USA300 methicillin-resistant Staphylococcus aureus (MRSA) is the most prevalent MRSA in the United States of America (USA) and a global epidemic threat. We investigated the prevalence of USA300 at a tertiary care hospital in Zurich, Switzerland, where all MRSA strains have been collected and PFGE typed since 1992. These strains were retrospectively compared to the PFGE pattern of USA300 strain JE2. Isolates with a respective PFGE pattern were spa-typed and tested for the presence of the arginine catabolic mobile element (ACME) arc gene cluster and Panton-Valentine Leucocidin (PVL) genes. The first MRSA strain with a USA300 PFGE pattern was isolated in 2001 from a patient visiting from the USA. USA300 strains represented between 0% (in 2002) and 9.2% (in 2012) of all MRSA isolates in our hospital. We identified various USA300 subtypes based on either the PFGE pattern, the spa-type or absence of either the PVL genes or ACME arc gene cluster. All the USA300 strains including the variants (n=47) accounted for 5.6% of all MRSA isolates typed between 2001 and 2013 and reached a maximum of 14.5% in 2009. They predominantly caused skin and soft tissue infections (74.4%). In conclusion, even though USA300 has been present in our hospital for over twelve years it has not become the predominant MRSA clone like in the USA. However, in light of the global burden of USA300, care must be taken to further contain the spread of this lineage and of MRSA in general in our hospital.
Clearance of invading pathogens is essential to preventing overwhelming inflammation and sepsis that are symptomatic of bacterial peritonitis. Macrophages participate in this innate immune response by engulfing and digesting pathogens, a process called phagocytosis. Oxidized phospholipids (OxPL) are danger-associated molecular patterns (DAMPs) generated in response to infection that can prevent the phagocytic clearance of bacteria. We investigated the mechanism underlying OxPL action in macrophages. Exposure to OxPL induced alterations in actin polymerization, resulting in spreading of peritoneal macrophages and diminished uptake of E. coli. Pharmacological and cell-based studies showed that an anchored pool of PKA mediates the effects of OxPL. Gene silencing approaches identified the A-kinase anchoring protein (AKAP) WAVE1 as an effector of OxPL action in vitro. Chimeric Wave1(-/-) mice survived significantly longer after infection with E. coli and OxPL treatment in vivo. Moreover, we found that endogenously generated OxPL in human peritoneal dialysis fluid from end-stage renal failure patients inhibited phagocytosis via WAVE1. Collectively, these data uncover an unanticipated role for WAVE1 as a critical modulator of the innate immune response to severe bacterial infections.
CD36 is a scavenger receptor that exhibits pleiotropic functions, including adhesion to thrombospondin, inhibition of angiogenesis, transport of long-chain fatty acids, and clearance of apoptotic cells. In addition, it has been implicated in the host immune response because it acts as a coreceptor for TLR2 and plays a role in Staphylococcus aureus infection. However, its role in other Gram-positive bacterial infections is unclear. In this study, using mice deficient in CD36, we sought to examine the role of CD36 in pneumococcal pneumonia, a major cause of morbidity and mortality worldwide. We show that CD36 is expressed on both alveolar macrophages and respiratory epithelial cells. Early in infection, CD36(-/-) mice have an exaggerated inflammatory response compared with wild-type littermate controls. In vitro studies using CD36(-/-) primary cells confirm the enhanced early inflammation in response to S. pneumoniae and its lipoteichoic acid, demonstrate that S. pneumoniae binds to cells via its phosphocholine residues, and suggest a role for CD36 in reducing inflammation induced by the phosphocholine residues of pneumococcal lipoteichoic acid. Later in infection, although CD36(-/-) mice exhibit impaired bacterial clearance, owing to a decreased capacity of CD36(-/-) macrophages to phagocytose S. pneumoniae, minor effects on mortality occur, in comparison with those in wild-type littermate control mice. These data show that CD36 contributes to the pulmonary host response during S. pneumoniae infection by virtue of its ability to act as a phagocytic receptor and as a modulator of the early innate immune response.
Group A Streptococcus (GAS) is a human pathogen causing a wide range of mild to severe and life-threatening diseases. The GAS M1 protein is a major virulence factor promoting GAS invasiveness and resistance to host innate immune clearance. M1 displays an irregular coiled-coil structure, including the B-repeats that bind fibrinogen. Previously, we found that B-repeat stabilisation generates an idealised version of M1 (M1) characterised by decreased fibrinogen binding in vitro. To extend these findings based on a soluble truncated version of M1, we now studied the importance of the B-repeat coiled-coil irregularities in full length M1 and M1 expressed in live GAS and tested whether the modulation of M1-fibrinogen interactions would open up novel therapeutic approaches. We found that altering either the M1 structure on the GAS cell surface or removing its target host protein fibrinogen blunted GAS virulence. GAS expressing M1 showed an impaired ability to adhere to and to invade human endothelial cells, was more readily killed by whole blood or neutrophils and most importantly was less virulent in a murine necrotising fasciitis model. M1-mediated virulence of wild-type GAS was strictly dependent on the presence and concentration of fibrinogen complementing our finding that M1-fibrinogen interactions are crucial for GAS virulence. Consistently blocking M1-fibrinogen interactions by fragment D reduced GAS virulence in vitro and in vivo. This supports our conclusion that M1-fibrinogen interactions are crucial for GAS virulence and that interference may open up novel complementary treatment options for GAS infections caused by the leading invasive GAS strain M1.
Streptococcus pyogenes is a Gram-positive human pathogen that is recognized by yet unknown pattern recognition receptors (PRRs). Engagement of these receptor molecules during infection with S. pyogenes, a largely extracellular bacterium with limited capacity for intracellular survival, causes innate immune cells to produce inflammatory mediators such as TNF, but also type I interferon (IFN). Here we show that signaling elicited by type I IFNs is required for successful defense of mice against lethal subcutaneous cellulitis caused by S. pyogenes. Type I IFN signaling was accompanied with reduced neutrophil recruitment to the site of infection. Mechanistic analysis revealed that macrophages and conventional dendritic cells (cDCs) employ different signaling pathways leading to IFN-beta production. Macrophages required IRF3, STING, TBK1 and partially MyD88, whereas in cDCs the IFN-beta production was fully dependent on IRF5 and MyD88. Furthermore, IFN-beta production by macrophages was dependent on the endosomal delivery of streptococcal DNA, while in cDCs streptococcal RNA was identified as the IFN-beta inducer. Despite a role of MyD88 in both cell types, the known IFN-inducing TLRs were individually not required for generation of the IFN-beta response. These results demonstrate that the innate immune system employs several strategies to efficiently recognize S. pyogenes, a pathogenic bacterium that succeeded in avoiding recognition by the standard arsenal of TLRs.
Resolution of inflammation is an important hallmark in the course of infectious diseases. Dysregulated inflammatory responses may have detrimental consequences for the affected organism. Therefore, tight regulation of inflammation is indispensable. Among numerous modulatory signaling pathways, the PI3K/PTEN signaling pathway has been proposed recently to be involved in the regulation of innate immune reactions. Here, we attempted to elucidate molecular mechanisms that contribute to the modulatory properties of the PI3K signaling pathway in inflammation. PTEN-deficient macrophages, which harbor constitutively active PI3Ks, were analyzed in response to gram-negative bacteria and PAMPs such as LPS. PTEN-deficient cells showed reduced inflammatory cytokine production, which was accompanied by reduced MAPK signaling activation in early- as well as late-phase activation. Simultaneously, we found increased levels of the MKP DUSP1, as well as the anti-inflammatory cytokine IL-10. Our data suggest that differential DUSP1 regulation coupled with enhanced IL-10 production contributes to the anti-inflammatory properties of the PI3K pathway.
Phosphatidylinositol 3-kinase has been described as an essential signaling component involved in the chemotactic cell influx that is required to eliminate pathogens. At the same time, PI3K was reported to modulate the immune response, thus limiting the magnitude of acute inflammation. The precise role of the PI3K pathway and its endogenous antagonist phosphatase and tensin homolog deleted on chromosome 10 (PTEN) during clinically relevant bacterial infections is still poorly understood. Utilizing mice lacking myeloid cell-specific PTEN, we studied the impact of PTEN on the immune response to Streptococcus pneumoniae. Survival analysis disclosed that PTEN-deficient mice displayed less severe signs of disease and prolonged survival. The inflammatory response to S. pneumoniae was greatly reduced in macrophages in vitro and in vivo. Unexpectedly, neutrophil influx to the lungs was significantly impaired in animals lacking myeloid-cell PTEN, whereas the additional observation of improved phagocytosis by alveolar macrophages lacking PTEN ultimately resulted in unaltered lung CFUs following bacterial infection. Together, the absence of myeloid cell-associated PTEN and consecutively enhanced PI3K activity dampened pulmonary inflammation, reduced neutrophil influx, and augmented phagocytic properties of macrophages, which ultimately resulted in decreased tissue injury and improved survival during murine pneumococcal pneumonia.
Acute lung injury (ALI) is a serious condition in critically ill patients that predisposes to secondary bacterial pneumonia. Vascular leak is a hallmark in the pathogenesis of ALI. The fibrin-derived peptide Bbeta(15-42) was shown to preserve endothelial barriers, thereby reducing vascular leak. The potential therapeutic role of Bbeta(15-42) in ALI has not been addressed so far.
Triggering receptor expressed on myeloid cells-1 (TREM-1) is an amplifier of TLR-mediated inflammation during bacterial infections. Thus far, TREM-1 is primarily associated with unwanted signs of overwhelming inflammation, rendering it an attractive target for conditions such as sepsis. Respiratory tract infections are the leading cause of sepsis, but the biological role of TREM-1 therein is poorly understood. To determine the function of TREM-1 in pneumococcal pneumonia, we first established TREM-1 up-regulation in infected lungs and human plasma together with augmented alveolar macrophage responsiveness toward Streptococcus pneumoniae. Mice treated with an agonistic TREM-1 Ab and infected with S. pneumoniae exhibited an enhanced early induction of the inflammatory response that was indirectly associated with lower levels of negative regulators of TLR signaling in lung tissue in vivo. Later in infection, TREM-1 engagement altered S. pneumoniae-induced IRAK-M (IL-1R-associated kinase-M) kinetics so as to promote the resolution of pneumonia and remarkably led to an accelerated elimination of bacteria and consequently improved survival. These data show that TREM-1 exerts a protective role in the innate immune response to a common bacterial infection and suggest that caution should be exerted in modulating TREM-1 activity during certain clinically relevant bacterial infections.
Protozoan parasites of the genus Leishmania are the causative agents of life-threatening visceral as well as cutaneous and mucocutaneous leishmaniasis. First-line drugs are antimonials, but toxicity and resistance in some endemic areas cause serious problems. In the current study, the antileishmanial activity of the weak oxidant N-chlorotaurine (NCT) was investigated. NCT is a derivative of the amino acid taurine produced by granulocytes and monocytes during oxidative burst, but can also be synthesized chemically and used topically as an antiseptic at a concentration of 1 % (55 mM) in vivo. NCT susceptibility tests were performed in vitro with promastigotes and amastigotes of Leishmania infantum and Leishmania donovani. As NH(4)Cl is known to increase the activity of NCT by the formation of monochloramine (NH(2)Cl), co-treatment assays were included in the study. Mean EC(50) values after 1 h of treatment were 5.94 mM for L. infantum and 9.8 mM for L. donovani promastigotes. Co-treatment with 5.5 mM NCT plus 19 mM NH(4)Cl led to complete killing of promastigotes of both strains within 15 min. Amastigotes were inactivated by treatment with 2 mM NCT alone. The results of this study indicate a high potential of NCT against Leishmania species.
Loss of vascular barrier function causes leak of fluid and proteins into tissues, extensive leak leads to shock and death. Barriers are largely formed by endothelial cell-cell contacts built up by VE-cadherin and are under the control of RhoGTPases. Here we show that a natural plasmin digest product of fibrin, peptide Bbeta15-42 (also called FX06), significantly reduces vascular leak and mortality in animal models for Dengue shock syndrome. The ability of Bbeta15-42 to preserve endothelial barriers is confirmed in rats i.v.-injected with LPS. In endothelial cells, Bbeta15-42 prevents thrombin-induced stress fiber formation, myosin light chain phosphorylation and RhoA activation. The molecular key for the protective effect of Bbeta15-42 is the src kinase Fyn, which associates with VE-cadherin-containing junctions. Following exposure to Bbeta15-42 Fyn dissociates from VE-cadherin and associates with p190RhoGAP, a known antagonists of RhoA activation. The role of Fyn in transducing effects of Bbeta15-42 is confirmed in Fyn(-/-) mice, where the peptide is unable to reduce LPS-induced lung edema, whereas in wild type littermates the peptide significantly reduces leak. Our results demonstrate a novel function for Bbeta15-42. Formerly mainly considered as a degradation product occurring after fibrin inactivation, it has now to be considered as a signaling molecule. It stabilizes endothelial barriers and thus could be an attractive adjuvant in the treatment of shock.
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