Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

University of Basel and University Hospital Basel

The virulence of SCCmec type IV hospital-acquired methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates belonging to the major sequence type 8 (ST8 [Lyon clone]) and to a minor upcoming clone, ST5, was compared with that of methicillin-susceptible S. aureus (MSSA) isolates of matching sequence types. In vitro adhesion to human airway epithelial cells (HAECs) as an indicator of dissemination and mortality in a murine sepsis model as an indicator of virulence were evaluated. Ten MRSA isolates and 8 MSSA isolates of ST8 and 8 MRSA isolates and 8 MSSA isolates of ST5 were characterized with respect to multilocus sequence type; agr, spa, and capsule typing; in vitro doubling time; toxin and adhesin gene profiles; and adherence to HAECs. Adherence was significantly lower in the MRSA ST5 group than in the ST8 groups. Infections with MRSA and MSSA isolates ST8 and ST5 were compared. No change in virulence related to the presence of SCCmec was observed, since ST8 but not ST5 caused a significantly lower mortality in its presence. Despite their similar genetic backgrounds, individual clonal MRSA and MSSA isolates were heterogeneous in adherence and virulence. No one of these specific virulence factors determined in vitro was related to mouse mortality. In conclusion, in a bacteremic model, mortality was dependent on the ST and was differentially modulated by SCCmec; within an ST, clonality was not associated with a homogenous outcome.

Comparison of adhesion and virulence of two predominant hospital-acquired methicillin-resistant Staphylococcus aureus clones and clonal methicillin-susceptible S. aureus isolates.

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.

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

Neutrophils kill invading pathogens by AMPs, including cathelicidins, ROS, and NETs. The human pathogen Staphylococcus aureus exhibits enhanced resistance to neutrophil AMPs, including the murine cathelicidin CRAMP, in part, as a result of alanylation of teichoic acids by the dlt operon. In this study, we took advantage of the hypersusceptible phenotype of S. aureus DeltadltA against cationic AMPs to study the impact of the murine cathelicidin CRAMP on staphylococcal killing and to identify its key site of action in murine neutrophils. We demonstrate that CRAMP remained intracellular during PMN exudation from blood and was secreted upon PMA stimulation. We show first evidence that CRAMP was recruited to phagolysosomes in infected neutrophils and exhibited intracellular activity against S. aureus. Later in infection, neutrophils produced NETs, and immunofluorescence revealed association of CRAMP with S. aureus in NETs, which similarly killed S. aureus wt and DeltadltA, indicating that CRAMP activity was reduced when associated with NETs. Indeed, the presence of DNA reduced the antimicrobial activity of CRAMP, and CRAMP localization in response to S. aureus was independent of the NADPH oxidase, whereas killing was partially dependent on a functional NADPH oxidase. Our study indicates that neutrophils use CRAMP in a timed and locally coordinated manner in defense against S. aureus.

Neutrophil antimicrobial defense against Staphylococcus aureus is mediated by phagolysosomal but not extracellular trap-associated cathelicidin.

Staphylococcus aureus expresses about 50 lipoproteins (Lpp), which are lipid-anchored in the membrane. The processing of the precursor to the mature Lpp is catalyzed by the phosphatidyl glycerol diacylglyceryl transferase (Lgt) and the lipoprotein-specific type II signal peptidase (LspA) leading to diacylated Lpp. Possibly another acyltransferase attaches a third fatty acid leading to triacylated Lpp. Lpp function as binding proteins for transport of nutrients across the microbial membrane and are involved in processing of other proteins, but most Lpp remain of predicted or unknown function. The di- or triacylated lipid structure is sensed by host pattern recognition receptor TLR2 and induces innate immune responses in professional and non-professional phagocytes. In the host, maturation of Lpp confers optimal metal ion - particularly iron - acquisition, it enhances staphylococcal invasion and phagocytosis, intracellular survival and persistence of infections. However, the advantages of Lpp maturation are counterbalanced by the capability to induce inflammation. In this review, we summarize the current knowledge about the role of Lpp in iron acquisition and TLR2 recognition in the host and describe the consequences of Lpp maturation for survival of S. aureus in the host.

Staphylococcal lipoproteins and their role in bacterial survival in mice.

Toll-like receptors play an essential role in the detection of invading pathogens. TLR2 is expressed in high concentrations on neutrophils and has been implicated as a critical mediator inducing host antimicrobial defenses against Gram-positive bacteria. Neutrophil responses induced via TLR2 are likely to have important clinical consequences, since Gram-positive organisms, such as Staphylococcus aureus, are an increasingly important source of severe infections. In the present study, we report that TLR2 has a central role in killing of S. aureus by murine PMN via enhancement of NADPH oxidase activity. PMN from TLR2-deficient mice showed a similar inability to kill S. aureus in vitro and under in vivo-like conditions as PMN with a non-functional NADPH oxidase. This defect in killing by TLR2-deficient PMN was not related to phagocytosis but caused by delayed and reduced NADPH oxidase-mediated production of superoxide anion in response to S. aureus and other Gram-positive bacteria. The cause of this was independent of PI3K- and p38 signaling. The TLR2-enhanced induction of superoxide was a defect in proper NADPH oxidase assembly. We hypothesize that early activation of TLR2-signaling may enhance p47(phox) phosphorylation subsequent to phagocytosis-mediated phosphorylation. Summarized, these data demonstrate a novel role of TLR2 in the killing of S. aureus by ensuring a rapid activation of the NADPH oxidase complex.

TLR2 enhances NADPH oxidase activity and killing of Staphylococcus aureus by PMN.

Staphylococcus aureus infection elicits through its mature lipoproteins an innate immune response by TLR2-MyD88 signaling, which improves bacterial clearing and disease outcome. The role of dendritic cells (DCs) and T cells in this immune activation and the function of T and B cells in defense against S. aureus infection remain unclear. Therefore, we first evaluated DC and T cell activation after infection with S. aureus wild type (WT) and its isogenic mutant, which is deficient in lipoprotein maturation, in vitro. Lipoproteins in viable S. aureus contributed via TLR2-MyD88 to activation of DCs, which promoted the release of IFN-γ and IL-17 in CD4(+) T cells. This strong effect was independent of superantigens and MHC class II. We next evaluated the function of T cells and their cytokines IFN-γ and IL-17 in infection in vivo. Six days after systemic murine infection IFN-γ, IL-17, and IL-10 production in total spleen cells were MyD88-dependent and their levels increased until day 21. The comparison of CD3(-/-), Rag2(-/-), and C57BL/6 mice after infection revealed that IFN-γ and IL-17 originated from T cells and IL-10 originated from innate immune cells. Furthermore, vaccination of mice to activate T and B cells did not improve eradication of S. aureus from organs. In conclusion, S. aureus enhances DC activation via TLR2-MyD88 and thereby promotes T(H)1 and T(H)17 cell differentiation. However, neither T cells and their MyD88-regulated products, IFN-γ and IL-17, nor B cells affected bacterial clearing from organs and disease outcome.

T and B cells are not required for clearing Staphylococcus aureus in systemic infection despite a strong TLR2-MyD88-dependent T cell activation.

Yersinia adhesin A (YadA) is an essential virulence factor for the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis. Surprisingly, 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 YadA(V₉₈D,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.

Oligomeric coiled-coil adhesin YadA is a double-edged sword.

Daptomycin (DAP) is bactericidal against methicillin-resistant Staphylococcus aureus (MRSA) in vitro, but it failed to eradicate MRSA in an experimental model of implant-associated infection. We therefore investigated various factors which could explain treatment failure by evaluating DAP activity, including the role of different cell wall components, adherence, biofilm, and calcium ions (Ca(2+)) in vitro and in vivo. In the tissue cage infection model, DAP was active only prophylactically and against low inocula. To identify the mechanisms of treatment failure, the in vitro activity of DAP against planktonic and adherent growing S. aureus and S. epidermidis mutants, differing in their capacity of biofilm formation and adherence, was determined. For planktonic staphylococci, the MIC was 0.625 μg/ml. For adherent staphylococci, DAP reduced biofilms at 30 μg/ml. However, it did not kill adherent bacteria up to 500 μg/ml, independent of biofilm biosynthesis (the ica mutant strain), nuclease (the nuc1/nuc2 mutant strain), LPXTG-anchored adhesin (the srtA mutant strain), autolysin (the atl mutant strain), or alanyl-LTA (the dltA mutant strain). Resistance of adherent staphylococci was not due to mutations of adherent bacteria, since staphylococci became DAP susceptible after detachment. Phenotypic tolerance was not explained by inactivation of DAP or inability of initial Ca(2+)-DAP complex formation. However, the addition of up to 100 mg/liter (2.5 mmol/liter) Ca(2+) gradually improved bactericidal activity toward adherent staphylococci in vitro and increased the prevention rate in the cage model from 40% to 60%. In summary, adherent staphylococci are resistant to DAP killing unless Ca(2+) is supplemented to physiologic concentrations.

Reversible daptomycin tolerance of adherent staphylococci in an implant infection model.

Capnocytophaga canimorsus are commensal Gram-negative bacteria from dog's mouth that cause rare but dramatic septicaemia in humans. C. canimorsus have the unusual property to feed on cultured mammalian cells, including phagocytes, by harvesting the glycan moiety of cellular glycoproteins. To understand the mechanism behind this unusual property, the genome of strain Cc5 was sequenced and analysed. In addition, Cc5 bacteria were cultivated onto HEK 293 cells and the surface proteome was determined. The genome was found to encode many lipoproteins encoded within 13 polysaccharide utilization loci (PULs) typical of the Flavobacteria-Bacteroides group. PULs encode surface exposed feeding complexes resembling the archetypal starch utilization system (Sus). The products of at least nine PULs were detected among the surface proteome and eight of them represented more than half of the total peptides detected from the surface proteome. Systematic deletions of the 13 PULs revealed that half of these Sus-like complexes contributed to growth on animal cells. The complex encoded by PUL5, one of the most abundant ones, was involved in foraging glycans from glycoproteins. It was essential for growth on cells and contributed to survival in mice. It thus represents a fitness factor during infection.

The genome and surface proteome of Capnocytophaga canimorsus reveal a key role of glycan foraging systems in host glycoproteins deglycosylation.

The strength of interactions between T cell receptors and the peptide-major histocompatibility complex (pMHC) directly modulates T cell fitness, clonal expansion, and acquisition of effector properties. Here we show that asymmetric T cell division is an important mechanistic link between increased signal strength, effector differentiation, and the ability to induce tissue pathology. Recognition of pMHC above a threshold affinity drove responding T cells into asymmetric cell division. The ensuing proximal daughters underwent extensive division and differentiated into short-lived effector cells expressing the integrin VLA-4, allowing the activated T cell to infiltrate and mediate destruction of peripheral target tissues. In contrast, T cells activated by below-threshold antigens underwent symmetric division, leading to abortive clonal expansion and failure to fully differentiate into tissue-infiltrating effector cells. Antigen affinity and asymmetric division are important factors that regulate fate specification in CD8(+) T cells and predict the potential of a self-reactive T cell to mediate tissue pathology.

Mathias Schmaler

Department of Biomedicine,
Immunoregulation,
Department of Biomedicine, Immunoregulation

University of Basel and University Hospital Basel

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

Isolation of Murine Lymph Node Stromal Cells

Mathias Schmaler

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biomedicine,Immunoregulation,Department of Biomedicine, Immunoregulation

University of Basel and University Hospital Basel

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

Isolation of Murine Lymph Node Stromal Cells

Department of Biomedicine,
Immunoregulation,
Department of Biomedicine, Immunoregulation