrier Dysfunction.

BACKGROUND: Streptococcus pneumoniae is a major etiologic agent of bacterial pneumonia. Autolysis and antibiotic-mediated lysis of pneumococci induce release of the pore-forming toxin, pneumolysin (PLY), their major virulence factor, which is a prominent cause of acute lung injury. PLY inhibits alveolar liquid clearance and severely compromises alveolar-capillary barrier function, leading to permeability edema associated with pneumonia. As a consequence, alveolar flooding occurs, which can precipitate lethal hypoxemia by impairing gas exchange. The α subunit of the epithelial sodium channel (ENaC) is crucial for promoting Na+ reabsorption across Na+-transporting epithelia. However, it is not known if human lung microvascular endothelial cells (HL-MVEC) also express ENaC-α and whether this subunit is involved in the regulation of their barrier function. METHODS: The presence of α, ß, and γ subunits of ENaC and protein phosphorylation status in HL-MVEC were assessed in western blotting. The role of ENaC-α in monolayer resistance of HL-MVEC was examined by depletion of this subunit by specific siRNA and by employing the TNF-derived TIP peptide, a specific activator that directly binds to ENaC-α. RESULTS: HL-MVEC express all three subunits of ENaC, as well as acid-sensing ion channel 1a (ASIC1a), which has the capacity to form hybrid non-selective cation channels with ENaC-α. Both TIP peptide, which specifically binds to ENaC-α, and the specific ASIC1a activator MitTx significantly strengthened barrier function in PLY-treated HL-MVEC. ENaC-α depletion significantly increased sensitivity to PLY-induced hyperpermeability and in addition, blunted the protective effect of both the TIP peptide and MitTx, indicating an important role for ENaC-α and for hybrid NSC channels in barrier function of HL-MVEC. TIP peptide blunted PLY-induced phosphorylation of both calmodulin-dependent kinase II (CaMKII) and of its substrate, the actin-binding protein filamin A (FLN-A), requiring the expression of both ENaC-α and ASIC1a. Since non-phosphorylated FLN-A promotes ENaC channel open probability and blunts stress fiber formation, modulation of this activity represents an attractive target for the protective actions of ENaC-α in both barrier function and liquid clearance. CONCLUSION: Our results in cultured endothelial cells demonstrate a previously unrecognized role for ENaC-α in strengthening capillary barrier function that may apply to the human lung. Strategies aiming to activate endothelial NSC channels that contain ENaC-α should be further investigated as a novel approach to improve barrier function in the capillary endothelium during pneumonia.

Pneumococcal hydrogen peroxide-induced stress signaling regulates inflammatory genes.

Microbial infections can induce aberrant responses in cellular stress pathways, leading to translational attenuation, metabolic restriction, and activation of oxidative stress, with detrimental effects on cell survival. Here we show that infection of human airway epithelial cells with Streptococcus pneumoniae leads to induction of endoplasmic reticulum (ER) and oxidative stress, activation of mitogen-associated protein kinase (MAPK) signaling pathways, and regulation of their respective target genes. We identify pneumococcal H2O2 as the causative agent for these responses, as both catalase-treated and pyruvate oxidase-deficient bacteria lacked these activities. Pneumococcal H2O2 induced nuclear NF-κB translocation and transcription of proinflammatory cytokines. Inhibition of translational arrest and ER stress by salubrinal or of MAPK signaling pathways attenuate cytokine transcription. These results provide strong evidence for the notion that inhibition of translation is an important host pathway in monitoring harmful pathogen-associated activities, thereby enabling differentiation between pathogenic and nonpathogenic bacteria.

Crystal structure of listeriolysin O reveals molecular details of oligomerization and pore formation.

Listeriolysin O (LLO) is an essential virulence factor of Listeria monocytogenes that causes listeriosis. Listeria monocytogenes owes its ability to live within cells to the pH- and temperature-dependent pore-forming activity of LLO, which is unique among cholesterol-dependent cytolysins. LLO enables the bacteria to cross the phagosomal membrane and is also involved in activation of cellular processes, including the modulation of gene expression or intracellular Ca(2+) oscillations. Neither the pore-forming mechanism nor the mechanisms triggering the signalling processes in the host cell are known in detail. Here, we report the crystal structure of LLO, in which we identified regions important for oligomerization and pore formation. Mutants were characterized by determining their haemolytic and Ca(2+) uptake activity. We analysed the pore formation of LLO and its variants on erythrocyte ghosts by electron microscopy and show that pore formation requires precise interface interactions during toxin oligomerization on the membrane.

Crystallization and X-ray crystallographic analysis of the cholesterol-dependent cytolysin listeriolysin O from Listeria monocytogenes.

The secreted pore-forming toxin listeriolysin O (LLO) from the intracellular pathogen Listeria monocytogenes is a member of the family of cholesterol-dependent cytolysins (CDC) with broad properties in pathogenesis. Its role as a virulence factor is enigmatic: it disrupts membranes and acts as an inductor of both pro- and anti-inflammatory responses in infected cells. In addition, LLO is also a potent target for immunogenicity during infection. Natively secreted LLO from a recombinant L. innocua strain was crystallized in its water-soluble monomeric form. The crystals obtained belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 26.7, b = 85.1, c = 230.0 Å, and diffracted to beyond 2.2 Šresolution. The Matthews coefficient and the solvent content were estimated to be 2.4 Å(3) Da(-1) and 49.2%, respectively. The structure with one molecule in the asymmetric unit was solved using Phaser employing the structure of the previously characterized CDC toxin perfringolysin O as a search model.