Staphylococcus aureus lipoproteins - TLR2-mediated activation of innate and adaptive immunity

Staphylococcus (S.) aureus is a very successful pathogen due to its immune evasion strategies. Besides toxins and adhesins, it expresses membrane lipoproteins (Lpp), which are bound by the pattern recognition receptor Toll-like receptor (TLR) 2 in the host. Recognition of Lpp activates the MyD88-signaling pathway, which allows mounting a strong inflammatory response. Interestingly, evolution did not select for S. aureus mutants deficient in lipid modification of proteins suggesting that Lpp, besides their signaling potential to the host, offer an advantage for S. aureus. The aim of this thesis was to identify the benefit of Lpp maturation for S. aureus and the contribution of Lpp-TLR2-signaling to the pathogenesis of staphylococcal disease in mouse infection models. In the first part, we demonstrate the strong cytokine-activating potential of Lpp in murine macrophages, which was associated with the presence of TLR2 in the host. In a systemic infection model, Lpp-TLR2 activation was less contributing to inflammation and S. aureus killing than MyD88-signaling. This indicates that other receptors signaling to MyD88 participate in the antimicrobial response. We further showed in systemic infection that maturation of Lpp facilitates survival of S. aureus in organs due to improved iron acquisition. Studies on growth, uptake, and intracellular storage of iron in vitro confirmed the iron dependence of S. aureus. It is long known, that the immunocompetent host restricts iron in an infection. We show that Lpp enhanced S. aureus growth in the iron-overloaded immunocompetent host, while they were not required in the iron rich environment in the MyD88-deficient host. Interestingly, iron-restricted S. aureus could not profit from Lpp for growth as long as the infected mice were fully immunocompetent. Only in mice deficient in MyD88-dependent inflammation iron-restricted S. aureus used Lpp for growth. In summary, the results in part 1 strongly suggest that Lpp confer a growth and survival advantage although allowing innate immune responses mediated through TLR2-MyD88-signaling. In the second part, data are presented showing that Lpp released during growth activate TLR2-signaling but engulfment of S. aureus enhances cytokine production. Lpp enhanced phagocytosis by macrophages and intracellular survival of S. aureus. Moreover, Lpp-TLR2-signaling induced cathepsin B-mediated cytotoxicity in macrophages. An effect of Lpp on various interactions of S. aureus with PMN was not found in vitro and in vivo, whereas Lpp enhanced invasion of S. aureus in endothelial cells in vitro. These results point to an additional survival advantage by maturation of Lpp in S. aureus due to improved evasion from extracellular killing, better intracellular survival, and escape from the phagosome. In the third part, we demonstrate that Lpp-TLR2-MyD88-signaling is important for activation of DCs to induce differentiation of na•ve CD4+ into IFN-g- and IL-17-producing T cells in vitro. Induction of Lpp-TLR2-signaling was also required to promote IFN-g release by na•ve CD8+ T cells. In systemic infection, restimulated spleen T cells produced MyD88-dependent IFN-g and TLR2-MyD88-dependent IL-17. Surprisingly, the presence of B and T cells diminished eradication of S. aureus from organs during early sepsis. These data show that detection of invading S. aureus by DCs leads to the development of adaptive immune responses, which are not always beneficial for eradication of S. aureus. In the fourth part, the role of other pattern recognition receptors (PRRs) in staphylococcal infection was examined. TLR9 and NOD2, in contrast to IL-1R, had a positive effect on cytokine induction in macrophages and in systemic infection, whereas killing was not affected. In inflammation and bacterial killing during S. aureus infection, TLR2 and TLR9, which both require the MyD88-adaptor, were found to cooperate. These data suggest that concurrent activation of different PRRs elicit a strong antimicrobial defense in response to various molecules of S. aureus

Disruption of Coronin 1 Signaling in T Cells Promotes Allograft Tolerance while Maintaining Anti-Pathogen Immunity

The ability of the immune system to discriminate self from non-self is essential for eradicating microbial pathogens but is also responsible for allograft rejection. Whether it is possible to selectively suppress alloresponses while maintaining anti-pathogen immunity remains unknown. We found that mice deficient in coronin 1, a regulator of naive T cell homeostasis, fully retained allografts while maintaining T cell-specific responses against microbial pathogens. Mechanistically, coronin 1-deficiency increased cyclic adenosine monophosphate (cAMP) concentrations to suppress allo-specific T cell responses. Costimulation induced on microbe-infected antigen presenting cells was able to overcome cAMP-mediated immunosuppression to maintain anti-pathogen immunity. In vivo pharmacological modulation of this pathway or a prior transfer of coronin 1-deficient T cells actively suppressed allograft rejection. These results define a coronin 1-dependent regulatory axis in T cells important for allograft rejection and suggest that modulation of this pathway may be a promising approach to achieve long-term acceptance of mismatched allografts

Oligomeric Coiled-Coil Adhesin YadA Is a Double-Edged Sword

Yersinia adhesin A (YadA) is an essential virulence factor for the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis. Suprisingly, it is a pseudogene in Yersinia pestis. Even more intriguing, the introduction of a functional yadA gene in Y. pestis EV76 was shown to correlate with a decrease in virulence in a mouse model. Here, we report that wild type (wt) Y. enterocolitica E40, as well as YadA-deprived E40 induced the synthesis of neutrophil extracellular traps (NETs) upon contact with neutrophils, but only YadA-expressing Y. enterocolitica adhered to NETs and were killed. As binding seemed to be a prerequisite for killing, we searched for YadA-binding substrates and detected the presence of collagen within NETs. E40 bacteria expressing V98D,N99A mutant YadA with a severely reduced ability to bind collagen were found to be more resistant to killing, suggesting that collagen binding contributes significantly to sensitivity to NETs. Wt Y. pestis EV76 were resistant to killing by NETs, while recombinant EV76 expressing YadA from either Y. pseudotuberculosis or Y. enterocolitica were sensitive to killing by NETs, outlining the importance of YadA for susceptibility to NET-dependent killing. Recombinant EV76 endowed with YadA from Y. enterocolitica were also less virulent for the mouse than wt EV76, as shown before. In addition, EV76 carrying wt YadA were less virulent for the mouse than EV76 expressing YadAV98D,N99A. The observation that YadA makes Yersinia sensitive to NETs provides an explanation as for why evolution selected for the inactivation of yadA in the flea-borne Y. pestis and clarifies an old enigma. Since YadA imposes the same cost to the food-borne Yersinia but was nevertheless conserved by evolution, this observation also illustrates the duality of some virulence functions

Human unconventional T cells in Plasmodium falciparum infection

Malaria is an old scourge of humankind and has a large negative impact on the economic development of affected communities. Recent success in malaria control and reduction of mortality seems to have stalled emphasizing that our current intervention tools need to be complemented by malaria vaccines. Different populations of unconventional T cells such as mucosal-associated invariant T (MAIT) cells, invariant natural killer T (iNKT) cells and γδ T cells are gaining attention in the field of malaria immunology. Significant advances in our basic understanding of unconventional T cell biology in rodent malaria models have been made, however, their roles in humans during malaria are less clear. Unconventional T cells are abundant in skin, gut and liver tissues, and long-lasting expansions and functional alterations were observed upon malaria infection in malaria naïve and malaria pre-exposed volunteers. Here, we review the current understanding of involvement of unconventional T cells in anti-Plasmodium falciparum immunity and highlight potential future research avenues

Skin Transplantation and Lymphoid Organ Analysis in Mice

Skin transplantation in mice is an important procedure to evaluate immune responses generated against heterologous grafts, especially given its highly immunogenic nature. In fact, skin is one of the most challenging organs in terms of allograft retention. In this protocol, we provide a detailed procedure for skin grafting using the tail skin as donor organ that is grafted on the dorsal site of thoracic cage in a recipient mouse. We also provide protocols for the systematic analysis of lymphoid organ analysis in transplanted mice. Together these protocols may be valuable for evaluation of parameters that affect skin grafting, including genetic factors, immune cell activation as well as the analysis of compounds that may be useful in allowing graft tolerance

Lipoproteins in Staphylococcus aureus mediate inflammation by TLR2 and iron-dependent growth in vivo

Lipoproteins (Lpp) are ligands of TLR2 and signal by the adaptor MyD88. As part of the bacterial cell envelope, Lpp are mainly involved in nutrient acquisition for Staphylococcus aureus. The impact of Lpp on TLR2-MyD88 activation for S. aureus in systemic infection is unknown. S. aureus strain SA113 deficient in the enzyme encoded by the prolipoprotein diacylglyceryl transferase gene (Deltalgt), which attaches the lipid anchor to pro-Lpp, was used to study benefits and costs of Lpp maturation. Lpp in S. aureus induced early and strong cytokines by TLR2-MyD88 signaling in murine peritoneal macrophages. Lpp contributed via TLR2 to pathogenesis of sepsis in C57BL/6 mice with IL-1beta, chemokine-mediated inflammation, and high bacterial numbers. In the absence of MyD88-mediated inflammation, Lpp allowed bacterial clearing from liver devoid of infiltrating cells, but still conferred a strong growth advantage in mice, which was shown to rely on iron uptake and storage in vitro and in vivo. With iron-restricted bacteria, the Lpp-related growth advantage was evident in infection of MyD88(-/-), but not of C57BL/6, mice. On the other hand, iron overload of the host restored the growth deficit of Deltalgt in MyD88(-/-), but not in immunocompetent C57BL/6 mice. These results indicate that iron acquisition is improved by Lpp of S. aureus but is counteracted by inflammation. Thus, lipid anchoring is an evolutionary advantage for S. aureus to retain essential proteins for better survival in infection