Hydrogen peroxide is responsible for the cytotoxic effects of Streptococcus pneumoniae on primary microglia in the absence of pneumolysin.

INTRODUCTION: Streptococcus pneumoniae is the most common cause of bacterial meningitis and meningoencephalitis in humans. The bacterium produces numerous virulence determinants, among them hydrogen peroxide (H2O2) and pneumolysin (Ply), which contribute to bacterial cytotoxicity. Microglia, the resident phagocytes in the brain, are distinct from other macrophages, and we thus compared their susceptibility to pneumococcal toxicity and their ability to phagocytose pneumococci with those of bone marrow-derived macrophages (BMDM). METHODS: Microglia and BMDM were co-incubated with S. pneumoniae D39 to analyze survival of phagocytes by fluorescence microscopy, bacterial growth by quantitative plating, and phagocytosis by an antibiotic protection assay. Ply was detected by hemolysis assay and Western blot analysis. RESULTS: We found that microglia were killed during pneumococcal infection with a wild-type and an isogenic ply-deficient mutant, whereas viability of BMDM was not affected by pneumococci. Treatment with recombinant Ply showed a dose-dependent cytotoxic effect on microglia and BMDM. However, high concentrations of recombinant Ply were required and under the chosen experimental conditions, Ply was not detectable in the supernatant during infection of microglia. Inactivation of H2O2 by exogenously added catalase abolished its cytotoxic effect. Consequently, infection of microglia with pneumococci deficient for the pyruvate oxidase SpxB, primarily producing H2O2, resulted in reduced killing of microglia. CONCLUSION: Taken together, in the absence of Ply, H2O2 caused cell death in primary phagocytes in concentrations produced by pneumococci.

The single D380 amino acid substitution increases pneumolysin cytotoxicity toward neuronal cells.

Bacterial meningitis, frequently caused by Streptococcus pneumoniae (pneumococcus), represents a substantial global health threat leading to long-term neurological disorders. This study focused on the cholesterol-binding toxin pneumolysin (PLY) released by pneumococci, specifically examining clinical isolates from patients with meningitis and comparing them to the PLY-reference S. pneumoniae strain D39. Clinical isolates exhibit enhanced PLY release, likely due to a significantly higher expression of the autolysin LytA. Notably, the same single amino acid (aa) D380 substitution in the PLY D4 domain present in all clinical isolates significantly enhances cholesterol binding, pore-forming activity, and cytotoxicity toward SH-SY5Y-derived neuronal cells. Scanning electron microscopy of human neuronal cells and patch clamp electrophysiological recordings on mouse brain slices confirm the enhanced neurotoxicity of the PLY variant carrying the single aa substitution. This study highlights how a single aa modification enormously alters PLY cytotoxic potential, emphasizing the importance of PLY as a major cause of the neurological sequelae associated with pneumococcal meningitis.

Combining antibiotic with anti-TLR2/TLR13 therapy prevents brain pathology in pneumococcal meningitis.

Despite effective antibiotic therapy, brain-destructive inflammation often cannot be avoided in pneumococcal meningitis. The causative signals are mediated predominantly through TLR-recruited myeloid differentiation primary response adaptor 88 (MyD88), as indicated by a dramatic pneumococcal meningitis phenotype of Myd88-/- mice. Because lipoproteins and single-stranded RNA are crucial for recognition of Gram-positive bacteria such as Streptococcus pneumoniae by the host immune system, we comparatively analyzed the disease courses of Myd88-/- and Tlr2-/- Tlr13-/- mice. Their phenotypic resemblance indicated TLR2 and -13 as master sensors of S. pneumoniae in the cerebrospinal fluid. A neutralizing anti-TLR2 antibody (T2.5) and chloroquine (CQ) - the latter applied here as an inhibitor of murine TLR13 and its human ortholog TLR8 - abrogated activation of murine and human primary immune cells exposed to antibiotic-treated S. pneumoniae. The inhibitory effect of the T2.5/CQ cocktail was stronger than that of dexamethasone, the current standard adjunctive drug for pneumococcal meningitis. Accordingly, TLR2/TLR13 blockade concomitant with ceftriaxone application significantly improved the clinical course of pneumococcal meningitis compared with treatment with ceftriaxone alone or in combination with dexamethasone. Our study indicates the importance of murine TLR13 and human TLR8, besides TLR2, in pneumococcal meningitis pathology, and suggests their blockade as a promising antibiotic therapy adjunct.

TLR4 sensing of IsdB of Staphylococcus aureus induces a proinflammatory cytokine response via the NLRP3-caspase-1 inflammasome cascade.

IMPORTANCE: The prevalence of multidrug-resistant Staphylococcus aureus is of global concern, and vaccines are urgently needed. The iron-regulated surface determinant protein B (IsdB) of S. aureus was investigated as a vaccine candidate because of its essential role in bacterial iron acquisition but failed in clinical trials despite strong immunogenicity. Here, we reveal an unexpected second function for IsdB in pathogen-host interaction: the bacterial fitness factor IsdB triggers a strong inflammatory response in innate immune cells via Toll-like receptor 4 and the inflammasome, thus acting as a novel pathogen-associated molecular pattern of S. aureus. Our discovery contributes to a better understanding of how S. aureus modulates the immune response, which is necessary for vaccine development against the sophisticated pathogen.

The type-2 Streptococcus canis M protein SCM-2 binds fibrinogen and facilitates antiphagocytic properties.

Streptococcus canis is a zoonotic agent that causes severe invasive diseases in domestic animals and humans, but little is known about its pathogenesis and virulence mechanisms so far. SCM, the M-like protein expressed by S. canis, is considered one of the major virulence determinants. Here, we report on the two distinct groups of SCM. SCM-1 proteins were already described to interact with its ligands IgG and plasminogen as well as with itself and confer antiphagocytic capability of SCM-1 expressing bacterial isolates. In contrast, the function of SCM-2 type remained unclear to date. Using whole-genome sequencing and subsequent bioinformatics, FACS analysis, fluorescence microscopy and surface plasmon resonance spectrometry, we demonstrate that, although different in amino acid sequence, a selection of diverse SCM-2-type S. canis isolates, phylogenetically representing the full breadth of SCM-2 sequences, were able to bind fibrinogen. Using targeted mutagenesis of an SCM-2 isolate, we further demonstrated that this strain was significantly less able to survive in canine blood. With respect to similar studies showing a correlation between fibrinogen binding and survival in whole blood, we hypothesize that SCM-2 has an important contribution to the pathogenesis of S. canis in the host.

Pericytes are protective in experimental pneumococcal meningitis through regulating leukocyte infiltration and blood-brain barrier function.

BACKGROUND: Brain pericytes participate in the regulation of cerebral blood flow and the maintenance of blood-brain barrier integrity. Because of their perivascular localization, their receptor repertoire, and their potential ability to respond to inflammatory and infectious stimuli by producing various cytokines and chemokines, these cells are also thought to play an active role in the immune response to brain infections. This assumption is mainly supported by in vitro studies, investigations in in vivo disease models are largely missing. Here, we analysed the role of brain pericytes in pneumococcal meningitis, in vitro and in vivo in two animal models of pneumococcal meningitis. METHODS: Primary murine and human pericytes were stimulated with increasing concentrations of different serotypes of Streptococcus pneumoniae in the presence or absence of Toll-like receptor inhibitors and their cell viability and cytokine production were monitored. To gain insight into the role of pericytes in brain infection in vivo, we performed studies in a zebrafish embryo model of pneumococcal meningitis in which pericytes were pharmacologically depleted. Furthermore, we analyzed the impact of genetically induced pericyte ablation on disease progression, intracranial complications, and brain inflammation in an adult mouse model of this disease. RESULTS: Both murine and human pericytes reacted to pneumococcal exposure with the release of selected cytokines. This cytokine release is pneumolysin-dependent, TLR-dependent in murine (but not human) pericytes and can be significantly increased by macrophage-derived IL-1b. Pharmacological depletion of pericytes in zebrafish embryos resulted in increased cerebral edema and mortality due to pneumococcal meningitis. Correspondingly, in an adult mouse meningitis model, a more pronounced blood-brain barrier disruption and leukocyte infiltration, resulting in an unfavorable disease course, was observed following genetic pericyte ablation. The degree of leukocyte infiltration positively correlated with an upregulation of chemokine expression in the brains of pericyte-depleted mice. CONCLUSIONS: Our findings show that pericytes play a protective role in pneumococcal meningitis by impeding leukocyte migration and preventing blood-brain barrier breaching. Thus, preserving the integrity of the pericyte population has the potential as a new therapeutic strategy in pneumococcal meningitis.

NAD+ metabolism is a key modulator of bacterial respiratory epithelial infections.

Lower respiratory tract infections caused by Streptococcus pneumoniae (Spn) are a leading cause of death globally. Here we investigate the bronchial epithelial cellular response to Spn infection on a transcriptomic, proteomic and metabolic level. We found the NAD+ salvage pathway to be dysregulated upon infection in a cell line model, primary human lung tissue and in vivo in rodents, leading to a reduced production of NAD+. Knockdown of NAD+ salvage enzymes (NAMPT, NMNAT1) increased bacterial replication. NAD+ treatment of Spn inhibited its growth while growth of other respiratory pathogens improved. Boosting NAD+ production increased NAD+ levels in immortalized and primary cells and decreased bacterial replication upon infection. NAD+ treatment of Spn dysregulated the bacterial metabolism and reduced intrabacterial ATP. Enhancing the bacterial ATP metabolism abolished the antibacterial effect of NAD+. Thus, we identified the NAD+ salvage pathway as an antibacterial pathway in Spn infections, predicting an antibacterial mechanism of NAD+.

The choline-binding proteins PspA, PspC, and LytA of Streptococcus pneumoniae and their interaction with human endothelial and red blood cells.

Streptococcus pneumoniae is a Gram-positive opportunistic pathogen that can colonize the upper respiratory tract. It is a leading cause of a wide range of infectious diseases, including community-acquired pneumonia and meningitis. Pneumococcal infections cause 1-2 million deaths per year, most of which occur in developing countries. Here, we focused on three choline-binding proteins (CBPs), i.e., PspC, PspA, and LytA. These pneumococcal proteins have different surface-exposed regions but share related choline-binding anchors. These surface-exposed pneumococcal proteins are in direct contact with host cells and have diverse functions. We explored the role of the three CBPs on adhesion and pathogenicity in a human host by performing relevant imaging and functional analyses, such as electron microscopy, confocal laser scanning microscopy, and functional quantitative assays, targeting biofilm formation and the hemolytic capacity of S. pneumoniae. In vitro biofilm formation assays and electron microscopy experiments were used to examine the ability of knockout mutant strains lacking the lytA, pspC, or pspA genes to adhere to surfaces. We found that LytA plays an important role in robust synthesis of the biofilm matrix. PspA and PspC appeared crucial for the hemolytic effects of S. pneumoniae on human red blood cells. Furthermore, all knockout mutants caused less damage to endothelial cells than wild-type bacteria, highlighting the significance of each CPB for the overall pathogenicity of S. pneumoniae. Hence, in addition to their structural function within the cell wall of S. pneumoniae, each of these three surface-exposed CBPs controls or mediates multiple steps during bacterial pathogenesis.

The role of pneumococcal extracellular vesicles on the pathophysiology of the kidney disease hemolytic uremic syndrome.

Streptococcus pneumoniae-induced hemolytic uremic syndrome (Sp-HUS) is a kidney disease characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. This disease is frequently underdiagnosed and its pathophysiology is poorly understood. In this work, we compared clinical strains, isolated from infant Sp-HUS patients, with a reference pathogenic strain D39, for host cytotoxicity and further explored the role of Sp-derived extracellular vesicles (EVs) in the pathogenesis of an HUS infection. In comparison with the wild-type strain, pneumococcal HUS strains caused significant lysis of human erythrocytes and increased the release of hydrogen peroxide. Isolated Sp-HUS EVs were characterized by performing dynamic light-scattering microscopy and proteomic analysis. Sp-HUS strain released EVs at a constant concentration during growth, yet the size of the EVs varied and several subpopulations emerged at later time points. The cargo of the Sp-HUS EVs included several virulence factors at high abundance, i.e., the ribosomal subunit assembly factor BipA, the pneumococcal surface protein A, the lytic enzyme LytC, several sugar utilization, and fatty acid synthesis proteins. Sp-HUS EVs strongly downregulated the expression of the endothelial surface marker platelet endothelial cell adhesion molecule-1 and were internalized by human endothelial cells. Sp-HUS EVs elicited the release of pro-inflammatory cytokines (interleukin [IL]-1ß, IL-6) and chemokines (CCL2, CCL3, CXCL1) by human monocytes. These findings shed new light on the overall function of Sp-EVs, in the scope of infection-mediated HUS, and suggest new avenues of research for exploring the usefulness of Sp-EVs as therapeutic and diagnostic targets. IMPORTANCE Streptococcus pneumoniae-associated hemolytic uremic syndrome (Sp-HUS) is a serious and underdiagnosed deadly complication of invasive pneumococcal disease. Despite the introduction of the pneumococcal vaccine, cases of Sp-HUS continue to emerge, especially in children under the age of 2. While a lot has been studied regarding pneumococcal proteins and their role on Sp-HUS pathophysiology, little is known about the role of extracellular vesicles (EVs). In our work, we isolate and initially characterize EVs from a reference pathogenic strain (D39) and a strain isolated from a 2-year-old patient suffering from Sp-HUS. We demonstrate that despite lacking cytotoxicity toward human cells, Sp-HUS EVs are highly internalized by endothelial cells and can trigger cytokine and chemokine production in monocytes. In addition, this work specifically highlights the distinct morphological characteristics of Sp-HUS EVs and their unique cargo. Overall, this work sheds new light into potentially relevant players contained in EVs that might elucidate about pneumococcal EVs biogenesis or pose as interesting candidates for vaccine design.

Impact of Pneumococcal and Viral Pneumonia on the Respiratory and Intestinal Tract Microbiomes of Mice.

With 2.56 million deaths worldwide annually, pneumonia is one of the leading causes of death. The most frequent causative pathogens are Streptococcus pneumoniae and influenza A virus. Lately, the interaction between the pathogens, the host, and its microbiome have gained more attention. The microbiome is known to promote the immune response toward pathogens; however, our knowledge on how infections affect the microbiome is still scarce. Here, the impact of colonization and infection with S. pneumoniae and influenza A virus on the structure and function of the respiratory and gastrointestinal microbiomes of mice was investigated. Using a meta-omics approach, we identified specific differences between the bacterial and viral infection. Pneumococcal colonization had minor effects on the taxonomic composition of the respiratory microbiome, while acute infections caused decreased microbial complexity. In contrast, richness was unaffected following H1N1 infection. Within the gastrointestinal microbiome, we found exclusive changes in structure and function, depending on the pathogen. While pneumococcal colonization had no effects on taxonomic composition of the gastrointestinal microbiome, increased abundance of Akkermansiaceae and Spirochaetaceae as well as decreased amounts of Clostridiaceae were exclusively found during invasive S. pneumoniae infection. The presence of Staphylococcaceae was specific for viral pneumonia. Investigation of the intestinal microbiomés functional composition revealed reduced expression of flagellin and rubrerythrin and increased levels of ATPase during pneumococcal infection, while increased amounts of acetyl coenzyme A (acetyl-CoA) acetyltransferase and enoyl-CoA transferase were unique after H1N1 infection. In conclusion, identification of specific taxonomic and functional profiles of the respiratory and gastrointestinal microbiome allowed the discrimination between bacterial and viral pneumonia. IMPORTANCE Pneumonia is one of the leading causes of death worldwide. Here, we compared the impact of bacterial- and viral-induced pneumonia on the respiratory and gastrointestinal microbiome. Using a meta-omics approach, we identified specific profiles that allow discrimination between bacterial and viral causative.

An innate pathogen sensing strategy involving ubiquitination of bacterial surface proteins.

Sensing of pathogens by ubiquitination is a critical arm of cellular immunity. However, universal ubiquitination targets on microbes remain unidentified. Here, using in vitro, ex vivo, and in vivo studies, we identify the first protein-based ubiquitination substrates on phylogenetically diverse bacteria by unveiling a strategy that uses recognition of degron-like motifs. Such motifs form a new class of intra-cytosolic pathogen-associated molecular patterns (PAMPs). Their incorporation enabled recognition of nonubiquitin targets by host ubiquitin ligases. We find that SCFFBW7 E3 ligase, supported by the regulatory kinase, glycogen synthase kinase 3ß, is crucial for effective pathogen detection and clearance. This provides a mechanistic explanation for enhanced risk of infections in patients with chronic lymphocytic leukemia bearing mutations in F-box and WD repeat domain containing 7 protein. We conclude that exploitation of this generic pathogen sensing strategy allows conservation of host resources and boosts antimicrobial immunity.

Competence remodels the pneumococcal cell wall exposing key surface virulence factors that mediate increased host adherence.

Competence development in the human pathogen Streptococcus pneumoniae controls several features such as genetic transformation, biofilm formation, and virulence. Competent bacteria produce so-called "fratricins" such as CbpD that kill noncompetent siblings by cleaving peptidoglycan (PGN). CbpD is a choline-binding protein (CBP) that binds to phosphorylcholine residues found on wall and lipoteichoic acids (WTA and LTA) that together with PGN are major constituents of the pneumococcal cell wall. Competent pneumococci are protected against fratricide by producing the immunity protein ComM. How competence and fratricide contribute to virulence is unknown. Here, using a genome-wide CRISPRi-seq screen, we show that genes involved in teichoic acid (TA) biosynthesis are essential during competence. We demonstrate that LytR is the major enzyme mediating the final step in WTA formation, and that, together with ComM, is essential for immunity against CbpD. Importantly, we show that key virulence factors PspA and PspC become more surface-exposed at midcell during competence, in a CbpD-dependent manner. Together, our work supports a model in which activation of competence is crucial for host adherence by increased surface exposure of its various CBPs.

Antimicrobial Activity of Peptide-Coupled Antisense Peptide Nucleic Acids in Streptococcus pneumoniae.

Streptococcus pneumoniae is the most common cause of community-acquired pneumonia and is responsible for multiple other infectious diseases, such as meningitis and otitis media, in children. Resistance to penicillins, macrolides, and fluoroquinolones is increasing and, since the introduction of pneumococcal conjugate vaccines (PCVs), vaccine serotypes have been replaced by non-vaccine serotypes. Antisense peptide nucleic acids (PNAs) have been shown to reduce the growth of several pathogenic bacteria in various infection models. PNAs are frequently coupled to cell-penetrating peptides (CPPs) to improve spontaneous cellular PNA uptake. In this study, different CPPs were investigated for their capability to support translocation of antisense PNAs into S. pneumoniae. HIV-1 TAT- and (RXR)4XB-coupled antisense PNAs efficiently reduced the viability of S. pneumoniae strains TIGR4 and D39 in vitro. Two essential genes, gyrA and rpoB, were used as targets for antisense PNAs. Overall, the antimicrobial activity of anti-gyrA PNAs was higher than that of anti-rpoB PNAs. Target gene transcription levels in S. pneumoniae were reduced following antisense PNA treatment. The effect of HIV-1 TAT- and (RXR)4XB-anti-gyrA PNAs on pneumococcal survival was also studied in vivo using an insect infection model. Treatment increased the survival of infected Galleria mellonella larvae. Our results represent a proof of principle and may provide a basis for the development of efficient antisense molecules for treatment of S. pneumoniae infections. IMPORTANCE Streptococcus pneumoniae is the most common cause of community-acquired pneumonia and is responsible for the deaths of up to 2 million children each year. Antibiotic resistance and strain replacement by non-vaccine serotypes are growing problems. For this reason, S. pneumoniae has been added to the WHO "global priority list" of antibiotic-resistant bacteria for which novel antimicrobials are most urgently needed. In this study, we investigated whether CPP-coupled antisense PNAs show antibacterial activity in S. pneumoniae. We demonstrated that HIV-1 TAT- and (RXR)4XB-coupled antisense PNAs were able to kill S. pneumoniae in vitro. The specificity of the antimicrobial effect was verified by reduced target gene transcription levels in S. pneumoniae. Moreover, CPP-antisense PNA treatment increased the survival rate of infected Galleria mellonella larvae in vivo. Based on these results, we believe that efficient antisense PNAs can be developed for the treatment of S. pneumoniae infections.

Specific associations between fungi and bacteria in broncho-alveolar aspirates from mechanically ventilated intensive care unit patients.

The detection of fungi in the human respiratory tract may represent contamination, colonization or a respiratory infection. To develop effective management strategies, a more accurate and comprehensive understanding of the lung fungal microbiome is required. Therefore, the objective of the present study was to define the "mycobiome" of mechanically ventilated patients admitted to an intensive care unit (ICU) using broncho-alveolar aspirate ("sputum") samples and correlate this with clinical parameters and the bacterial microbiota. To this end, the mycobiome of 33 sputum samples was analyzed by Internal Transcribed Spacer2 (ITS2) amplicon sequencing of the ribosomal operons. The results show that in the investigated sputa of mechanically ventilated patients Candida spp. were most frequently detected, independent of pneumonia or antimicrobial therapy. The presence of Candida excluded in most cases the presence of Malassezia, which was the second most-frequently encountered fungus. Moreover, a hierarchical clustering of the sequence data indicated a patient-specific mycobiome. Fungi detected by culturing (Candida and Aspergillus) were also detected through ITS2 sequencing, but other yeasts and fungi were only detectable by sequencing. While Candida showed no correlations with identified bacterial groups, the presence of Malassezia and Rhodotorula correlated with oral bacteria associated with periodontal disease. Likewise, Cladosporium correlated with other oral bacteria, whereas Saccharomyces correlated more specifically with dental plaque bacteria and Alternaria with the nasal-throat-resident bacteria Neisseria, Haemophilus and Moraxella. In conclusion, ITS2 sequencing of sputum samples uncovered patient-specific lung mycobiomes, which were only partially detectable by culturing, and which could be correlated to specific nasal-oral-pharyngeal niches.

Molecular Characterization and Antibiotic Susceptibility of Non-PCV13 Pneumococcal Serotypes among Vaccinated Children in Cape Coast, Ghana.

Preventive strategies involving the use of pneumococcal conjugate vaccines (PCVs) are known to drastically reduce pneumococcal disease. However, PCV vaccination has been plagued with serotype replacement by non-PCV serotypes. In this study, we describe the prevalence and molecular characteristics of non-PCV13 serotypes (non-vaccine serotypes, NVTs) from pneumococcal carriage isolates obtained from children < 5 years old in Cape Coast, Ghana, after PCV introduction. The isolates were subjected to antibiotic susceptibility testing and multilocus sequence typing (MLST), and molecular techniques were used to detect the presence of virulence genes. Serotypes 11A, 13, 15B, 23B, and 34 formed the top five of the 93 NVT isolates. As such, 20 (21.5%), 49 (48.4%), and 70 (74.3%) isolates were non-susceptible to penicillin, tetracycline, and cotrimoxazole, respectively. Sixteen (17.2%) multidrug-resistant isolates were identified. However, non-susceptibility to ceftriaxone and erythromycin was low and all isolates were fully susceptible to levofloxacin, linezolid, and vancomycin. Whereas pcpA, pavB, lytA, and psrP genes were detected in nearly all serotypes, pilus islet genes were limited to serotypes 11A, 13, and 23B. MLST for predominant serotype 23B isolates revealed three known and seven novel sequence types (STs). ST172 and novel ST15111 were the most dominant and both STs were related to PMEN clone Columbia23F-26 (ST338). In conclusion, non-PCV13 serotype 23B was the most prevalent, with characteristics of rapid clonal expansion of ST172 and ST15111, which are related to international clones of the pneumococcus. Continuous monitoring of NVTs in Ghana is, therefore, essential, as they have the potential to cause invasive disease, show high antibiotic resistance, and attenuate the effects of PCV vaccination.

Procoagulant Activity of Blood and Microvesicles Is Disturbed by Pneumococcal Pneumolysin, Which Interacts with Coagulation Factors.

The coagulation and contact systems are parts of the innate immune system as they prevent bleeding and dissemination of pathogens and also contribute to microbial killing by inflammatory reactions and the release of antimicrobial peptides. Here, we investigated the influence of Streptococcus pneumoniae on the coagulation and contact system. S. pneumoniae (pneumococci), but no other investigated streptococcal species, impairs coagulation of blood by autolysis and release of pneumolysin. Defective blood coagulation results from the lysis of tissue factor-producing mononuclear cells and their procoagulant microvesicles, which are the main trigger for blood coagulation during sepsis. In addition, pneumolysin binds coagulation and contact system factors, but this does not result in activation. Thus, pneumococci modulate activation of the coagulation system by releasing pneumolysin, which could potentiate lung injury during pneumonia.

Bioactive lipid screening during respiratory tract infections with bacterial and viral pathogens in mice.

INTRODUCTION: Respiratory tract infections are a worldwide health problem for humans and animals. Different cell types produce lipid mediators in response to infections, which consist of eicosanoids like hydroxyeicosatetraenoic acids (HETEs) or oxylipins like hydroxydocosahexaenoic acids (HDHAs). Both substance classes possess immunomodulatory functions. However, little is known about their role in respiratory infections. OBJECTIVES: Here, we aimed to analyze the lipid mediator imprint of different organs of C57BL/6J mice after intranasal mono-infections with Streptococcus pneumoniae (pneumococcus), Staphylococcus aureus or Influenza A virus (IAV) as wells as pneumococcal-IAV co-infection. METHODS: C57BL/6J mice were infected with different pathogens and lungs, spleen, and plasma were collected. Lipid mediators were analyzed using HPLC-MS/MS. In addition, spatial-distribution of sphingosine 1-phosphate (S1P) and ceramide 1-phosphates (C1P) in tissue samples was examined using MALDI-MS-Imaging. The presence of bacterial pathogens in the lung was confirmed via immunofluorescence staining. RESULTS: We found IAV specific changes for different HDHAs and HETEs in mouse lungs as well as enhanced levels of 20-HETE in severe S. aureus infection. Moreover, MALDI-MS-Imaging analysis showed an accumulation of C1P and a decrease of S1P during co-infection in lung and spleen. Long chain C1P was enriched in the red and not in the white pulp of the spleen. CONCLUSIONS: Lipid mediator analysis showed that host synthesis of bioactive lipids is in part specific for a certain pathogen, in particular for IAV infection. Furthermore, MS-Imaging displayed great potential to study infections and revealed changes of S1P and C1P in lungs and spleen of co-infected animals, which was not described before.

Group B Streptococcal Hemolytic Pigment Impairs Platelet Function in a Two-Step Process.

Group B streptococci (GBS) cause a range of invasive maternal-fetal diseases during pregnancy and post-partum. However, invasive infections in non-pregnant adults are constantly increasing. These include sepsis and streptococcal toxic shock syndrome, which are often complicated by systemic coagulation and thrombocytopenia. GBS express a hyper-hemolytic ornithine rhamnolipid pigment toxin with cytolytic and coagulatory activity. Here, we investigated the effects of GBS pigment on human platelets. Infections of platelets with pigmented GBS resulted initially in platelet activation, followed by necrotic cell death. Thus, this study shows that GBS pigment kills human platelets.

Streptococcus pneumoniae Affects Endothelial Cell Migration in Microfluidic Circulation.

Bloodstream infections caused by Streptococcus pneumoniae induce strong inflammatory and procoagulant cellular responses and affect the endothelial barrier of the vascular system. Bacterial virulence determinants, such as the cytotoxic pore-forming pneumolysin, increase the endothelial barrier permeability by inducing cell apoptosis and cell damage. As life-threatening consequences, disseminated intravascular coagulation followed by consumption coagulopathy and low blood pressure is described. With the aim to decipher the role of pneumolysin in endothelial damage and leakage of the vascular barrier in more detail, we established a chamber-separation cell migration assay (CSMA) used to illustrate endothelial wound healing upon bacterial infections. We used chambered inlets for cell cultivation, which, after removal, provide a cell-free area of 500 µm in diameter as a defined gap in primary endothelial cell layers. During the process of wound healing, the size of the cell-free area is decreasing due to cell migration and proliferation, which we quantitatively determined by microscopic live cell monitoring. In addition, differential immunofluorescence staining combined with confocal microscopy was used to morphologically characterize the effect of bacterial attachment on cell migration and the velocity of gap closure. In all assays, the presence of wild-type pneumococci significantly inhibited endothelial gap closure. Remarkably, even in the presence of pneumolysin-deficient pneumococci, cell migration was significantly retarded. Moreover, the inhibitory effect of pneumococci on the proportion of cell proliferation versus cell migration within the process of endothelial gap closure was assessed by implementation of a fluorescence-conjugated nucleoside analogon. We further combined the endothelial CSMA with a microfluidic pump system, which for the first time enabled the microscopic visualization and monitoring of endothelial gap closure in the presence of circulating bacteria at defined vascular shear stress values for up to 48 h. In accordance with our CSMA results under static conditions, the gap remained cell free in the presence of circulating pneumococci in flow. Hence, our combined endothelial cultivation technique represents a complex in vitro system, which mimics the vascular physiology as close as possible by providing essential parameters of the blood flow to gain new insights into the effect of pneumococcal infection on endothelial barrier integrity in flow.