Enhanced surface colonisation and competition during bacterial adaptation to a fungus.

Bacterial-fungal interactions influence microbial community performance of most ecosystems and elicit specific microbial behaviours, including stimulating specialised metabolite production. Here, we use a co-culture experimental evolution approach to investigate bacterial adaptation to the presence of a fungus, using a simple model of bacterial-fungal interactions encompassing the bacterium Bacillus subtilis and the fungus Aspergillus niger. We find in one evolving population that B. subtilis was selected for enhanced production of the lipopeptide surfactin and accelerated surface spreading ability, leading to inhibition of fungal expansion and acidification of the environment. These phenotypes were explained by specific mutations in the DegS-DegU two-component system. In the presence of surfactin, fungal hyphae exhibited bulging cells with delocalised secretory vesicles possibly provoking an RlmA-dependent cell wall stress. Thus, our results indicate that the presence of the fungus selects for increased surfactin production, which inhibits fungal growth and facilitates the competitive success of the bacterium.

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.

Primary Retroperitoneal Smooth Muscle Tumor of Uncertain Malignant Potential (STUMP): A Diagnostic Enigma.

Retroperitoneal sarcomas represent a group of rare malignant neoplasms with complex clinical management and often a poor prognosis. An elderly male presented with a slowly progressive, right-sided abdominal lump for four months associated with loss of appetite and abdominal discomfort. Abdominal examination revealed an apparent retroperitoneal lump in the right lumbar and umbilical region, which was well-defined, and firm in consistency with the bosselated surface. Contrast-enhanced computed tomography (CECT) of the abdomen and pelvis revealed a heterogenous lobulated malignant appearing retroperitoneal lesion arising from the right anterior perirenal space with a differential of retroperitoneal sarcoma. Wide local excision of the tumor was done. Histopathology of the lesion revealed a smooth muscle tumor of uncertain malignant potential (STUMP). The patient is asymptomatic and recurrence-free after 24 months of follow-up.

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.

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.

16HBE Cell Lipid Mediator Responses to Mono and Co-Infections with Respiratory Pathogens.

Respiratory tract infections are a global health problem. The main causative agents of these infections are influenza A virus (IAV), Staphylococcus aureus (S. aureus), and Streptococcus pneumoniae (S. pneumoniae). Major research focuses on genetics and immune responses in these infections. Eicosanoids and other oxylipins are host-derived lipid mediators that play an important role in the activation and resolution of inflammation. In this study, we assess, for the first time, the different intracellular profiles of these bioactive lipid mediators during S. aureus LUG2012, S. pneumoniae TIGR4, IAV, and corresponding viral and bacterial co-infections of 16HBE cells. We observed a multitude of altered lipid mediators. Changes in the amount of 5-hydroxyeicosatetraenoic acid (5-HETE) were prominent for all bacterial infections. The infection with S. pneumoniae showed the strongest impact on bioactive lipid production and led to alterations in the amount of PPARγ ligands and precursors of pro-resolving lipid mediators.

The Role of NLRP3 Inflammasome in Pneumococcal Infections.

Inflammasomes are innate immune sensors that regulate caspase-1 mediated inflammation in response to environmental, host- and pathogen-derived factors. The NLRP3 inflammasome is highly versatile as it is activated by a diverse range of stimuli. However, excessive or chronic inflammasome activation and subsequent interleukin-1ß (IL-1ß) release are implicated in the pathogenesis of various autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, and diabetes. Accordingly, inflammasome inhibitor therapy has a therapeutic benefit in these diseases. In contrast, NLRP3 inflammasome is an important defense mechanism against microbial infections. IL-1ß antagonizes bacterial invasion and dissemination. Unfortunately, patients receiving IL-1ß or inflammasome inhibitors are reported to be at a disproportionate risk to experience invasive bacterial infections including pneumococcal infections. Pneumococci are typical colonizers of immunocompromised individuals and a leading cause of community-acquired pneumonia worldwide. Here, we summarize the current limited knowledge of inflammasome activation in pneumococcal infections of the respiratory tract and how inflammasome inhibition may benefit these infections in immunocompromised patients.