Microenvironmental acidification by pneumococcal sugar consumption fosters barrier disruption and immune suppression in the human alveolus.

Streptococcus pneumoniae (S.p.) is the most common causative agent of community-acquired pneumonia worldwide. A key pathogenic mechanism that exacerbates severity of disease is the disruption of the alveolar-capillary barrier. However, the specific virulence mechanisms responsible for this in the human lung are not yet fully understood.In this study, we infected living human lung tissue with S.p. and observed a significant degradation of the central junctional proteins occludin and VE-cadherin, indicating barrier disruption. Surprisingly, neither pneumolysin, bacterial hydrogen peroxide nor pro-inflammatory activation were sufficient to cause this junctional degradation. Instead, pneumococcal infection led to a significant decrease of pH (approximately 6), resulting in acidification of the alveolar microenvironment, which was linked to junctional degradation. Stabilising the pH at physiological levels during infection reversed this effect, even in a therapeutic-like approach.Further analysis of bacterial metabolites and RNA sequencing revealed sugar consumption and subsequent lactate production were the major factors contributing to bacterially induced alveolar acidification, which also hindered the release of critical immune factors.Our findings highlight bacterial metabolite-induced acidification as an independent virulence mechanism for barrier disruption and inflammatory dysregulation in pneumonia. Thus, our data suggest that strictly monitoring and buffering alveolar pH during infections caused by fermentative bacteria could serve as an adjunctive therapeutic strategy for sustaining barrier integrity and immune response.

Pneumococcal Neuraminidases Increase Platelet Killing by Pneumolysin.

BACKGROUND: Platelets prevent extravasation of capillary fluids into the pulmonary interstitial tissue by sealing gaps in inflamed endothelium. This reduces respiratory distress associated with pneumonia. Streptococcus pneumoniae is the leading cause of severe community-acquired pneumonia. Pneumococci produce pneumolysin (PLY), which forms pores in membranes of eukaryotic cells including platelets. Additionally, pneumococci express neuraminidases, which cleave sialic acid residues from eukaryotic glycoproteins. In this study, we investigated the effect of desialylation on PLY binding and pore formation on platelets. MATERIALS AND METHODS: We incubated human platelets with purified neuraminidases and PLY, or nonencapsulated S. pneumoniae D39/TIGR4 and isogenic mutants deficient in PLY and/or NanA. We assessed platelet desialylation, PLY binding, and pore formation by flow cytometry. We also analyzed the inhibitory potential of therapeutic immunoglobulin G preparations (IVIG [intravenous immunoglobulin]). RESULTS: Wild-type pneumococci cause desialylation of platelet glycoproteins by neuraminidases, which is reduced by 90 to 100% in NanA-deficient mutants. NanC, cleaving only α2,3-linked sialic acid, induced platelet desialylation. PLY binding to platelets then x2doubled (p = 0.0166) and pore formation tripled (p = 0.0373). A neuraminidase cleaving α2,3-, α2,6-, and α2,8-linked sialic acid like NanA was even more efficient. Addition of polyvalent IVIG (5 mg/mL) decreased platelet desialylation induced by NanC up to 90% (p = 0.263) and reduced pore formation >95% (p < 0.0001) when incubated with pneumococci. CONCLUSION: Neuraminidases are key virulence factors of pneumococci and desialylate platelet glycoproteins, thereby unmasking PLY-binding sites. This enhances binding of PLY and pore formation showing that pneumococcal neuraminidases and PLY act in concert to kill platelets. However, human polyvalent immunoglobulin G preparations are promising agents for therapeutic intervention during severe pneumococcal pneumonia.

Lipoteichoic Acids Are Essential for Pneumococcal Colonization and Membrane Integrity.

INTRODUCTION: The hydrophilic, polymeric chain of the lipoteichoic acid (LTA) of the Gram-positive pathobiont Streptococcus pneumoniae is covalently linked to the glycosylglycerolipid α-d-glucopyranosyl-(1,3)-diacylglycerol by the LTA ligase TacL, leading to its fixation in the cytoplasmic membrane. Pneumococcal LTA, sharing identical repeating units with the wall teichoic acids (WTA), is dispensable for normal growth but required for full virulence in invasive infections. METHODS: Mutants deficient in TacL and complemented strains constructed were tested for their growth, resistance against oxidative stress, and susceptibility against antimicrobial peptides. Further, the membrane fluidity of pneumococci, their capability to adhere to lung epithelial cells, and virulence in a Galleria mellonella as well as intranasal mouse infection model were assessed. RESULTS: In the present study, we indicate that LTA is already indispensable for pneumococcal adherence to human nasopharyngeal cells and colonization in an intranasal mouse infection model. Mutants deficient for TacL did not show morphological defects. However, our analysis of pneumococcal membranes in different serotypes showed an altered membrane fluidity and surface protein abundance of lipoproteins in mutants deficient for LTA but not WTA. These mutants had a decreased membrane fluidity, exhibited higher amounts of lipoproteins, and showed an increased susceptibility to antimicrobial peptides. In complemented mutant strains, this defect was fully restored. CONCLUSION: Taken together, LTA is crucial for colonization and required to effectively protect pneumococci from innate immune defence mechanisms by maintaining the membrane integrity.

Molecular and structural basis of oligopeptide recognition by the Ami transporter system in pneumococci.

ATP-binding cassette (ABC) transport systems are crucial for bacteria to ensure sufficient uptake of nutrients that are not produced de novo or improve the energy balance. The cell surface of the pathobiont Streptococcus pneumoniae (pneumococcus) is decorated with a substantial array of ABC transporters, critically influencing nasopharyngeal colonization and invasive infections. Given the auxotrophic nature of pneumococci for certain amino acids, the Ami ABC transporter system, orchestrating oligopeptide uptake, becomes indispensable in host compartments lacking amino acids. The system comprises five exposed Oligopeptide Binding Proteins (OBPs) and four proteins building the ABC transporter channel. Here, we present a structural analysis of all the OBPs in this system. Multiple crystallographic structures, capturing both open and closed conformations along with complexes involving chemically synthesized peptides, have been solved at high resolution providing insights into the molecular basis of their diverse peptide specificities. Mass spectrometry analysis of oligopeptides demonstrates the unexpected remarkable promiscuity of some of these proteins when expressed in Escherichia coli, displaying affinity for a wide range of peptides. Finally, a model is proposed for the complete Ami transport system in complex with its various OBPs. We further disclosed, through in silico modelling, some essential structural changes facilitating oligopeptide transport into the cellular cytoplasm. Thus, the structural analysis of the Ami system provides valuable insights into the mechanism and specificity of oligopeptide binding by the different OBPs, shedding light on the intricacies of the uptake mechanism and the in vivo implications for this human pathogen.

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.

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.

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.

Platelets, Bacterial Adhesins and the Pneumococcus.

Systemic infections with pathogenic or facultative pathogenic bacteria are associated with activation and aggregation of platelets leading to thrombocytopenia and activation of the clotting system. Bacterial proteins leading to platelet activation and aggregation have been identified, and while platelet receptors are recognized, induced signal transduction cascades are still often unknown. In addition to proteinaceous adhesins, pathogenic bacteria such as Staphylococcus aureus and Streptococcus pneumoniae also produce toxins such as pneumolysin and alpha-hemolysin. They bind to cellular receptors or form pores, which can result in disturbance of physiological functions of platelets. Here, we discuss the bacteria-platelet interplay in the context of adhesin-receptor interactions and platelet-activating bacterial proteins, with a main emphasis on S. aureus and S. pneumoniae. More importantly, we summarize recent findings of how S. aureus toxins and the pore-forming toxin pneumolysin of S. pneumoniae interfere with platelet function. Finally, the relevance of platelet dysfunction due to killing by toxins and potential treatment interventions protecting platelets against cell death are summarized.

α-hemolysin of Staphylococcus aureus impairs thrombus formation.

BACKGROUND: Toxins are key virulence determinants of pathogens and can impair the function of host immune cells, including platelets. Insights into pathogen toxin interference with platelets will be pivotal to improve treatment of patients with bacterial bloodstream infections. MATERIALS AND METHODS: In this study, we deciphered the effects of Staphylococcus aureus toxins α-hemolysin, LukAB, LukDE, and LukSF on human platelets and compared the effects with the pore forming toxin pneumolysin of Streptococcus pneumoniae. Activation of platelets and loss of platelet function were investigated by flow cytometry, aggregometry, platelet viability, fluorescence microscopy, and intracellular calcium release. Thrombus formation was assessed in whole blood. RESULTS: α-hemolysin (Hla) is known to be a pore-forming toxin. Hla-induced calcium influx initially activates platelets as indicated by CD62P and αIIbß3 integrin activation, but also induces finally alterations in the phenotype of platelets. In contrast to Hla and pneumolysin, S. aureus bicomponent pore-forming leukocidins LukAB, LukED, and LukSF do not bind to platelets and had no significant effect on platelet activation and viability. The presence of small amounts of Hla (0.2 µg/ml) in whole blood abrogates thrombus formation indicating that in systemic infections with S. aureus the stability of formed thrombi is impaired. Damage of platelets by Hla was not neutralized by intravenous immune globulins. CONCLUSION: Our findings might be of clinical relevance for S. aureus induced endocarditis. Stabilizing the aortic-valve thrombi by inhibiting Hla-induced impairment of platelets might reduce the risk for septic (micro-)embolization.

Pneumococcal Extracellular Serine Proteases: Molecular Analysis and Impact on Colonization and Disease.

The pathobiont Streptococcus pneumoniae causes life-threatening diseases, including pneumonia, sepsis, meningitis, or non-invasive infections such as otitis media. Serine proteases are enzymes that have been emerged during evolution as one of the most abundant and functionally diverse group of proteins in eukaryotic and prokaryotic organisms. S. pneumoniae expresses up to four extracellular serine proteases belonging to the category of trypsin-like or subtilisin-like family proteins: HtrA, SFP, PrtA, and CbpG. These serine proteases have recently received increasing attention because of their immunogenicity and pivotal role in the interaction with host proteins. This review is summarizing and focusing on the molecular and functional analysis of pneumococcal serine proteases, thereby discussing their contribution to pathogenesis.

Innate immune responses at the asymptomatic stage of influenza A viral infections of Streptococcus pneumoniae colonized and non-colonized mice.

Seasonal Influenza A virus (IAV) infections can promote dissemination of upper respiratory tract commensals such as Streptococcus pneumoniae to the lower respiratory tract resulting in severe life-threatening pneumonia. Here, we aimed to compare innate immune responses in the lungs of healthy colonized and non-colonized mice after IAV challenge at the initial asymptomatic stage of infection. Responses during a severe bacterial pneumonia were profiled for comparison. Cytokine and innate immune cell imprints of the lungs were analyzed. Irrespective of the colonization status, mild H1N1 IAV infection was characterized by a bi-phasic disease progression resulting in full recovery of the animals. Already at the asymptomatic stage of viral infection, the pro-inflammatory cytokine response was as high as in pneumococcal pneumonia. Flow cytometry analyses revealed an early influx of inflammatory monocytes into the lungs. Neutrophil influx was mostly limited to bacterial infections. The majority of cells, except monocytes, displayed an activated phenotype characterized by elevated CCR2 and MHCII expression. In conclusion, we show that IAV challenge of colonized healthy mice does not automatically result in severe co-infection. However, a general local inflammatory response was noted at the asymptomatic stage of infection irrespective of the infection type.

The Two-Component System 09 of Streptococcus pneumoniae Is Important for Metabolic Fitness and Resistance during Dissemination in the Host.

The two-component regulatory system 09 of Streptococcus pneumoniae has been shown to modulate resistance against oxidative stress as well as capsule expression. These data and the implication of TCS09 in cell wall integrity have been shown for serotype 2 strain D39. Other data have suggested strain-specific regulatory effects of TCS09. Contradictory data are known on the impact of TCS09 on virulence, but all have been explored using only the rr09-mutant. In this study, we have therefore deleted one or both components of the TCS09 (SP_0661 and SP_0662) in serotype 4 S. pneumoniae TIGR4. In vitro growth assays in chemically defined medium (CDM) using sucrose or lactose as a carbon source indicated a delayed growth of nonencapsulated tcs09-mutants, while encapsulated wild-type TIGR4 and tcs09-mutants have reduced growth in CDM with glucose. Using a set of antigen-specific antibodies, immunoblot analysis showed that only the pilus 1 backbone protein RrgB is significantly reduced in TIGR4ΔcpsΔhk09. Electron microscopy, adherence and phagocytosis assays showed no impact of TCS09 on the TIGR4 cell morphology and interaction with host cells. In contrast, in vivo infections and in particular competitive co-infection experiments demonstrated that TCS09 enhances robustness during dissemination in the host by maintaining bacterial fitness.

Crystal Structure and Pathophysiological Role of the Pneumococcal Nucleoside-binding Protein PnrA.

Nucleotides are important for RNA and DNA synthesis and, despite a de novo synthesis by bacteria, uptake systems are crucial. Streptococcus pneumoniae, a facultative human pathogen, produces a surface-exposed nucleoside-binding protein, PnrA, as part of an ABC transporter system. Here we demonstrate the binding affinity of PnrA to nucleosides adenosine, guanosine, cytidine, thymidine and uridine by microscale thermophoresis and indicate the consumption of adenosine and guanosine by 1H NMR spectroscopy. In a series of five crystal structures we revealed the PnrA structure and provide insights into how PnrA can bind purine and pyrimidine ribonucleosides but with preference for purine ribonucleosides. Crystal structures of PnrA:nucleoside complexes unveil a clear pattern of interactions in which both the N- and C- domains of PnrA contribute. The ribose moiety is strongly recognized through a conserved network of H-bond interactions, while plasticity in loop 27-36 is essential to bind purine- or pyrimidine-based nucleosides. Further, we deciphered the role of PnrA in pneumococcal fitness in infection experiments. Phagocytosis experiments did not show a clear difference in phagocytosis between PnrA-deficient and wild-type pneumococci. In the acute pneumonia infection model the deficiency of PnrA attenuated moderately virulence of the mutant, which is indicated by a delay in the development of severe lung infections. Importantly, we confirmed the loss of fitness in co-infections, where the wild-type out-competed the pnrA-mutant. In conclusion, we present the PnrA structure in complex with individual nucleosides and show that the consumption of adenosine and guanosine under infection conditions is required for virulence.

Pneumolysin induces platelet destruction, not platelet activation, which can be prevented by immunoglobulin preparations in vitro.

Community-acquired pneumonia by primary or superinfections with Streptococcus pneumoniae can lead to acute respiratory distress requiring mechanical ventilation. The pore-forming toxin pneumolysin alters the alveolar-capillary barrier and causes extravasation of protein-rich fluid into the interstitial pulmonary tissue, which impairs gas exchange. Platelets usually prevent endothelial leakage in inflamed pulmonary tissue by sealing inflammation-induced endothelial gaps. We not only confirm that S pneumoniae induces CD62P expression in platelets, but we also show that, in the presence of pneumolysin, CD62P expression is not associated with platelet activation. Pneumolysin induces pores in the platelet membrane, which allow anti-CD62P antibodies to stain the intracellular CD62P without platelet activation. Pneumolysin treatment also results in calcium efflux, increase in light transmission by platelet lysis (not aggregation), loss of platelet thrombus formation in the flow chamber, and loss of pore-sealing capacity of platelets in the Boyden chamber. Specific anti-pneumolysin monoclonal and polyclonal antibodies inhibit these effects of pneumolysin on platelets as do polyvalent human immunoglobulins. In a post hoc analysis of the prospective randomized phase 2 CIGMA trial, we show that administration of a polyvalent immunoglobulin preparation was associated with a nominally higher platelet count and nominally improved survival in patients with severe S pneumoniae-related community-acquired pneumonia. Although, due to the low number of patients, no definitive conclusion can be made, our findings provide a rationale for investigation of pharmacologic immunoglobulin preparations to target pneumolysin by polyvalent immunoglobulin preparations in severe community-acquired pneumococcal pneumonia, to counteract the risk of these patients becoming ventilation dependent. This trial was registered at www.clinicaltrials.gov as #NCT01420744.

Activated platelets kill Staphylococcus aureus, but not Streptococcus pneumoniae-The role of FcγRIIa and platelet factor 4/heparinantibodies.

BACKGROUND: Heparin induced thrombocytopenia (HIT) is likely a misdirected bacterial host defense mechanism. Platelet factor 4 (PF4) binds to polyanions on bacterial surfaces exposing neo-epitopes to which HIT antibodies bind. Platelets are activated by the resulting immune complexes via FcγRIIA, release bactericidal substances, and kill Gram-negative Escherichia coli. OBJECTIVES: To assess the role of PF4, anti-PF4/H antibodies and FcγRIIa in killing of Gram-positive bacteria by platelets. METHODS: Binding of PF4 to protein-A deficient Staphylococcus aureus (SA113Δspa) and non-encapsulated Streptococcus pneumoniae (D39Δcps) and its conformational change were assessed by flow cytometry using monoclonal (KKO,5B9) and patient derived anti-PF4/H antibodies. Killing of bacteria was quantified by counting colony forming units (cfu) after incubation with platelets or platelet releasate. Using flow cytometry, platelet activation (CD62P-expression, PAC-1 binding) and phosphatidylserine (PS)-exposure were analyzed. RESULTS: Monoclonal and patient-derived anti-PF4/H antibodies bound in the presence of PF4 to both S. aureus and S. pneumoniae (1.6-fold increased fluorescence signal for human anti-PF4/H antibodies to 24.0-fold increase for KKO). Staphylococcus aureus (5.5 × 104 cfu/mL) was efficiently killed by platelets (2.7 × 104 cfu/mL) or their releasate (2.9 × 104 cfu/mL). Killing was not further enhanced by PF4 or anti-PF4/H antibodies. Blocking FcγRIIa had no impact on killing of S. aureus by platelets. In contrast, S. pneumoniae was not killed by platelets or releasate. Instead, after incubation with pneumococci platelets were unresponsive to TRAP-6 stimulation and exposed high levels of PS. CONCLUSIONS: Anti-PF4/H antibodies seem to have only a minor role for direct killing of Gram-positive bacteria by platelets. Staphylococcus aureus is killed by platelets or platelet releasate. In contrast, S. pneumoniae affects platelet viability.

Proteomic Adaptation of Streptococcus pneumoniae to the Human Antimicrobial Peptide LL-37.

Secreted antimicrobial peptides (AMPs) are an important part of the human innate immune system and prevent local and systemic infections by inhibiting bacterial growth in a concentration-dependent manner. In the respiratory tract, the cationic peptide LL-37 is one of the most abundant AMPs and capable of building pore complexes in usually negatively charged bacterial membranes, leading to the destruction of bacteria. However, the adaptation mechanisms of several pathogens to LL-37 are already described and are known to weaken the antimicrobial effect of the AMP, for instance, by repulsion, export or degradation of the peptide. This study examines proteome-wide changes in Streptococcus pneumoniae D39, the leading cause of bacterial pneumonia, in response to physiological concentrations of LL-37 by high-resolution mass spectrometry. Our data indicate that pneumococci may use some of the known adaptation mechanisms to reduce the effect of LL-37 on their physiology, too. Additionally, several proteins seem to be involved in resistance to AMPs which have not been related to this process before, such as the teichoic acid flippase TacF (SPD_1128). Understanding colonization- and infection-relevant adaptations of the pneumococcus to AMPs, especially LL-37, could finally uncover new drug targets to weaken the burden of this widespread pathogen.

Extracellular Pneumococcal Serine Proteases Affect Nasopharyngeal Colonization.

Streptococcus pneumoniae has evolved versatile strategies to colonize the nasopharynx of humans. Colonization is facilitated by direct interactions with host cell receptors or via binding to components of the extracellular matrix. In addition, pneumococci hijack host-derived extracellular proteases such as the serine protease plasmin(ogen) for ECM and mucus degradation as well as colonization. S. pneumoniae expresses strain-dependent up to four serine proteases. In this study, we assessed the role of secreted or cell-bound serine proteases HtrA, PrtA, SFP, and CbpG, in adherence assays and in a mouse colonization model. We hypothesized that the redundancy of serine proteases compensates for the deficiency of a single enzyme. Therefore, double and triple mutants were generated in serotype 19F strain EF3030 and serotype 4 strain TIGR4. Strain EF3030 produces only three serine proteases and lacks the SFP encoding gene. In adherence studies using Detroit-562 epithelial cells, we demonstrated that both TIGR4Δcps and 19F mutants without serine proteases or expressing only CbpG, HtrA, or PrtA have a reduced ability to adhere to Detroit-562 cells. Consistent with these results, we show that the mutants of strain 19F, which preferentially colonizes mice, abrogate nasopharyngeal colonization in CD-1 mice after intranasal infection. The bacterial load in the nasopharynx was monitored for 14 days. Importantly, mutants showed significantly lower bacterial numbers in the nasopharynx two days after infection. Similarly, we detected a significantly reduced pneumococcal colonization on days 3, 7, and 14 post-inoculations. To assess the impact of pneumococcal serine proteases on acute infection, we infected mice intranasally with bioluminescent and invasive TIGR4 or isogenic triple mutants expressing only CbpG, HtrA, PrtA, or SFP. We imaged the acute lung infection in real-time and determined the survival of the mice. The TIGR4lux mutant expressing only PrtA showed a significant attenuation and was less virulent in the acute pneumonia model. In conclusion, our results showed that pneumococcal serine proteases contributed significantly to pneumococcal colonization but played only a minor role in pneumonia and invasive diseases. Because colonization is a prerequisite for invasive diseases and transmission, these enzymes could be promising candidates for the development of antimicrobials to reduce pneumococcal transmission.

Von Willebrand Factor Mediates Pneumococcal Aggregation and Adhesion in Blood Flow.

Streptococcus pneumoniae is a major cause of community acquired pneumonia and septicaemia in humans. These diseases are frequently associated with thromboembolic cardiovascular complications. Pneumococci induce the exocytosis of endothelial Weibel-Palade Bodies and thereby actively stimulate the release of von Willebrand factor (VWF), which is an essential glycoprotein of the vascular hemostasis. Both, the pneumococcus induced pulmonary inflammation and the thromboembolytic complications are characterized by a dysbalanced hemostasis including a marked increase in VWF plasma concentrations. Here, we describe for the first time VWF as a novel interaction partner of capsulated and non-encapsulated pneumococci. Moreover, cell culture infection analyses with primary endothelial cells characterized VWF as bridging molecule that mediates bacterial adherence to endothelial cells in a heparin-sensitive manner. Due to the mechanoresponsive changes of the VWF protein conformation and multimerization status, which occur in the blood stream, we used a microfluidic pump system to generate shear flow-induced multimeric VWF strings on endothelial cell surfaces and analyzed attachment of RFP-expressing pneumococci in flow. By applying immunofluorescence visualization and additional electron microscopy, we detected a frequent and enduring bacterial attachment to the VWF strings. Bacterial attachment to the endothelium was confirmed in vivo using a zebrafish infection model, which is described in many reports and acknowledged as suitable model to study hemostasis mechanisms and protein interactions of coagulation factors. Notably, we visualized the recruitment of zebrafish-derived VWF to the surface of pneumococci circulating in the blood stream and detected a VWF-dependent formation of bacterial aggregates within the vasculature of infected zebrafish larvae. Furthermore, we identified the surface-exposed bacterial enolase as pneumococcal VWF binding protein, which interacts with the VWF domain A1 and determined the binding kinetics by surface plasmon resonance. Subsequent epitope mapping using an enolase peptide array indicates that the peptide 181YGAEIFHALKKILKS195 might serve as a possible core sequence of the VWF interaction site. In conclusion, we describe a VWF-mediated mechanism for pneumococcal anchoring within the bloodstream via surface-displayed enolase, which promotes intravascular bacterial aggregation.

Homophilic protein interactions facilitate bacterial aggregation and IgG-dependent complex formation by the Streptococcus canis M protein SCM.

Streptococcus canis is a zoonotic agent that causes serious invasive diseases in domestic animals and humans, but knowledge about its pathogenic potential and underlying virulence mechanisms is limited. Here, we report on the ability of certain S. canis isolates to form large bacterial aggregates when grown in liquid broth. Bacterial aggregation was attributed to the presence and the self-binding activity of SCM, the M protein of S. canis, as evaluated by bacterial sedimentation assays, immunofluorescence- and electron microscopic approaches. Using a variety of truncated recombinant SCM fragments, we demonstrated that homophilic SCM interactions occur via the N-terminal, but not the C-terminal part, of the mature M protein. Interestingly, when incubated in human plasma, SCM forms soluble protein complexes comprising its known ligands, immunoglobulin G (IgG) and plasminogen (Plg). Co-incubation studies with purified host proteins revealed that SCM-mediated complex formation is based on the interaction of SCM with itself and with IgG, but not with Plg or fibrinogen (Fbg), well-established constituents of M protein-mediated protein complexes in human-associated streptococci. Notably, these soluble, SCM-mediated plasma complexes harbored complement factor C1q, which can induce complement breakdown in the periphery and therefore represent another immune evasion mechanism of SCM.