Elisabet Josefsson

Protection against Experimental Staphylococcus aureus Arthritis by Vaccination with Clumping Factor A, a Novel Virulence Determinant

The importance of the fibrinogen-binding adhesin clumping factor A (ClfA) in the pathogenesis of Staphylococcus aureus septic arthritis was examined in an animal model. The protective effect of active and passive immunization with ClfA also was investigated in S. aureus infection models. The severity of arthritis was markedly reduced in mice challenged intravenously with a clfA mutant, compared with mice infected with the wild-type strain. Mice immunized with recombinant ClfA and challenged with S. aureus developed less-severe arthritis than did mice immunized with a control antigen. Passive immunization of mice with rat and rabbit anti-ClfA antibodies protected against S. aureus arthritis and sepsis-induced death, indicating that the protection by active immunization is antibody mediated. Taken together, these data strongly suggest that ClfA is a crucial virulence determinant for septic arthritis and an excellent target for the generation of immune therapies directed against S. aureus.

Catecholamines are synthesized by mouse lymphocytes and regulate function of these cells by induction of apoptosis.

The immune and the nervous systems are anatomically closely related and interact with each other by molecules common to both systems, such as cytokines and neurotransmitters. The purpose of this study was to investigate the participation of catecholamines in the neuroimmunological network. The ability of immune cells to produce catecholamines was examined by a highly sensitive capillary electrophoresis assay, which permits detection of easily oxidized catecholamines in the zeptomole (10−21) range. In addition, the effects of catecholamines on in vitro proliferation, differentiation and apoptosis of lymphocytes were assessed. Mouse spleen cells and macrophages contained on average 7 × 10−17 and 2 × 10−17 mole dopamine per cell, respectively. In the former cell population also norepinephrine was found. Several mouse B‐ and T‐cell hybridomas were also shown to contain endogenously produced dopamine in levels ranging from 7 × 10−20 to 2 × 10−18 mole dopamine per cell. In addition, one of the T‐cell hybridomas proved to synthesize norepinephrine. The dopamine production of lymphocytes was blocked by the tyrosine hydroxylase inhibitor α‐methyl‐p‐tyrosine, whereas incubation with the precursor L‐DOPA increased the dopamine content. Incubation with L‐DOPA, dopamine and norepinephrine dose‐dependently suppressed mitogen induced proliferation and differentiation of mouse lymphocytes. Even short‐time pretreatment of lymphocytes with L‐DOPA and dopamine strongly suppressed lymphocyte proliferation and cytokine production. Incubation of lymphoid cells with L‐DOPA, dopamine and norepinephrine dose‐dependently induced apoptosis which, at least partly, explains the suppressive effects of catecholamines on lymphocyte function. Our results demonstrate that catecholamines: (i) are actively produced by lymphocytes and (ii) have the capacity to act as auto‐ and/or paracrine regulators of lymphocyte activity through induction of apoptosis.

Three new members of the serine-aspartate repeat protein multigene family of Staphylococcus aureus

Three new genes encoding the serine-aspartate (SD) repeat-containing proteins SdrC, SdrD and SdrE were found in Staphylococcus aureus strain Newman. The SD repeats had earlier been found in the S. aureus fibrinogen-binding clumping factors ClfA and ClfB. The clfA and clfB genes encode high-molecular-mass fibrinogen-binding proteins that are anchored to the cell surface of S. aureus. The sdr genes now reported are closely linked and tandemly arrayed. The putative Sdr proteins have both organizational and sequence similarity to ClfA and ClfB. At the N-terminus, putative secretory signal sequences precede approximately 500 residue A regions. The A regions of the Sdr and Clf proteins exhibit only 20-30% residue identity when aligned with any other member of the family. The only conserved sequence is the consensus motif TYTFTDYVD. The Sdr proteins differ from ClfA and ClfB by having two to five additional 110-113 residue repeated sequences (B-motifs) located between region A and the R-region. Each B-motif contains a consensus Ca2+-binding EF-hand loop normally found in eukaryotic proteins. The structural integrity of recombinant SdrD(B1-B5) protein comprising the five B-repeats of SdrD was shown by bisANS fluorescence analysis to be Ca2+-dependent, suggesting that the EF-hands are functional. When Ca2+ was removed the structure collapsed to an unfolded conformation. The original structure was restored by addition of Ca2+. The C-terminal R-domains of the Sdr proteins contain 132-170 SD residues. These are followed by conserved wall-anchoring regions characteristic of many surface proteins of Gram-positive bacteria. The sdr locus was present in all 31 S. aureus strains from human and bovine sources tested by Southern hybridization, although in a few strains it contained two rather than three genes.

Anti-inflammatory properties of estrogen: I. In vivo suppression of leukocyte production in bone marrow and redistribution of peripheral blood neutrophils

We have recently demonstrated that estrogen has differential immunomodulatory properties in mice because it stimulates antibody responses but inhibits T cell-mediated inflammation. In the present study we have examined the influence of estrogen on T cell-independent inflammatory responses. A local inflammation was induced by intradermal injection of olive oil and cholera toxin. Treatment of castrated mice with pharmacological and even physiological doses of estradiol significantly suppressed the inflammatory response, as measured by footpad swelling and documented by histologic examination. Even a single injection of a low dose of estradiol (3.2 micrograms/mouse) given up to 4 days prior to the administration of the phlogistic compound reduced the inflammatory response. Our results demonstrate that the mechanisms whereby estrogen exerts its anti-inflammatory property are probably not mediated by modulation of corticosteroid production. Instead, it is evidenced that estrogen significantly suppresses the bone marrow production of leukocytes and affects the distribution of polymorphonuclear cells in peripheral blood. The possible implications of our results are discussed in the context of the modulation of inflammatory rheumatic diseases.

Staphylococcus aureus Host Cell Invasion and Virulence in Sepsis Is Facilitated by the Multiple Repeats within FnBPA

Entry of Staphylococcus aureus into the bloodstream can lead to metastatic abscess formation and infective endocarditis. Crucial to the development of both these conditions is the interaction of S. aureus with endothelial cells. In vivo and in vitro studies have shown that the staphylococcal invasin FnBPA triggers bacterial invasion of endothelial cells via a process that involves fibronectin (Fn) bridging to α5β1 integrins. The Fn-binding region of FnBPA usually contains 11 non-identical repeats (FnBRs) with differing affinities for Fn, which facilitate the binding of multiple Fn molecules and may promote integrin clustering. We thus hypothesized that multiple repeats are necessary to trigger the invasion of endothelial cells by S. aureus. To test this we constructed variants of fnbA containing various combinations of FnBRs. In vitro assays revealed that endothelial cell invasion can be facilitated by a single high-affinity, but not low-affinity FnBR. Studies using a nisin-inducible system that controlled surface expression of FnBPA revealed that variants encoding fewer FnBRs required higher levels of surface expression to mediate invasion. High expression levels of FnBPA bearing a single low affinity FnBR bound Fn but did not invade, suggesting that FnBPA affinity for Fn is crucial for triggering internalization. In addition, multiple FnBRs increased the speed of internalization, as did higher expression levels of FnBPA, without altering the uptake mechanism. The relevance of these findings to pathogenesis was demonstrated using a murine sepsis model, which showed that multiple FnBRs were required for virulence. In conclusion, multiple FnBRs within FnBPA facilitate efficient Fn adhesion, trigger rapid bacterial uptake and are required for pathogenesis.

The Staphylococcus aureus response to unsaturated long chain free fatty acids: survival mechanisms and virulence implications.

Staphylococcus aureus is an important human commensal and opportunistic pathogen responsible for a wide range of infections. Long chain unsaturated free fatty acids represent a barrier to colonisation and infection by S. aureus and act as an antimicrobial component of the innate immune system where they are found on epithelial surfaces and in abscesses. Despite many contradictory reports, the precise anti-staphylococcal mode of action of free fatty acids remains undetermined. In this study, transcriptional (microarrays and qRT-PCR) and translational (proteomics) analyses were applied to ascertain the response of S. aureus to a range of free fatty acids. An increase in expression of the σB and CtsR stress response regulons was observed. This included increased expression of genes associated with staphyloxanthin synthesis, which has been linked to membrane stabilisation. Similarly, up-regulation of genes involved in capsule formation was recorded as were significant changes in the expression of genes associated with peptidoglycan synthesis and regulation. Overall, alterations were recorded predominantly in pathways involved in cellular energetics. In addition, sensitivity to linoleic acid of a range of defined (sigB, arcA, sasF, sarA, agr, crtM) and transposon-derived mutants (vraE, SAR2632) was determined. Taken together, these data indicate a common mode of action for long chain unsaturated fatty acids that involves disruption of the cell membrane, leading to interference with energy production within the bacterial cell. Contrary to data reported for other strains, the clinically important EMRSA-16 strain MRSA252 used in this study showed an increase in expression of the important virulence regulator RNAIII following all of the treatment conditions tested. An adaptive response by S. aureus of reducing cell surface hydrophobicity was also observed. Two fatty acid sensitive mutants created during this study were also shown to diplay altered pathogenesis as assessed by a murine arthritis model. Differences in the prevalence and clinical importance of S. aureus strains might partly be explained by their responses to antimicrobial fatty acids.

Predicting the virulence of MRSA from its genome sequence

Microbial virulence is a complex and often multifactorial phenotype, intricately linked to a pathogens evolutionary trajectory. Toxicity, the ability to destroy host cell membranes, and adhesion, the ability to adhere to human tissues, are the major virulence factors of many bacterial pathogens, including Staphylococcus aureus. Here, we assayed the toxicity and adhesiveness of 90 MRSA (methicillin resistant S. aureus) isolates and found that while there was remarkably little variation in adhesion, toxicity varied by over an order of magnitude between isolates, suggesting different evolutionary selection pressures acting on these two traits. We performed a genome-wide association study (GWAS) and identified a large number of loci, as well as a putative network of epistatically interacting loci, that significantly associated with toxicity. Despite this apparent complexity in toxicity regulation, a predictive model based on a set of significant single nucleotide polymorphisms (SNPs) and insertion and deletions events (indels) showed a high degree of accuracy in predicting an isolates toxicity solely from the genetic signature at these sites. Our results thus highlight the potential of using sequence data to determine clinically relevant parameters and have further implications for understanding the microbial virulence of this opportunistic pathogen.

The binding of calcium to the B-repeat segment of SdrD, a cell surface protein of Staphylococcus aureus.

In the Sdr family of Staphylococcus aureus cell surface proteins, three recently cloned members (Josefsson, E., McCrea, K., Ni Eidhin, D., O’Connell, D., Cox, J. A., Hook, M., and Foster, T. (1998) Microbiology, in press) display variable numbers of B-repeats, i.e. segments of 110–113 residues that probably make up one folding unit. Each B-repeat contains one conserved EF-hand motif and two acidic stretches. Equilibrium dialysis revealed that segment B1–B5 of SrdD contains 14 Ca2+-binding sites with high affinity ([Ca2+]0.5, 4 μm), whereas flow dialysis yielded 5 sites of high affinity (class I) and 10 of low affinity (class II). The discrepancy could be explained by the slow induction of high affinity in the class II sites. Kinetic experiments using fluorescent Ca2+ indicators corroborated slow binding of Ca2+ at the latter sites. Circular dichroism and Trp fluorescence showed that, whereas the Ca2+ form is well folded, the metal-free form seems strongly disorganized. The Ca2+-induced conformational changes comprise both fast and slow steps, giving thus a structural support for the induction of class II Ca2+-binding sites. The B-repeats may act as rulers or springs that modulate the distance between the interactive A region and the bacterial cell surface.

Fibrinogen Binding Sites P336 and Y338 of Clumping Factor A Are Crucial for Staphylococcus aureus Virulence

We have earlier shown that clumping factor A (ClfA), a fibrinogen binding surface protein of Staphylococcus aureus, is an important virulence factor in septic arthritis. When two amino acids in the ClfA molecule, P336 and Y338, were changed to serine and alanine, respectively, the fibrinogen binding property was lost. ClfAP336Y338 mutants have been constructed in two virulent S. aureus strains Newman and LS-1. The aim of this study was to analyze if these two amino acids which are vital for the fibrinogen binding of ClfA are of importance for the ability of S. aureus to generate disease. Septic arthritis or sepsis were induced in mice by intravenous inoculation of bacteria. The clfAP336Y338 mutant induced significantly less arthritis than the wild type strain, both with respect to severity and frequency. The mutant infected mice developed also a much milder systemic inflammation, measured as lower mortality, weight loss, bacterial growth in kidneys and lower IL-6 levels. The data were verified with a second mutant where clfAP336 and Y338 were changed to alanine and serine respectively. When sepsis was induced by a larger bacterial inoculum, the clfAP336Y338 mutants induced significantly less septic death. Importantly, immunization with the recombinant A domain of ClfAP336SY338A mutant but not with recombinant ClfA, protected against septic death. Our data strongly suggest that the fibrinogen binding activity of ClfA is crucial for the ability of S. aureus to provoke disease manifestations, and that the vaccine potential of recombinant ClfA is improved by removing its ability to bind fibrinogen.

Immune evasion by Staphylococcus aureus conferred by iron-regulated surface determinant protein IsdH.

The ability of Staphylococcus aureus to avoid innate immune responses including neutrophil-mediated phagocytosis is crucial for the organism to cause infection. This multifactorial process involves several secreted and cell-surface-associated proteins. In this paper we report a novel mechanism of combating neutrophils that involves iron-regulated surface determinant protein H (IsdH). The IsdH protein is part of a complex that is only expressed under iron-restricted conditions in order to bind haemoglobin and extract and transport haem into the cytoplasm. A null mutant defective in expression of IsdH, and mutants expressing variants of IsdH with substitutions in residues predicted to be involved in ligand binding, were generated from S. aureus 8325-4. The IsdH-defective mutants were shown by several measures to have reduced virulence compared with the wild-type. The mutant was engulfed more rapidly by human neutrophils in the presence of serum opsonins, survived poorly in fresh whole human blood and was less virulent in a mouse model of sepsis. The protective mechanism seems to stem from an accelerated degradation of the serum opsonin C3b.