Biomedical subjects

Muzaffar Hussain

Publications and source records attributed to Muzaffar Hussain.

9 recordsLinked to original sources

PubMed2006

The extracellular adherence protein from Staphylococcus aureus abrogates angiogenic responses of endothelial cells by blocking Ras activation.

The extracellular adherence protein (Eap), a broad-spectrum adhesin secreted by Staphylococcus aureus, was previously shown to curb acute inflammatory responses, presumably through its binding to endothelial cell (EC) ICAM-1. Examining the effect of Eap on endothelial function in more detail, we here show that, in addition, Eap functions as a potent angiostatic agent. Concomitant treatment of EC with purified Eap resulted in the complete blockage of the mitogenic and sprouting responses elicited by vascular endothelial growth factor (VEGF)165 or basic fibroblast growth factor (bFGF). Moreover, the induction of tissue factor and decay-accelerating factor were repressed by Eap, as determined by qRT-polymerase chain reaction (qRT-PCR), with a corresponding reduction in Egr-1 protein up-regulation seen. This angiostatic activity was accompanied by a corresponding inhibition in ERK1/2 phosphorylation, while activation of p38 was not affected. Inhibition occurred downstream of tyrosine kinase receptor activation, as comparable effects were seen on TPA-induced ERK1/2 phosphorylation. Similar to previously described angiostatic agents like angiopoietin-1 or the 16-kDa prolactin fragment, Eap blockage of the Ras/Raf/MEK/ERK cascade was localized by pull-down assay at the level of Ras activation. Eap's combined anti-inflammatory and antiangiogenic properties render this bacterial protein not only an important virulence factor during S. aureus infection but open new perspectives for therapeutic applications in pathological neovascularization.

Bacterial Proteins↗

PubMed2006

The anchorless adhesin Eap (extracellular adherence protein) from Staphylococcus aureus selectively recognizes extracellular matrix aggregates but binds promiscuously to monomeric matrix macromolecules.

Besides a number of cell wall-anchored adhesins, the majority of Staphylococcus aureus strains produce anchorless, cell wall-associated proteins, such as Eap (extracellular adherence protein). Eap contains four to six tandem repeat (EAP)-domains. Eap mediates diverse biological functions, including adherence and immunomodulation, thus contributing to S. aureus pathogenesis. Eap binding to host macromolecules is unusually promiscuous and includes matrix or matricellular proteins as well as plasma proteins. The structural basis of this promiscuity is poorly understood. Here, we show that in spite of the preferential location of the binding epitopes within triple helical regions in some collagens there is a striking specificity of Eap binding to different collagen types. Collagen I, but not collagen II, is a binding substrate in monomolecular form. However, collagen I is virtually unrecognized by Eap when incorporated into banded fibrils. By contrast, microfibrils containing collagen VI as well as basement membrane-associated networks containing collagen IV, or aggregates containing fibronectin bound Eap as effectively as the monomeric proteins. Therefore, Eap-binding to extracellular matrix ligands is promiscuous at the molecular level but not indiscriminate with respect to supramolecular structures containing the same macromolecules. In addition, Eap bound to banded fibrils after their partial disintegration by matrix-degrading proteinases, including matrix metalloproteinase 1. Therefore, adherence to matrix suprastructures by S. aureus can be supported by inflammatory reactions.

Bacterial Adhesion↗

PubMed2005

The extracellular adherence protein (Eap) of Staphylococcus aureus inhibits wound healing by interfering with host defense and repair mechanisms.

Staphylococcus aureus is a major human pathogen interfering with host-cell functions. Impaired wound healing is often observed in S aureus-infected wounds, yet, the underlying mechanisms are poorly defined. Here, we identify the extracellular adherence protein (Eap) of S aureus to be responsible for impaired wound healing. In a mouse wound-healing model wound closure was inhibited in the presence of wild-type S aureus and this effect was reversible when the wounds were incubated with an isogenic Eap-deficient strain. Isolated Eap also delayed wound closure. In the presence of Eap, recruitment of inflammatory cells to the wound site as well as neovascularization of the wound were prevented. In vitro, Eap significantly reduced intercellular adhesion molecule 1 (ICAM-1)-dependent leukocyte-endothelial interactions and diminished the consequent activation of the proinflammatory transcription factor nuclear factor kappaB (NFkappaB) in leukocytes associated with a decrease in expression of tissue factor. Moreover, Eap blocked alphav-integrin-mediated endothelial-cell migration and capillary tube formation, and neovascularization in matrigels in vivo. Collectively, the potent anti-inflammatory and antiangiogenic properties of Eap provide an underlying mechanism that may explain the impaired wound healing in S aureus-infected wounds. Eap may also serve as a lead compound for new anti-inflammatory and antiangiogenic therapies in several pathologies.

Animals↗

PubMed2005

Induction of Staphylococcus epidermidis biofilm formation via proteolytic processing of the accumulation-associated protein by staphylococcal and host proteases.

Because of its biofilm forming potential Staphylococcus epidermidis has evolved as a leading cause of device-related infections. The polysaccharide intercellular adhesin (PIA) is significantly involved in biofilm accumulation. However, infections because of PIA-negative strains are not uncommon, suggesting the existence of PIA-independent biofilm accumulation mechanisms. Here we found that biofilm formation in the clinically significant S. epidermidis 5179 depended on the expression of a truncated 140 kDa isoform of the 220 kDa accumulation-associated protein Aap. As expression of the truncated Aap isoform leads to biofilm formation in aap-negative S. epidermidis 1585, this domain mediates intercellular adhesion in a polysaccharide-independent manner. In contrast, expression of full-length Aap did not lead to a biofilm-positive phenotype. Obviously, to gain adhesive function, full-length Aap has to be proteolytically processed through staphylococcal proteases as demonstrated by inhibition of biofilm formation by alpha(2)-macroglobulin. Importantly, also exogenously added granulocyte proteases activated Aap, thereby inducing biofilm formation in S. epidermidis 5179 and four additional, independent clinical S. epidermidis strains. It is therefore reasonable to assume that in vivo effector mechanisms of the innate immunity can directly induce protein-dependent S. epidermidis cell aggregation and biofilm formation, thereby enabling the pathogen to evade clearance by phagocytes.

Adhesins, Bacterial↗

PubMed2004

Truncation of fibronectin-binding proteins in Staphylococcus aureus strain Newman leads to deficient adherence and host cell invasion due to loss of the cell wall anchor function.

Staphylococcus aureus fibronectin-binding proteins (FnBPs) play a critical role in S. aureus pathogenesis. FnBPs mediate adhesion to fibronectin and invasion of mammalian cells, including epithelial, endothelial, and fibroblastic cells, by fibronectin bridging to the host cell fibronectin receptor integrin (alpha(5))beta(1). Strain Newman is a laboratory strain frequently used for genetic, functional, and in vivo studies. However, despite pronounced production of FnBPs, strain Newman is only weakly adherent to immobilized Fn and weakly invasive. We examined whether these effects are due to a structural difference of FnBPs. Here, we show that both fnbA(Newman) and fnbB(Newman) contain a centrally located point mutation resulting in a stop codon. This leads to a truncation of both FnBPs at the end of the C domain at identical positions. Most likely, the stop codon occurred first in fnbB(Newman) and was subsequently transferred to fnbA(Newman) by replacement of the entire region encompassing the C, D, and W domains with the respective sequence of fnbB(Newman). Using heterologous expression in Staphylococcus carnosus, we found that truncated FnBPs were completely secreted into the culture medium and not anchored to the cell wall, since they lack the sortase motif (LPETG). Consequently, this led to a loss of FnBP-dependent functions, such as strong adhesion to immobilized fibronectin, binding of fibrinogen, and host cell invasion. This mutation may explain some of the earlier reported conflicting data with strain Newman. Thus, care should be taken when drawing negative conclusions about the role of FnBPs as a virulence factor in a given model.

Adhesins, Bacterial↗

PubMed2003

The adhesive and immunomodulating properties of the multifunctional Staphylococcus aureus protein Eap.

Adherence of Staphylococcus aureus to the host tissue is an important step in the initiation of pathogenesis. At least 10 adhesins produced by S. aureus have been described and it is becoming clear that the expression of these adhesins and their interactions with eukaryotic cells involve complex processes. Some of these, such as the fibronectin-binding proteins (FnBPs) and Clumping Factor A, are well characterized. However, in the last 10 years a number of novel S. aureus adhesins have been described. Functional analyses of these proteins, one of which is Eap (extracellular adherence protein, also known as Map and p70), are revealing important information on the pathogenesis of staphylococcal disease. More than 10 years after the first report of Eap, we are beginning to understand that this protein, which has a broad spectrum of functions, may be a critical factor in the pathogenesis of S. aureus. This review will focus on the interactions of Eap with eukaryotic cells, plasma proteins and the extracellular matrix as well as on the recently recognized role of Eap as an important mediator in the immune response to staphylococcal infection.

Adhesins, Bacterial↗

PubMed2003

Extracellular adherence protein from Staphylococcus aureus enhances internalization into eukaryotic cells.

In this study we have shown that Eap (extracellular adherence protein) plays a role in the internalization process of Staphylococcus aureus into eukaryotic cells. Eap is a protein that is mostly extracellularly and to a lesser extent is bound to the bacterial surface as a result of rebinding. Eap is able to bind to several plasma proteins, such as fibronectin, fibrinogen, and prothrombin. It has the capacity to form oligomers and is able to agglutinate S. aureus. A mutant strain, Newman mAH12 (eap:: Ery(r)), with a deficient eap gene was used in the present study. We have demonstrated that (i) strain Newman mAH12 could adhere to and become internalized to a higher extent by eukaryotic cells than the isogenic mutant, (ii) strain Newman mAH12 complemented with the eap gene displayed restoration of the internalization level, (iii) externally added Eap enhanced the internalization of laboratory and clinical S. aureus strains as well as of S. carnosus (a coagulase-negative species devoid of proteins important for internalization), and (iv) antibodies against Eap were able to block the internalization process in strain Newman mAH12 and clinical isolates. Eap, with its broad binding capacity and its surface localization, thus seems to contribute to the internalization of S. aureus into eukaryotic cells. We therefore propose a novel internalization pathway for S. aureus in which Eap plays an enhancing role.

Adhesins, Bacterial↗

PubMed2002

Staphylococcus aureus extracellular adherence protein serves as anti-inflammatory factor by inhibiting the recruitment of host leukocytes.

Staphylococcus aureus is a human pathogen that secretes proteins that contribute to bacterial colonization. Here we describe the extracellular adherence protein (Eap) as a novel anti-inflammatory factor that inhibits host leukocyte recruitment. Due to its direct interactions with the host adhesive proteins intercellular adhesion molecule 1 (ICAM-1), fibrinogen or vitronectin, Eap disrupted beta(2)-integrin and urokinase receptor mediated leukocyte adhesion in vitro. Whereas Eap-expressing S. aureus induced a 2 3-fold lower neutrophil recruitment in bacterial peritonitis in mice as compared with an Eap-negative strain, isolated Eap prevented beta(2)-integrin-dependent neutrophil recruitment in a mouse model of acute thioglycollate-induced peritonitis. Thus, the specific interactions with ICAM-1 and extracellular matrix proteins render Eap a potent anti-inflammatory factor, which may serve as a new therapeutic substance to block leukocyte extravasation in patients with hyperinflammatory pathologies.

Animals↗

PubMed2002

Insertional inactivation of Eap in Staphylococcus aureus strain Newman confers reduced staphylococcal binding to fibroblasts.

To initiate invasive infection, Staphylococcus aureus must adhere to host substrates, such as the extracellular matrix or eukaryotic cells, by virtue of different surface proteins (adhesins). Recently, we identified a 60-kDa cell-secreted extracellular adherence protein (Eap) of S. aureus strain Newman with broad-spectrum binding characteristics (M. Palma, A. Haggar, and J. I. Flock, J. Bacteriol. 181:2840-2845, 1999), and we have molecularly confirmed Eap to be an analogue of the previously identified major histocompatibility complex class II analog protein (Map) (M. Hussain, K. Becker, C. von Eiff, G. Peter, and M. Herrmann, Clin. Diagn. Lab. Immunol. 8:1281-1286, 2001). Previous analyses of the Eap/Map function performed with purified protein did not allow dissection of its precise role in the complex situation of the staphylococcal whole cell presenting several secreted and wall-bound adhesins. Therefore, the role of Eap was investigated by constructing a stable eap::ermB deletion in strain Newman and by complementation of the mutant. Patterns of extracted cell surface proteins analyzed both by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western ligand assays with various adhesive matrix molecules clearly confirmed the absence of Eap in the mutant. However, binding and adhesion tests using whole staphylococcal cells demonstrated that both the parent and mutant strains bound equally well to fibronectin- and fibrinogen-coated surfaces, possibly due to their recognition by other staphylococcal adhesins. Furthermore, Eap mediated staphylococcal agglutination of both wild-type and mutant cells. In contrast, the mutant adhered to a significantly lesser extent to cultured fibroblasts (P < 0.001) than did the wild type, while adherence was restorable upon complementation. Furthermore, adherence to both epithelial cells (P < 0.05) and fibroblasts (not significant) could be blocked with antibodies against Eap, whereas preimmune serum was not active. In conclusion, Eap may contribute to pathogenicity by promoting adhesion of whole staphylococcal cells to complex eukaryotic substrates.

Adhesins, Bacterial↗