Silke Niemann

Staphylococcus aureus phenotype switching: an effective bacterial strategy to escape host immune response and establish a chronic infection

Staphylococcus aureus is a frequent cause for serious, chronic and therapy‐refractive infections in spite of susceptibility to antibiotics in vitro. In chronic infections, altered bacterial phenotypes, such as small colony variants (SCVs), have been found. Yet, it is largely unclear whether the ability to interconvert from the wild‐type to the SCV phenotype is only a rare clinical and/or just laboratory phenomenon or is essential to sustain an infection. Here, we performed different long‐term in vitro and in vivo infection models with S. aureus and we show that viable bacteria can persist within host cells and/or tissues for several weeks. Persistence induced bacterial phenotypic diversity, including SCV phenotypes, accompanied by changes in virulence factor expression and auxotrophism. However, the recovered SCV phenotypes were highly dynamic and rapidly reverted to the fully virulent wild‐type form when leaving the intracellular location and infecting new cells. Our findings demonstrate that bacterial phenotype switching is an integral part of the infection process that enables the bacteria to hide inside host cells, which can be a reservoir for chronic and therapy‐refractive infections.

Staphylococcus aureus Panton-Valentine leukocidin induces an inflammatory response in human phagocytes via the NLRP3 inflammasome.

The Staphylococcus aureus pore‐forming toxin PVL is most likely causative for life‐threatening necrotizing infections, which are characterized by massive tissue inflammation and necrosis. Whereas the cytotoxic action of PVL on human neutrophils is already well established, the PVL effects on other sensitive cell types, such as monocytes and macrophages, are less clear. In this study, we used different types of human leukocytes (neutrophils, monocytes, macrophages, lymphocytes) to investigate cell‐specific binding of PVL subunits and subsequent proinflammatory and cytotoxic effects. In all PVL‐sensitive cells, we identified the binding of the subunit LukS‐PV as the critical factor for PVL‐induced cytotoxicity, which was followed by binding of LukF‐PV. LukS‐PV binds to monocytes, macrophages, and neutrophils but not to lymphocytes. Additionally, we showed that PVL binding to monocytes and macrophages leads to release of caspase‐1‐dependent proinflammatory cytokines IL‐1β and IL‐18. PVL activates the NLRP3 inflammasome, a signaling complex of myeloid cells that is involved in caspase‐1‐dependent IL‐1β processing in response to pathogens and endogenous danger signals. Specific inhibition of this pathway at several steps significantly reduced inflammasome activation and subsequent pyronecrosis. Furthermore, we found that PAMPs and DAMPs derived from dying neutrophils can dramatically enhance this response by up‐regulating pro‐IL‐1β in monocytes/macrophages. This study analyzes a specific host signaling pathway that mediates PVL‐induced inflammation and cytotoxicity, which has high relevance for CA‐MRSA‐associated and PVL‐mediated pathogenic processes, such as necrotizing infections.

Thrombospondin-1 promotes cellular adherence of Gram-positive pathogens via recognition of peptidoglycan

Thrombospondin‐1 (TSP1) is a matri‐cellular glycoprotein that has key roles in interactions between human cells and components of the extracellular matrix. Here we report a novel role for the lectin TSP1 in pathogen‐host interactions. Binding assays and flow cytometric analysis demonstrate that Streptococcus pneumoniae and other Gram‐positive pathogens including S. pyogenes, Staphylococcus aureus, and Listeria monocytogenes interact specifically with human TSP1. We also show for the first time that host cell‐bound TSP1 promotes adherence of Gram‐positive pathogens to human epithelial and endothelial cell lines. Pretreat‐ment of bacteria with sodium periodate but not Pronase E substantially reduced TSP1‐mediated bacterial adherence to host cells, suggesting that a glycoconju‐gate on the bacterial cell surface functions as the receptor for TSP1. Lipoteichoic acids did not affect TSP1‐mediated adherence of S. pneumoniae to host cells. In contrast, attachment of S. pneumoniae and other Gram‐positive pathogens to host cells via TSP1 was blocked by soluble peptidoglycan, indicating recognition of bacterial peptidoglycan by TSP1. In conclusion, our results demonstrate that recognition of Grampositive pathogens by TSP1 promotes bacterial colonization of host tissue cells. In this scenario, pep‐tidoglycan functions as adhesin and TSP1 acts as a molecular bridge linking Gram‐positive bacteria with receptors on the host cell.—Rennemeier, C., Hammerschmidt, S., Niemann, S., Inamura, S., Zähringer, U., Kehrel, B. E. Thrombospondin‐1 promotes cellular adherence of Gram‐positive pathogens via recognition of peptidoglycan. FASEB J. 21, 3118–3132 (2007)

Staphylococcus aureus fibronectin-binding protein (FnBP)-mediated adherence to platelets, and aggregation of platelets induced by FnBPA but not by FnBPB.

BACKGROUND The ability of Staphylococcus aureus to adhere to platelets and to induce aggregation of platelets is considered to be a critical factor in S. aureus-associated infective endocarditis. METHODS To identify and characterize further bacterial factors involved in the S. aureus-platelet interaction, we generated a phage-display library of S. aureus genomic DNA by use of the improved phagemid vector pG8SAET. The library was affinity-panned against gel-filtered, immobilized platelets. RESULTS Repeatedly isolated clones contained overlapping DNA fragments encoding a portion of the S. aureus fibronectin (Fn)-binding proteins (FnBPs). In a flow cytometric adherence assay, Staphylococcus carnosus that heterologously expressed either fnbA or fnbB, which encode FnBPA and FnBPB, respectively, showed increased adherence to activated, gel-filtered platelets. Adherence was promoted by the addition of Fn or fibrinogen (Fg), which most likely act as bridging molecules. Interestingly, promotion of adherence mediated by Fn was in the same range with S. carnosus producing either FnBPA or FnBPB, whereas promotion of adherence mediated by Fg was significantly more pronounced with S. carnosus that produce FnBPA than with S. carnosus that produce FnBPB. Furthermore, FnBPA, but not FnBPB, mediated aggregation of platelets when present on S. carnosus cells. CONCLUSION Our results indicate a substantial functional difference between FnBPA and FnBPB in the S. aureus-platelet interaction.

Soluble Fibrin Is the Main Mediator of Staphylococcus aureus Adhesion to Platelets

Background—Infective endocarditis (IE) caused by Staphylococcus aureus is associated with significant morbidity and mortality rates. Platelets play a dual role as adhesive cells forming associates with bacteria as well as specialized inflammatory cells. The specific role of the various factors involved in bacteria-platelet association has not yet been fully elucidated. Methods and Results—We observed a dramatic increase in the capability to bind S aureus when platelets were activated with thrombin (from 5% to 30%, P <0.001). To pinpoint platelet-binding sites involved in the interaction, platelets from knockout mice and from patients with selective inherited deficiency of membrane proteins or of granules were used. CD36, GPIIb/IIIa, and P-selectin were excluded as receptors for S aureus. Platelets from patients with &agr;-δ–storage pool disease and Gray platelet syndrome indicate the requirement of &agr;-granule contents. Platelet activation by ADP did not promote platelet–S aureus associate formation, although these platelets were covered with bound fibrinogen. Only small numbers of associates between fibrinogen-covered bacteria and ADP-activated platelets were observed. Formation of fibrin alone was also not sufficient to induce association. Only when fibrin formation and platelet activation occurred together were large numbers of associates formed (P <0.001). A potential receptor for fibrin on S aureus is clumping factor A. Addition of thrombospondin-1 to control platelets increased the number of associates (P =0.02). Conclusions—Soluble fibrin but not fibrinogen is the main mediator of platelet–S aureus association. In addition, platelet activation and the release of &agr;-granule contents, particularly thrombospondin-1, is a requirement for platelet–S aureus association.

Mechanisms of infective endocarditis: pathogen–host interaction and risk states

Patients with infective endocarditis (IE) form a heterogeneous group, ranging from those who are successfully treated with no adverse events, to those with severe complications and a high mortality. In this Review, we highlight pathogen–host interactions and the mechanisms underlying various risk factors for patients with IE. A temporal trend in the pattern of IE has been observed in high-income countries within the past 5 decades, with patients contracting IE at an increasingly old age, and a growing incidence of health-care-associated staphylococcal IE. Consequently, prevention strategies should no longer focus on prophylaxis of streptococcal bacteraemia during dental procedures, but instead encourage a more-general approach to reduce the incidence of health-care-associated IE. Much knowledge has been gained about the mechanisms of vegetation formation, growth, and embolization on damaged or inflamed cardiac valves, and on cardiac devices. Improved understanding of these mechanisms will help to combat the increasing problem of antimicrobial resistance. Two mechanisms of IE should increasingly be the focus of future research: the role of immunosenescence in elderly patients with IE, particularly after transcatheter aortic valve implantation, and the mechanisms that trigger septic shock, a condition that leads to a substantial increase in the risk of death in patients with IE.

Staphylococcus aureus persistence in non-professional phagocytes

S. aureus is a frequent cause of chronic and therapy-refractory infections. The ability of S. aureus to invade different types of non-professional phagocytes, to escape from the host lysosomal degradation machinery and to persist within the intracellular location for long time periods are most likely essential steps in pathogenesis. During the course from acute to chronic infection the bacteria need to dynamically react to the environmental changes and to adapt to the intracellular environment. In this context the bacteria change to SCV-like phenotypes that exhibit some characteristics of stable SCV-mutants, like upregulation of adhesins and downregulation of toxins. The exact formation mechanism and further typical features of these dynamically forming SCVs are largely unknown. In this review, recent data on the essential steps to establish chronic infections will be summarized and the clinical consequences of the dynamic bacterial adaptation mechanisms will be discussed.

Sigma Factor SigB Is Crucial to Mediate Staphylococcus aureus Adaptation during Chronic Infections

Staphylococcus aureus is a major human pathogen that causes a range of infections from acute invasive to chronic and difficult-to-treat. Infection strategies associated with persisting S. aureus infections are bacterial host cell invasion and the bacterial ability to dynamically change phenotypes from the aggressive wild-type to small colony variants (SCVs), which are adapted for intracellular long-term persistence. The underlying mechanisms of the bacterial switching and adaptation mechanisms appear to be very dynamic, but are largely unknown. Here, we analyzed the role and the crosstalk of the global S. aureus regulators agr, sarA and SigB by generating single, double and triple mutants, and testing them with proteome analysis and in different in vitro and in vivo infection models. We were able to demonstrate that SigB is the crucial factor for adaptation in chronic infections. During acute infection, the bacteria require the simultaneous action of the agr and sarA loci to defend against invading immune cells by causing inflammation and cytotoxicity and to escape from phagosomes in their host cells that enable them to settle an infection at high bacterial density. To persist intracellularly the bacteria subsequently need to silence agr and sarA. Indeed agr and sarA deletion mutants expressed a much lower number of virulence factors and could persist at high numbers intracellularly. SigB plays a crucial function to promote bacterial intracellular persistence. In fact, ΔsigB-mutants did not generate SCVs and were completely cleared by the host cells within a few days. In this study we identified SigB as an essential factor that enables the bacteria to switch from the highly aggressive phenotype that settles an acute infection to a silent SCV-phenotype that allows for long-term intracellular persistence. Consequently, the SigB-operon represents a possible target to develop preventive and therapeutic strategies against chronic and therapy-refractory infections.

Combined Action of Influenza Virus and Staphylococcus aureus Panton–Valentine Leukocidin Provokes Severe Lung Epithelium Damage

Staphylococcus aureus necrotizing pneumonia is a life-threatening disease that is frequently preceded by influenza infection. The S. aureus toxin Panton–Valentine leukocidin (PVL) is most likely causative for necrotizing diseases, but the precise pathogenic mechanisms of PVL and a possible contribution of influenza virus remain to be elucidated. In this study, we present a model that explains how influenza virus and PVL act together to cause necrotizing pneumonia: an influenza infection activates the lung epithelium to produce chemoattractants for neutrophils. Upon superinfection with PVL-expressing S. aureus, the recruited neutrophils are rapidly killed by PVL, resulting in uncontrolled release of neutrophil proteases that damage the airway epithelium. The host counteracts this pathogen strategy by generating PVL-neutralizing antibodies and by neutralizing the released proteases via protease inhibitors present in the serum. These findings explain why necrotizing infections mainly develop in serum-free spaces (eg, pulmonary alveoli) and open options for new therapeutic approaches.

Pathogenesis of Staphylococcus aureus necrotizing pneumonia: the role of PVL and an influenza coinfection

Only recently necrotizing pneumonia was defined as a specific disease entity that is caused by a Panton-Valentine leukocidin (PVL)-producing Staphylococcus aureus strain and is frequently preceded by an influenza infection. Necrotizing pneumonia is characterized by a sudden onset and rapid worsening of symptoms, leukopenia, airway hemorrhages, severe respiratory failure and a high mortality rate. Despite clear epidemiological data, the function of PVL in necrotizing pneumonia has been controversially discussed due to conflicting results from different disease models. Furthermore, there are many proposed mechanisms how a viral infection could facilitate and interact with a bacterial superinfection. In this review, we summarize current data from 43 clinical cases and results from various infection models on necrotizing pneumonia. We discuss the contribution of S. aureus PVL and a preceding influenza infection and present a concept of the pathogenesis of necrotizing pneumonia.