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The iron-regulated surface determinant protein B (IsdB) of Staphylococcus aureus is involved in the acquisition of iron from hemoglobin. Moreover, IsdB elicits an adaptive immune response in mice and humans. Here, we show that IsdB also has impact on innate immunity. IsdB induces the release of proinflammatory cytokines, including IL-6 and IL-1β, in innate immune cells of humans and mice. In silico analysis and thermophoresis show that IsdB directly binds to TLR4 with high affinity. TLR4 sensing was essential for the IsdB-mediated production of IL-6, IL-1β, and other cytokines as it was abolished by blocking of TLR4-MyD88-IRAK1/4-NF-κB signaling. The release of IL-1β additionally required activation of the NLRP3 inflammasome. In human monocytes infected with live S. aureus, IsdB was necessary for maximal IL-1β release. Our studies identify S. aureus IsdB as a novel pathogen-associated molecular pattern that triggers innate immune defense mechanisms.
Epithelial cells are an important line of defense within the lung. Disruption of the epithelial barrier by pathogens enables the systemic dissemination of bacteria or viruses within the host leading to severe diseases with fatal outcomes. Thus, the lung epithelium can be damaged by seasonal and pandemic influenza A viruses. Influenza A virus infection induced dysregulation of the immune system is beneficial for the dissemination of bacteria to the lower respiratory tract, causing bacterial and viral co-infection. Host cells regulate protein homeostasis and the response to different perturbances, for instance provoked by infections, by post translational modification of proteins. Aside from protein phosphorylation, ubiquitination of proteins is an essential regulatory tool in virtually every cellular process such as protein homeostasis, host immune response, cell morphology, and in clearing of cytosolic pathogens. Here, we analyzed the proteome and ubiquitinome of A549 alveolar lung epithelial cells in response to infection by either Streptococcus pneumoniae D39Δcps or influenza A virus H1N1 as well as bacterial and viral co-infection. Pneumococcal infection induced alterations in the ubiquitination of proteins involved in the organization of the actin cytoskeleton and Rho GTPases, but had minor effects on the abundance of host proteins. H1N1 infection results in an anti-viral state of A549 cells. Finally, co-infection resembled the imprints of both infecting pathogens with a minor increase in the observed alterations in protein and ubiquitination abundance.
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.
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.
Antibiotic resistance in pneumococci contributes to the high pneumococcal deaths in children. We assessed the molecular characteristics of multidrug-resistant (MDR) pneumococci isolated from healthy vaccinated children under five years of age in Cape Coast, Ghana. A total of 43 MDR isolates were selected from 151 pneumococcal strains obtained from nasopharyngeal carriage. All isolates were previously serotyped by multiplex PCR and Quellung reaction. Susceptibility testing was performed using either the E-test or disk diffusion method. Virulence and antibiotic resistance genes were identified by PCR. Molecular epidemiology was analyzed using multilocus sequence typing (MLST). Vaccine-serotypes 23F and 19F were predominant. The lytA and pavB virulence genes were present in all isolates, whiles 14–86% of the isolates carried pilus-islets 1 and 2, pcpA, and psrP genes. Penicillin, tetracycline, and cotrimoxazole resistance were evident in >90% of the isolates. The ermB, mefA, and tetM genes were detected in (n = 7, 16.3%), (n = 4, 9.3%) and (n = 43, 100%) of the isolates, respectively. However, >60% showed alteration in the pbp2b gene. MLST revealed five novel and six known sequence types (STs). ST156 (Spain9V-3) and ST802 were identified as international antibiotic-resistant clones. The emergence of international-MDR clones in Ghana requires continuous monitoring of the pneumococcus through a robust surveillance system.
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.
Influenza A Virus (IAV), Staphylococcus aureus (staphylococci), and Streptococcus pneumoniae (pneumococci) are leading viral and bacterial causes of pneumonia. Dendritic cells (DCs) are present in the lower respiratory tract. They are characterized by low expression of co-stimulatory molecules, including CD80 and CD86 and high capacity of antigen uptake. Subsequently, DCs upregulate co-stimulatory signals and cytokine secretion to effectively induce T-cell priming. Here, we investigated these processes in response to bacterial and viral single as well as coinfections using human monocyte-derived (mo)DCs. Irrespective of single or coinfections, moDCs matured in response to IAV and/or staphylococcal infections, secreted a wide range of cytokines, and activated CD4+, CD8+ as well as double-negative T cells. In contrast, pneumococcal single and coinfections impaired moDC maturation, which was characterized by low expression of CD80 and CD86, downregulated expression of CD40, and a mild cytokine release resulting in abrogated CD4+ T-cell activation. These actions were attributed to the cholesterol-dependent cytotoxin pneumolysin (Ply). Infections with a ply-deficient mutant resulted in restored moDC maturation and exclusive CD4+ T-cell activation. These findings show that Ply has important immunomodulatory functions, supporting further investigations in specific modalities of Ply-DC interplay.
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.
Abstract
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 αIIbβ3 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.
The Two-Component System 09 Regulates Pneumococcal Carbohydrate Metabolism and Capsule Expression
(2021)
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.
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.
Respiratory infection caused by Streptococcus pneumoniae is a leading cause of morbidity and mortality in older adults. Acquired CD4+ T cell mechanism are essential for the protection against colonization and subsequent development of infections by S. pneumoniae. In this study, we hypothesized that age-related changes within the CD4+ T-cell population compromise CD4+ T-cell specific responses to S. pneumoniae, thereby contributing to increased susceptibility at older age. To this end, we interrogated the CD4+ T-cell response against the immunogenic pneumococcal protein AliB, part of the unique oligopeptide ABC transporter system responsible for the uptake of nutrients for the bacterium and crucial for the development of pneumococcal meningitis, in healthy young and older adults. Specifically, proliferation of CD4+ T cells as well as concomitant cytokine profiles and phenotypic markers implied in immunosenescence were studied. Older adults showed decreased AliB-induced CD4+ T-cell proliferation that is associated with an increased frequency of regulatory T cells and lower levels of active CD25+CD127+CTLA-4−TIGIT-CD4+T cells. Additionally, levels of pro-inflammatory cytokines IFNy and IL-17F were decreased at older age. Our findings indicate that key features of a pneumococcal-specific CD4+ T-cell immune response are altered at older age, which may contribute to enhanced susceptibility for pneumococcal infections.
Like eukaryotes, different bacterial species express one or more Ser/Thr kinases and phosphatases that operate in various signaling networks by catalyzing phosphorylation and dephosphorylation of proteins that can immediately regulate biochemical pathways by altering protein function. The human pathogen Streptococcus pneumoniae encodes a single Ser/Thr kinase-phosphatase couple known as StkP-PhpP, which has shown to be crucial in the regulation of cell wall synthesis and cell division. In this study, we applied proteomics to further understand the physiological role of pneumococcal PhpP and StkP with an emphasis on phosphorylation events on Ser and Thr residues. Therefore, the proteome of the non-encapsulated D39 strain (WT), a kinase (ΔstkP), and phosphatase mutant (ΔphpP) were compared in a mass spectrometry based label-free quantification experiment. Results show that a loss of function of PhpP causes an increased abundance of proteins in the phosphate uptake system Pst. Quantitative proteomic data demonstrated an effect of StkP and PhpP on the two-component systems ComDE, LiaRS, CiaRH, and VicRK. To obtain further information on the function, targets and target sites of PhpP and StkP we combined the advantages of phosphopeptide enrichment using titanium dioxide and spectral library based data evaluation for sensitive detection of changes in the phosphoproteome of the wild type and the mutant strains. According to the role of StkP in cell division we identified several proteins involved in cell wall synthesis and cell division that are apparently phosphorylated by StkP. Unlike StkP, the physiological function of the co-expressed PhpP is poorly understood. For the first time we were able to provide a list of previously unknown putative targets of PhpP. Under these new putative targets of PhpP are, among others, five proteins with direct involvement in cell division (DivIVA, GpsB) and peptidoglycan biosynthesis (MltG, MreC, MacP).
Streptococcus pneumoniaeinfections lead to high morbidity and mortality rates worldwide.Pneumococcal polysaccharide conjugate vaccines significantly reduce the burden of disease but havea limited range of protection, which encourages the development of a broadly protective protein-basedalternative. We and others have shown that immunization with pneumococcal lipoproteins that lackthe lipid anchor protects against colonization. Since immunity againstS. pneumoniaeis mediatedthrough Toll-like receptor 2 signaling induced by lipidated proteins, we investigated the effects ofa lipid modification on the induced immune responses in either intranasally or subcutaneouslyvaccinated mice. Here, we demonstrate that lipidation of recombinant lipoproteins DacB and PnrAstrongly improves their immunogenicity. Mice immunized with lipidated proteins showed enhancedantibody concentrations and different induction kinetics. The induced humoral immune responsewas modulated by lipidation, indicated by increased IgG2/IgG1 subclass ratios related to Th1-typeimmunity. In a mouse model of colonization, immunization with lipidated antigens led to a moderatebut consistent reduction of pneumococcal colonization as compared to the non-lipidated proteins,indicating that protein lipidation can improve the protective capacity of the coupled antigen. Thus,protein lipidation represents a promising approach for the development of a serotype-independentpneumococcal vaccine.
Abstract
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 × 104cfu/mL) was efficiently killed by platelets (2.7 × 104cfu/mL) or their releasate (2.9 × 104cfu/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.