Cobalt and Chromium Ions Impair Macrophage Response to Staphylococcus aureus Infection.

Cobalt-chromium-molybdenum (CoCrMo) alloys are routinely used in arthroplasty. CoCrMo wear particles and ions derived from arthroplasty implants lead to macrophage-driven adverse local tissue reactions, which have been linked to an increased risk of periprosthetic joint infection after revision arthroplasty. While metal-induced cytotoxicity is well characterized in human macrophages, direct effects on their functionality have remained elusive. Synchrotron radiation X-ray microtomography and X-ray fluorescence mapping indicated that peri-implant tissues harvested during aseptic revision of different arthroplasty implants are exposed to Co and Cr in situ. Confocal laser scanning microscopy revealed that macrophage influx is predominant in patient tissue. While in vitro exposure to Cr3+ had only minor effects on monocytes/macrophage phenotype, pathologic concentrations of Co2+ significantly impaired both, monocyte/macrophage phenotype and functionality. High concentrations of Co2+ led to a shift in macrophage subsets and loss of surface markers, including CD14 and CD16. Both Co2+ and Cr3+ impaired macrophage responses to Staphylococcus aureus infection, and particularly, Co2+-exposed macrophages showed decreased phagocytic activity. These findings demonstrate the immunosuppressive effects of locally elevated metal ions on the innate immune response and support further investigations, including studies exploring whether Co2+ and Cr3+ or CoCrMo alloys per se expose the patients to a higher risk of infections post-revision arthroplasty.

The global proteome and ubiquitinome of bacterial and viral co-infected bronchial epithelial cells.

Viral infections facilitate bacterial trafficking to the lower respiratory tract resulting in bacterial-viral co-infections. Bacterial dissemination to the lower respiratory tract is enhanced by influenza A virus induced epithelial cell damage and dysregulation of immune responses. Epithelial cells act as a line of defense and detect pathogens by a high variety of pattern recognition receptors. The post-translational modification ubiquitin is involved in almost every cellular process. Moreover, ubiquitination contributes to the regulation of host immune responses, influenza A virus uncoating and transport within host cells. We applied proteomics with a special focus on ubiquitination to assess the impact of single bacterial and viral as well as bacterial-viral co-infections on bronchial epithelial cells. We used Tandem Ubiquitin Binding Entities to enrich polyubiquitinated proteins and assess changes in the ubiquitinome. Infecting 16HBE cells with Streptococcus pyogenes led to an increased abundance of proteins related to mitochondrial translation and energy metabolism in proteome and ubiquitinome. In contrast, influenza A virus infection mainly altered the ubiquitinome. Co-infections had no additional impact on protein abundances or affected pathways. Changes in protein abundance and enriched pathways were assigned to imprints of both infecting pathogens. SIGNIFICANCE: Viral and bacterial co-infections of the lower respiratory tract are a burden for health systems worldwide. Therefore, it is necessary to elucidate the complex interplay between the host and the infecting pathogens. Thus, we analyzed the proteome and the ubiquitinome of co-infected bronchial epithelial cells to elaborate a potential synergism of the two infecting organisms. The results presented in this work can be used as a starting point for further analyses.

Hydrogen Peroxide Is Crucial for NLRP3 Inflammasome-Mediated IL-1ß Production and Cell Death in Pneumococcal Infections of Bronchial Epithelial Cells.

Epithelial cells play a crucial role in detection of the pathogens as well as in initiation of the host immune response. Streptococcus pneumoniae (pneumococcus) is a typical colonizer of the human nasopharynx, which can disseminate to the lower respiratory tract and subsequently cause severe invasive diseases such as pneumonia, sepsis, and meningitis. Hydrogen peroxide (H2O2) is produced by pneumococci as a product of the pyruvate oxidase SpxB. However, its role as a virulence determinant in pneumococcal infections of the lower respiratory tract is not well understood. In this study, we investigated the role of pneumococcal-derived H2O2 in initiating epithelial cell death by analyzing the interplay between 2 key cell death pathways, namely, apoptosis and pyroptosis. We demonstrate that H2O2 primes as well as activates the NLRP3 inflammasome and thereby mediates IL-1ß production and release. Furthermore, we show that pneumococcal H2O2 causes cell death via the activation of both apoptotic as well as pyroptotic pathways which are mediated by the activation of caspase-3/7 and caspase-1, respectively. However, H2O2-mediated IL-1ß release itself occurs mainly via apoptosis.

Bronchial Epithelial Cells Accumulate Citrate Intracellularly in Response to Pneumococcal Hydrogen Peroxide.

Community-acquired pneumonia is an infection of the lower respiratory tract caused by various viral and bacterial pathogens, including influenza A virus (IAV), Streptococcus pneumoniae, and Staphylococcus aureus. To understand the disease pathology, it is important to delineate host metabolic responses to an infection. In this study, metabolome profiling of mono- and coinfected human bronchial epithelial cells was performed. We show that IAV and S. aureus silently survive within the cells with almost negligible effects on the host metabolome. In contrast, S. pneumoniae significantly altered various host pathways such as glycolysis, tricarboxylic acid cycle, and amino acid metabolism. Intracellular citrate accumulation was the most prominent signature of pneumococcal infections and was directly attributed to the action of pneumococci-derived hydrogen peroxide. No coinfection specific metabolome signatures were observed.

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.

Adenosine Triphosphate Neutralizes Pneumolysin-Induced Neutrophil Activation.

BACKGROUND: In tissue infections, adenosine triphosphate (ATP) is released into extracellular space and contributes to purinergic chemotaxis. Neutrophils are important players in bacterial clearance and are recruited to the site of tissue infections. Pneumococcal infections can lead to uncontrolled hyperinflammation of the tissue along with substantial tissue damage through excessive neutrophil activation and uncontrolled granule release. We aimed to investigate the role of ATP in neutrophil response to pneumococcal infections. METHODS: Primary human neutrophils were exposed to the pneumococcal strain TIGR4 and its pneumolysin-deficient mutant or directly to different concentrations of recombinant pneumolysin. Neutrophil activation was assessed by measurement of secreted azurophilic granule protein resistin and profiling of the secretome, using mass spectrometry. RESULTS: Pneumococci are potent inducers of neutrophil degranulation. Pneumolysin was identified as a major trigger of neutrophil activation. This process is partially lysis independent and inhibited by ATP. Pneumolysin and ATP interact with each other in the extracellular space leading to reduced neutrophil activation. Proteome analyses of the neutrophil secretome confirmed that ATP inhibits pneumolysin-dependent neutrophil activation. CONCLUSIONS: Our findings suggest that despite its cytolytic activity, pneumolysin serves as a potent neutrophil activating factor. Extracellular ATP mitigates pneumolysin-induced neutrophil activation.

Prothrombotic and Proinflammatory Activities of the ß-Hemolytic Group B Streptococcal Pigment.

A prominent feature of severe streptococcal infections is the profound inflammatory response that contributes to systemic toxicity. In sepsis the dysregulated host response involves both immunological and nonimmunological pathways. Here, we report a fatal case of an immunocompetent healthy female presenting with toxic shock and purpura fulminans caused by group B streptococcus (GBS; serotype III, CC19). The strain (LUMC16) was pigmented and hyperhemolytic. Stimulation of human primary cells with hyperhemolytic LUMC16 and STSS/NF-HH strains and pigment toxin resulted in a release of proinflammatory mediators, including tumor necrosis factor, interleukin (IL)-1ß, and IL-6. In addition, LUMC16 induced blood clotting and showed factor XII activity on its surface, which was linked to the presence of the pigment. The expression of pigment was not linked to a mutation within the CovR/S region. In conclusion, our study shows that the hemolytic lipid toxin contributes to the ability of GBS to cause systemic hyperinflammation and interferes with the coagulation system.

Phosphoglycerate Kinase-A Novel Streptococcal Factor Involved in Neutrophil Activation and Degranulation.

BACKGROUND: Neutrophils have been proposed as important contributors to the hyperinflammatory responses that are associated with severe invasive Streptococcus pyogenes infections. In particular, streptococcal surface proteins have been implicated as potent neutrophil activators. Here we explore the impact of streptococcus-secreted factors on neutrophil activation and degranulation. METHODS: Primary human neutrophils were exposed to supernatants prepared from cultures of invasive S. pyogenes strains of varying serotypes in the stationary growth phase. Neutrophil activation was assessed by measurement of secreted resistin, an azurophilic granule marker, and by determination of the secretome profile, using mass spectrometry. RESULTS: Marked variation in resistin release and the neutrophil secretome profile were observed following exposure to different strains. A high resistin response was triggered exclusively by SpeB-negative strains, suggesting that at least 1 stimulatory factor is susceptible to SpeB proteolytic degradation. Further analysis, including proteomics and stimulation analyses, identified phosphoglycerate kinase as a stimulatory factor for neutrophils. CONCLUSIONS: Taken together, results of this study reveal a novel secreted streptococcal factor that, in the absence of SpeB, can trigger neutrophil activation and degranulation. This finding is of interest in light of reports of hypervirulent SpeB-negative S. pyogenes variants present during invasive infections.

A point mutation in AgrC determines cytotoxic or colonizing properties associated with phenotypic variants of ST22 MRSA strains.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of skin and soft tissue infections. One of the highly successful and rapidly disseminating clones is MRSA ST22 commonly associated with skin tropism. Here we show that a naturally occurring single amino acid substitution (tyrosine to cysteine) at position 223 of AgrC determines starkly different ST22 S. aureus virulence phenotypes, e.g. cytotoxic or colonizing, as evident in both in vitro and in vivo skin infections. Y223C amino acid substitution destabilizes AgrC-AgrA interaction leading to a colonizing phenotype characterized by upregulation of bacterial surface proteins. The colonizing phenotype strains cause less severe skin tissue damage, show decreased susceptibility towards the antimicrobial LL-37 and induce autophagy. In contrast, cytotoxic strains with tyrosine at position 223 of AgrC cause infections characterized by inflammasome activation and severe skin tissue pathology. Taken together, the study demonstrates how a single amino acid substitution in the histidine kinase receptor AgrC of ST22 strains determines virulence properties and infection outcome.

Differential neutrophil responses to bacterial stimuli: Streptococcal strains are potent inducers of heparin-binding protein and resistin-release.

Neutrophils are critical for the control of bacterial infections, but they may also contribute to disease pathology. Here we explore neutrophil responses, in particular the release of sepsis-associated factors heparin-binding protein (HBP) and resistin in relation to specific bacterial stimuli and sepsis of varying aetiology. Analyses of HBP and resistin in plasma of septic patients revealed elevated levels as compared to non-infected critically ill patients. HBP and resistin correlated significantly in septic patients, with the strongest association seen in group A streptococcal (GAS) cases. In vitro stimulation of human neutrophils revealed that fixed streptococcal strains induced significantly higher release of HBP and resistin, as compared to Staphylococcus aureus or Escherichia coli. Similarly, neutrophils stimulated with the streptococcal M1-protein showed a significant increase in co-localization of HBP and resistin positive granules as well as exocytosis of these factors, as compared to LPS. Using a GAS strain deficient in M1-protein expression had negligible effect on neutrophil activation, while a strain deficient in the stand-alone regulator MsmR was significantly less stimulatory as compared to its wild type strain. Taken together, the findings suggest that the streptococcal activation of neutrophils is multifactorial and involves, but is not limited to, proteins encoded by the FCT-locus.

LL-37 Triggers Formation of Streptococcus pyogenes Extracellular Vesicle-Like Structures with Immune Stimulatory Properties.

Reports have shown that the antimicrobial peptide LL-37 is abundantly expressed but has limited bactericidal effect in Streptococcus pyogenes infections. At sub-inhibitory concentrations, LL-37 has been reported to alter virulence gene expression. Here, we explored the interaction of S. pyogenes strains with LL-37, focusing on bacterial growth, cell surface alterations and pro-inflammatory responses. Bioscreen turbidity measurements of strain 5448 cultured in the presence or absence of LL-37 confirmed the poor antimicrobial effect, and revealed a significant increase in turbidity of bacterial cultures exposed to sub-inhibitory concentrations of LL-37. However, this was not linked to increased bacterial counts. Electron microscopy of LL-37-exposed bacteria revealed the presence of vesicle-like structures on the bacterial surface. The vesicles stained positive for LL-37 and were released from the bacterial surface. Concentrated supernatants enriched in these structures had a broader protein content, including several virulence factors, compared to supernatants from untreated bacteria. The supernatants from LL-37-exposed bacteria were pro-inflammatory and elicited resistin and myeloperoxidase release from neutrophils. This is the first report on S. pyogenes extracellular vesicle-like structures formed at the bacterial surface in response to LL-37. The associated increased pro-inflammatory activity further implicates LL-37 as a potential factor involved in S. pyogenes pathogenesis.

Modelling staphylococcal pneumonia in a human 3D lung tissue model system delineates toxin-mediated pathology.

Staphylococcus aureus necrotizing pneumonia is recognized as a toxin-mediated disease, yet the tissue-destructive events remain elusive, partly as a result of lack of mechanistic studies in human lung tissue. In this study, a three-dimensional (3D) tissue model composed of human lung epithelial cells and fibroblasts was used to delineate the role of specific staphylococcal exotoxins in tissue pathology associated with severe pneumonia. To this end, the models were exposed to the mixture of exotoxins produced by S. aureus strains isolated from patients with varying severity of lung infection, namely necrotizing pneumonia or lung empyema, or to purified toxins. The necrotizing pneumonia strains secreted high levels of α-toxin and Panton-Valentine leukocidin (PVL), and triggered high cytotoxicity, inflammation, necrosis and loss of E-cadherin from the lung epithelium. In contrast, the lung empyema strain produced moderate levels of PVL, but negligible amounts of α-toxin, and triggered limited tissue damage. α-toxin had a direct damaging effect on the epithelium, as verified using toxin-deficient mutants and pure α-toxin. Moreover, PVL contributed to pathology through the lysis of neutrophils. A combination of α-toxin and PVL resulted in the most severe epithelial injury. In addition, toxin-induced release of pro-inflammatory mediators from lung tissue models resulted in enhanced neutrophil migration. Using a collection of 31 strains from patients with staphylococcal pneumonia revealed that strains producing high levels of α-toxin and PVL were cytotoxic and associated with fatal outcome. Also, the strains that produced the highest toxin levels induced significantly greater epithelial disruption. Of importance, toxin-mediated lung epithelium destruction could be inhibited by polyspecific intravenous immunoglobulin containing antibodies against α-toxin and PVL. This study introduces a novel model system for study of staphylococcal pneumonia in a human setting. The results reveal that the combination and levels of α-toxin and PVL correlate with tissue pathology and clinical outcome associated with pneumonia.

Heterologous expression of Ralp3 in Streptococcus pyogenes M2 and M6 strains affects the virulence characteristics.

BACKGROUND: Ralp3 is a transcriptional regulator present in a serotype specific fashion on the chromosome of the human pathogen Streptococcus pyogenes (group A streptococci, GAS). In serotypes harbouring the ralp3 gene either positive or negative effects on important metabolic and virulence genes involved in colonization and immune evasion in the human host were observed. A previous study revealed that deletion of ralp3 in a GAS M49 serotype significantly attenuated many virulence traits and caused metabolic disadvantages. This leads to two questions: (i) which kind of consequences could Ralp3 expression have in GAS serotypes naturally lacking this gene, and (ii) is Ralp3 actively lost during evolution in these serotypes. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the role of Ralp3 in GAS M2 and M6 pathogenesis. Both serotypes lack ralp3 on their chromosome. The heterologous expression of ralp3 in both serotypes resulted in reduced attachment to and internalization into the majority of tested epithelial cells. Both ralp3 expression strains showed a decreased ability to survive in human blood and exclusively M2::ralp3 showed decreased survival in human serum. Both mutants secreted more active SpeB in the supernatant, resulting in a higher activity compared to wild type strains. The respective M2 and M6 wild type strains outcompeted the ralp3 expression strains in direct metabolic competition assays. The phenotypic changes observed in the M2:ralp3 and M6:ralp3 were verified on the transcriptional level. Consistent with the virulence data, tested genes showed transcript level changes in the same direction. CONCLUSIONS/SIGNIFICANCE: Together these data suggest that Ralp3 can take over transcriptional control of virulence genes in serotypes lacking the ralp3 gene. Those serotypes most likely lost Ralp3 during evolution since obviously expression of this gene is disadvantageous for metabolism and pathogenesis.

The extracellular protein factor Epf from Streptococcus pyogenes is a cell surface adhesin that binds to cells through an N-terminal domain containing a carbohydrate-binding module.

Streptococcus pyogenes is an exclusively human pathogen. Streptococcal attachment to and entry into epithelial cells is a prerequisite for a successful infection of the human host and requires adhesins. Here, we demonstrate that the multidomain protein Epf from S. pyogenes serotype M49 is a streptococcal adhesin. An epf-deficient mutant showed significantly decreased adhesion to and internalization into human keratinocytes. Cell adhesion is mediated by the N-terminal domain of Epf (EpfN) and increased by the human plasma protein plasminogen. The crystal structure of EpfN, solved at 1.6 Å resolution, shows that it consists of two subdomains: a carbohydrate-binding module and a fibronectin type III domain. Both fold types commonly participate in ligand receptor and protein-protein interactions. EpfN is followed by 18 repeats of a domain classified as DUF1542 (domain of unknown function 1542) and a C-terminal cell wall sorting signal. The DUF1542 repeats are not involved in adhesion, but biophysical studies show they are predominantly α-helical and form a fiber-like stalk of tandem DUF1542 domains. Epf thus conforms with the widespread family of adhesins known as MSCRAMMs (microbial surface components recognizing adhesive matrix molecules), in which a cell wall-attached stalk enables long range interactions via its adhesive N-terminal domain.

Effects of the ERES pathogenicity region regulator Ralp3 on Streptococcus pyogenes serotype M49 virulence factor expression.

Streptococcus pyogenes (group A streptococcus [GAS]) is a highly virulent Gram-positive bacterium. For successful infection, GAS expresses many virulence factors, which are clustered together with transcriptional regulators in distinct genomic regions. Ralp3 is a central regulator of the ERES region. In this study, we investigated the role of Ralp3 in GAS M49 pathogenesis. The inactivation of Ralp3 resulted in reduced attachment to and internalization into human keratinocytes. The Δralp3 mutant failed to survive in human blood and serum, and the hyaluronic acid capsule was slightly decreased. In addition, the mutant showed a lower binding capacity to human plasminogen, and the SpeB activity was significantly decreased. Complementation of the Δralp3 mutant restored the wild-type phenotype. The transcriptome and quantitative reverse transcription-PCR analysis of the serotype M49 GAS strain and its isogenic Δralp3 mutant identified 16 genes as upregulated, and 43 genes were found to be downregulated. Among the downregulated genes, there were open reading frames encoding proteins involved in metabolism (e.g., both lac operons and the fru operon), genes encoding lantibiotics (e.g., the putative salivaricin operon), and ORFs encoding virulence factors (such as the whole Mga core regulon and further genes under Mga control). In summary, the ERES region regulator Ralp3 is an important serotype-specific transcriptional regulator for virulence and metabolic control.

Streptococcus pyogenes M49 plasminogen/plasmin binding facilitates keratinocyte invasion via integrin-integrin-linked kinase (ILK) pathways and protects from macrophage killing.

The entry into epithelial cells and the prevention of primary immune responses are a prerequisite for a successful colonization and subsequent infection of the human host by Streptococcus pyogenes (group A streptococci, GAS). Here, we demonstrate that interaction of GAS with plasminogen promotes an integrin-mediated internalization of the bacteria into keratinocytes, which is independent from the serine protease activity of potentially generated plasmin. α(1)ß(1)- and α(5)ß(1)-integrins were identified as the major keratinocyte receptors involved in this process. Inhibition of integrin-linked kinase (ILK) expression by siRNA silencing or blocking of PI3K and Akt with specific inhibitors, reduced the GAS M49-plasminogen/plasmin-mediated invasion of keratinocytes. In addition, blocking of actin polymerization significantly reduced GAS internalization into keratinocytes. Altogether, these results provide a first model of plasminogen-mediated GAS invasion into keratinocytes. Furthermore, we demonstrate that plasminogen binding protects the bacteria against macrophage killing.

Characterization of three lactic acid bacteria and their isogenic ldh deletion mutants shows optimization for YATP (cell mass produced per mole of ATP) at their physiological pHs.

Several lactic acid bacteria use homolactic acid fermentation for generation of ATP. Here we studied the role of the lactate dehydrogenase enzyme on the general physiology of the three homolactic acid bacteria Lactococcus lactis, Enterococcus faecalis, and Streptococcus pyogenes. Of note, deletion of the ldh genes hardly affected the growth rate in chemically defined medium under microaerophilic conditions. However, the growth rate was affected in rich medium. Furthermore, deletion of ldh affected the ability for utilization of various substrates as a carbon source. A switch to mixed acid fermentation was observed during glucose-limited continuous growth and was dependent on the growth rate for S. pyogenes and on the pH for E. faecalis. In S. pyogenes and L. lactis, a change in pH resulted in a clear change in Y(ATP) (cell mass produced per mole of ATP). The pH that showed the highest Y(ATP) corresponded to the pH of the natural habitat of the organisms.