Heterologously expressed Staphylococcus aureus fibronectin-binding proteins are sufficient for invasion of host cells

Staphylococcus aureus invasion of mammalian cells, including epithelial, endothelial, and fibroblastic cells, critically depends on fibronectin bridging between S. aureus fibronectin-binding proteins (FnBPs) and the host fibronectin receptor integrin alpha(5)beta(1) (B. Sinha et al., Cell. Microbiol. 1:101-117, 1999). However, it is unknown whether this mechanism is sufficient for S. aureus invasion. To address this question, various S. aureus adhesins (FnBPA, FnBPB, and clumping factor [ClfA]) were expressed in Staphylococcus carnosus and Lactococcus lactis subsp. cremoris. Both noninvasive gram-positive microorganisms are genetically distinct from S. aureus, lack any known S. aureus surface protein, and do not bind fibronectin. Transformants of S. carnosus and L. lactis harboring plasmids coding for various S. aureus surface proteins (FnBPA, FnBPB, and ClfA) functionally expressed adhesins (as determined by bacterial clumping in plasma, specific latex agglutination, Western ligand blotting, and binding to immobilized and soluble fibronectin). FnBPA or FnBPB but not of ClfA conferred invasiveness to S. carnosus and L. lactis. Invasion of 293 cells by transformants was comparable to that of strongly invasive S. aureus strain Cowan 1. Binding of soluble and immobilized fibronectin paralleled invasiveness, demonstrating that the amount of accessible surface FnBPs is rate limiting. Thus, S. aureus FnBPs confer invasiveness to noninvasive, apathogenic gram-positive cocci. Furthermore, FnBP-coated polystyrene beads were internalized by 293 cells, demonstrating that FnBPs are sufficient for invasion of host cells without the need for (S. aureus-specific) coreceptors.     

Endovascular Infections Caused by Methicillin-Resistant Staphylococcus aureus Are Linked to Clonal Complex-Specific Alterations in Binding and Invasion Domains of Fibronectin-Binding Protein A as Well as the Occurrence of fnbB

Endovascular infections caused by Staphylococcus aureus involve interactions with fibronectin present as extracellular matrix or surface ligand on host cells. We examined the expression, structure, and binding activity of the two major S. aureus fibronectin-binding proteins (FnBPA, FnBPB) in 10 distinct, methicillin-resistant clinical isolates from patients with either persistent or resolving bacteremia. The persistent bacteremia isolates (n = 5) formed significantly stronger bonds with immobilized fibronectin as determined by dynamic binding measurements performed with atomic force microscopy. Several notable differences were also observed when the results were grouped by clonal complex 5 (CC5) strains (n = 5) versus CC45 strains (n = 5). Fibronectin-binding receptors on CC5 formed stronger bonds with immobilized fibronectin (P < 0.001). The fnbA gene was expressed at higher levels in CC45, whereas fnbB was found in only CC5 isolates. The fnbB gene was not sequenced because all CC45 isolates lacked this gene. Instead, comparisons were made for fnbA, which was present in all 10 isolates. Sequencing of fnbA revealed discrete differences within high-affinity, fibronectin-binding repeats (FnBRs) of FnBPA that included (i) 5-amino-acid polymorphisms in FnBR-9, FnBR-10, and FnBR-11 involving charged or polar side chains, (ii) an extra, 38-amino-acid repeat inserted between FnBR-9 and FnBR-10 exclusively seen in CC45 isolates, and (iii) CC5 isolates had the SVDFEED epitope in FnBR-11 (a sequence shown to be essential for fibronectin binding), while this sequence was replaced in all CC45 isolates with GIDFVED (a motif known to favor host cell invasion at the cost of reduced fibronectin binding). These complementary sequence and binding data suggest that differences in fnbA and fnbB, particularly polymorphisms and duplications in FnBPA, give S. aureus two distinct advantages in human endovascular infections: (i) FnBPs similar to that of CC5 enhance ligand binding and foster initiation of disease, and (ii) CC45-like FnBPs promote cell invasion, a key attribute in persistent endovascular infections.     

More than one tandem repeat domain of the extracellular adherence protein of Staphylococcus aureus is required for aggregation, adherence, and host cell invasion but not for leukocyte activation

The extracellular adherence protein (Eap) is a multifunctional Staphylococcus aureus protein and broad-spectrum adhesin for several host matrix and plasma proteins. We investigated the interactions of full-length Eap and five recombinant tandem repeat domains with host proteins by use of surface plasmon resonance (BIAcore) and ligand overlay assays. In addition, agglutination and host cell interaction, namely, adherence, invasion, and stimulation of proliferation, were determined. With plasmon resonance, the interaction of full-length Eap isoforms (from strains Newman and Wood 46) with fibrinogen, fibronectin, vitronectin, and thrombospondin-1 was found to be specific but with different affinities for the ligands tested. In the ligand overlay assay, the interactions of five single tandem repeat domains (D1 to D5) of Eap-7 (from strain CI-7) with fibronectin, fibrinogen, vitronectin, thrombospondin-1, and collagen I differed substantially. Most prominently, D3 bound most strongly to fibronectin and fibrinogen. Full-length Eap, but none of the single tandem repeat domains, agglutinated S. aureus and enhanced adherence to and invasion of host cells by S. aureus. Constructs D3-4 and D1-3 (in cis) increased adherence and invasiveness compared to what was seen for single Eap tandem repeat domains. By contrast, single Eap tandem repeat domains and full-length Eap similarly modulated the proliferation of peripheral blood mononuclear cells (PBMCs): low concentrations stimulated, whereas high concentrations inhibited, proliferation. Taken together, the data indicate that Eap tandem repeat domains appear to have distinct characteristics for the binding of soluble ligands, despite a high degree of sequence similarity. In addition, more than one Eap tandem repeat domain is required for S. aureus agglutination, adherence, and cellular invasion but not for the stimulation of PBMC proliferation.     

Role of fibronectin-binding proteins A and B in in vitro cellular infections and in vivo septic infections by Staphylococcus aureus

Fibronectin-binding protein A (FnBPA) and FnBPB are important adhesins for Staphylococcus aureus infection. We constructed fnbA and/or fnbB mutant strains from S. aureus SH1000, which possesses intact rsbU, and studied the role of these adhesins in in vitro and in vivo infections. In intravenous infection, all fnb mutants caused a remarkable reduction in the colonization rate in kidneys and the mortality rate of mice. fnbB mutant caused a more severe decrease in body weight than that caused by fnbA mutant. Serum levels of interleukin-6 and nuclear factor κB (NF-κB) activation in spleen cells were remarkably reduced in fnbA or fnbA fnbB mutant infections; however, there was no significant reduction in fnbB mutant infections. In in vitro cellular infection, FnBPA was shown to be indispensable for adhesion to and internalization by nonprofessional phagocytic cells upon ingestion by inflammatory macrophages and NF-κB activation. However, both FnBPs were required for efficient cellular responses. The results showed that FnBPA is more important for in vitro and in vivo infections; however, cooperation between FnBPA and FnBPB is indispensable for the induction of severe infection resulting in septic death.     

Physiological osmotic induction of Leptospira interrogans adhesion: LigA and LigB bind extracellular matrix proteins and fibrinogen

Transmission of leptospirosis occurs through contact of mucous membranes and abraded skin with freshwater contaminated by pathogenic Leptospira spp. Exposure to physiological osmolarity induces leptospires to express high levels of the Lig surface proteins containing imperfect immunoglobulin-like repeats that are shared or differ between LigA and LigB. We report that osmotic induction of Lig is accompanied by 1.6- to 2.5-fold increases in leptospiral adhesion to immobilized extracellular matrix and plasma proteins, including collagens I and IV, laminin, and especially fibronectin and fibrinogen. Recombinant LigA-unique and LigB-unique repeat proteins bind to these same host ligands. We found that the avidity of LigB in binding fibronectin is comparable to that of the Staphylococcus aureus FnBPA D repeats. Both LigA- and LigB-unique repeats interact with the amino-terminal fibrin- and gelatin-binding domains of fibronectin, which are also recognized by fibronectin-binding proteins mediating the adhesion of other microbial pathogens. In contrast, repeats common to both LigA and LigB do not bind these host proteins, and nonrepeat sequences in the carboxy-terminal domain of LigB show only weak interaction with fibronectin and fibrinogen. A functional role for the binding activity of LigA and LigB is suggested by the ability of the recombinants to inhibit leptospiral adhesion to fibronectin by 28% and 21%, respectively. The binding of LigA and LigB to multiple ligands present in different tissues suggests that these adhesins may be involved in the initial colonization and dissemination stages of leptospirosis. The characterization of the Lig adhesin function should aid the design of Lig-based vaccines and serodiagnostic tests.     

Heparan sulfate proteoglycans mediate Staphylococcus aureus interactions with intestinal epithelium

Staphylococcus aureus can be internalized by non-professional phagocytes, and may colonize the intestine in normal and antibiotic-treated individuals. Intestinal colonization may depend on the interactions of S. aureus with the intestinal epithelium. The best described mechanism of S. aureus binding to eukaryotic cells involves S. aureus fibronectin binding proteins (FnBPs), using fibronectin as a bridging molecule to β1 integrins on the eukaryotic cell surface. Because S. aureus can be internalized by enterocytes, and because S. aureus is known to bind heparan sulfate (HS), we hypothesized that heparan sulfate proteoglycans (HSPGs) widely expressed on epithelia may mediate S. aureus interactions with intestinal epithelial cells. Internalization of S. aureus RN6390 by cultured intestinal epithelial cells was inhibited in a dose-dependent fashion by the HS mimic heparin, and by HS itself. Internalization of S. aureus DU5883, which lacks expression of staphylococcal FnBPs, was also inhibited by heparin. S. aureus adherence to ARH-77 cells, transfected to express the HSPG syndecan-1, was greatly increased when compared to adherence to plasmid control ARH-77 cells which have little detergent extractable HS. In addition, compared to wild-type HS-expressing Chinese hamster ovary (CHO) cells, internalization of S. aureus was decreased using mutant CHO cells with decreased HS expression. These findings are consistent with a model wherein S. aureus internalization by intestinal epithelial cells (and perhaps other epithelia) is mediated by S. aureus binding to the HS moiety of cell-surface HSPGs, and this interaction appears independent of fibronectin binding.     

The fibrinogen- and fibronectin-binding domains of Staphylococcus aureus fibronectin-binding protein A synergistically promote endothelial invasion and experimental endocarditis

Staphylococcus aureus experimental endocarditis relies on sequential fibrinogen binding (for valve colonization) and fibronectin binding (for endothelial invasion) conferred by peptidoglycan-attached adhesins. Fibronectin-binding protein A (FnBPA) reconciles these two properties—as well as elastin binding—and promotes experimental endocarditis by itself. Here we attempted to delineate the minimal subdomain of FnBPA responsible for fibrinogen and fibronectin binding, cell invasion, and in vivo endocarditis. A large library of truncated constructs of FnBPA was expressed in Lactococcus lactis and tested in vitro and in animals. A 127-amino-acid subdomain spanning the hinge of the FnBPA fibrinogen-binding and fibronectin-binding regions appeared necessary and sufficient to confer the sum of these properties. Competition with synthetic peptides could not delineate specific fibrinogen- and fibronectin-binding sites, suggesting that dual binding arose from protein folding, irrespective of clearly defined binding domains. Moreover, coexpressing the 127-amino-acid subdomain with remote domains of FnBPA further increased fibrinogen binding by ≥10 times, confirming the importance of domain interactions for binding efficacy. In animals, fibrinogen binding (but not fibronectin binding) was significantly associated with endocarditis induction, whereas both fibrinogen binding and fibronectin binding were associated with disease severity. Moreover, fibrinogen binding also combined with fibronectin binding to synergize the invasion of cultured cell lines significantly, a feature correlating with endocarditis severity. Thus, while fibrinogen binding and fibronectin binding were believed to act sequentially in colonization and invasion, they appeared unexpectedly intertwined in terms of both functional anatomy and pathogenicity (in endocarditis). This unforeseen FnBPA subtlety might bear importance for the development of antiadhesin strategies.     

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.     

Identification of a fibronectin-binding protein from Staphylococcus epidermidis

Staphylococcus epidermidis has been reported to bind to a number of host cell extracellular matrix proteins, including fibronectin. Here we report the identification of a fibronectin-binding protein from S. epidermidis. A phage display library of S. epidermidis genomic DNA was constructed and panned against immobilized fibronectin. A number of phagemid clones containing overlapping inserts were identified, and one of these clones, pSE109FN, contained a 1.4-kb insert. Phage pSE109FN was found to bind to fibronectin but not to collagen, fibrinogen, laminin, or vitronectin. However, pSE109FN also bound to heparin, hyaluronate, and plasminogen, although to a lesser extent than it bound to fibronectin. Analysis of The Institute for Genomic Research S. epidermidis genome sequence database revealed a 1.85-kb region within a putative 30.5-kb open reading frame, to which the overlapping DNA inserts contained within the fibronectin-binding phagemids mapped. We have designated the gene encoding the fibronectin-binding domain embp. A recombinant protein, Embp32, which encompassed the fibronectin-binding domain of Embp, blocked the binding of S. epidermidis, but not the binding of Staphylococcus aureus, to fibronectin. In contrast, a recombinant protein, FnBPB[D1-D4], spanning the fibronectin-binding domain of the S. aureus fibronectin-binding protein FnBPB, blocked binding of S. aureus to fibronectin but had a negligible effect on the binding of S. epidermidis.     

Internalization of Staphylococcus aureus by human keratinocytes

Staphylococcus aureus is among the most important human pathogens and causes various superficial and systemic infections. The ability of S. aureus to be internalized by, and survive within, host cells, such as keratinocytes, may contribute to the development of persistent or chronic infections and may finally lead to deeper tissue infections or dissemination. To examine the mechanisms of internalization of S. aureus by keratinocytes, isogenic mutants lacking fibronectin-binding proteins (FnBPs), a recombinant protein consisting of the fibronectin-binding domain of S. aureus FnBPs, and an anti-alpha5beta1 antibody were used in cocultures with immortalized keratinocytes and primary keratinocytes. We found that internalization of S. aureus by immortalized keratinocytes requires bacterial FnBPs and is mediated by the major fibronectin-binding integrin alpha5beta1. In contrast to internalization by immortalized keratinocytes, internalization of S. aureus by primary keratinocytes could occur through FnBP-dependent and -independent pathways. S. aureus clumping factor B (ClfB), which was recently determined to bind to epithelial cells, was not involved in the uptake of this bacterium by keratinocytes. The identification of an alternate uptake pathway, which is independent of S. aureus FnBPs and host cell alpha5beta1, has important implications for the design of therapies targeted to bacterial uptake by host cells.     

Fibronectin Binding Protein and Host Cell Tyrosine Kinase Are Required for Internalization of Staphylococcus aureus by Epithelial Cells

Staphylococcus aureus expresses several surface proteins that promote adherence to host cell extracellular matrix proteins, including fibronectin (Fn). Since this organism has recently been shown to be internalized by nonprofessional phagocytes, a process that typically requires high-affinity binding to host cell receptors, we investigated the role of its Fn binding proteins (FnBPs) and other surface proteins in internalization by the bovine mammary gland epithelial cell line (MAC-T). Efficient internalization of S. aureus 8325-4 required expression of FnBPs; an isogenic mutant (DU5883), not expressing FnBPs, was reduced by more than 95% in its ability to invade MAC-T cells. Moreover, D3, a synthetic peptide derived from the ligand binding domain of FnBP, inhibited the internalization of the 8325-4 strain in a dose-dependent fashion and the efficiency of staphylococcal internalization was partially correlated with Fn binding ability. Interestingly, Fn also inhibited the internalization and adherence of S. aureus 8325-4 in a dose-dependent manner. In contrast to internalization, adherence of DU5883 to MAC-T was reduced by only approximately 40%, suggesting that surface binding proteins, other than FnBPs, can mediate bacterial adherence to cells. Adherence via these proteins, however, does not necessarily result in internalization of the staphylococci. An inhibitor of protein tyrosine kinase, genistein, reduced MAC-T internalization of S. aureus by 95%, indicating a requirement for a host signal transduction system in this process. Taken together, these results indicate that S. aureus invades nonprofessional phagocytes by a mechanism requiring interaction between FnBP and the host cell, leading to signal transduction and subsequent rearrangement of the host cell cytoskeleton.     

Effect of deoxymannojirimycin and Brefeldin A onYersinia pseudotuberculosisinvasin–eukaryotic cell interaction

The cell surface receptors of theYersinia pseudotuberculosisinvasin protein are theα_3–6β₁integrins. Invasin and the extracellular matrix protein fibronectin bind to the same or closely located sites onα₅β₁integrin, although invasin bind with a much greater affinity. Invasin-integrin interaction promotes bacterial penetration into eukaryotic cells. Binding of fibronectin to its integrin receptor seems to be dependent on terminal oligosaccharides processing. In this paper, we have examined the effect of 1-deoxymannojirimycin (dMNJ), an inhibitor of Golgiα-mannosidases involved in processing of N-glycan precursors, and of Brefeldin A (BFA), a natural product of fungi which has profound effects on the structure and function of the Golgi apparatus, on invasin–cell interaction. We found that unlike fibronectin, the interaction of invasin with cells was resistance to dMNJ. However, preincubating cells with BFA caused a dose dependent inhibition of invasin-mediated cell entry, while cell invasion bySalmonella typhimuriumwas not affected.     

Fibronectin bound to the surface of Staphylococcus aureus induces association of very late antigen 5 and intracellular signaling factors with macrophage cytoskeleton

Staphylococcus aureus Cowan I and a clinically isolated coagulase-negative Staphylococcus strain, S. saprophyticus 10312, were found to have two fibronectin binding proteins, FnBPA and FnBPB. While both staphylococci bound to serum fibronectin to a similar extent, fibronectin binding significantly increased the phagocytic activity of macrophages against S. aureus (by ca. 150%) but not against S. saprophyticus. This enhancing effect of fibronectin was inhibited by an RGD sequence-containing peptide and also by anti-very late antigen 5 antibody. This suggests that the effect is mediated by very late antigen 5 expressed on macrophages. In macrophages ingesting fibronectin-bound Cowan I, alpha(5) and beta(1) chains were associated with the cytoskeleton. Cytosolic signaling factors such as paxillin, c-Src, and c-Csk were also associated with the cytoskeleton. On the contrary, beta(3) integrin transiently disappeared from the cytoskeleton when macrophages ingested the fibronectin-treated S. aureus Cowan I. Furthermore, the Src kinase family tyrosine kinase Lyn dissociated from the cytoskeleton. These cellular components did not respond in a fibronectin-dependent manner when macrophages phagocytosed S. saprophyticus. This means that only fibronectin-treated S. aureus Cowan I induces the accumulation of very late antigen 5, which in turn induces the association of paxillin and tyrosine kinases. It is thought that the phagocytic activity of macrophages against fibronectin-treated S. aureus was increased by signaling via the activation of very late antigen 5.     

Natural Variability of In Vitro Adherence to Fibrinogen and Fibronectin Does Not Correlate with In Vivo Infectivity of Staphylococcus aureus

Adherence to fibrinogen and fibronectin plays a crucial role in Staphylococcus aureus experimental endocarditis. Previous genetic studies have shown that infection and carriage isolates do not systematically differ in their virulence-related genes, including genes conferring adherence, such as clfA and fnbA. We set out to determine the range of adherence phenotypes in carriage isolates of S. aureus, to compare the adherence of these isolates to the adherence of infection isolates, and to determine the relationship between adherence and infectivity in a rat model of experimental endocarditis. A total of 133 healthy carriage isolates were screened for in vitro adherence to fibrinogen and fibronectin, and 30 isolates were randomly chosen for further investigation. These 30 isolates were compared to 30 infective endocarditis isolates and 30 blood culture isolates. The infectivities of the carriage isolates, which displayed either extremely low or high adherence to fibrinogen and fibronectin, were tested using a rat model of experimental endocarditis. The levels of adherence to both fibrinogen and fibronectin were very similar for isolates from healthy carriers and members of the two groups of infection isolates. All three groups of isolates showed a wide range of adherence to fibrinogen and fibronectin. Moreover, the carriage isolates that showed minimal adherence and the carriage isolates that showed strong adherence had the same infectivity in experimental endocarditis. Adherence was proven to be important for pathogenesis in experimental endocarditis, but even the least adherent carriage strains had the ability to induce infection. We discuss the roles of differential gene expression, human host factors, and gene redundancy in resolving this apparent paradox.Staphylococcus aureus is a human commensal, but at the same time it is one of the most important bacterial pathogens that cause community-acquired and nosocomial infections. It can produce a wide variety of diseases, from benign skin infections, such as folliculitis or furunculosis, to life-threatening conditions, like osteomyelitis, septic arthritis, sepsis, pneumonia, and endocarditis (12). About 20% of humans carry S. aureus permanently in their noses, and another 60% are intermittent carriers (16). The association between S. aureus nasal carriage and staphylococcal disease has been reported for several decades (43, 44). More recently, it has been unambiguously shown that carriers have a higher risk of infection, at least when they are hospitalized (41, 45).The pathogenicity of S. aureus involves a wide range of cell wall-associated adhesins and extracellular toxins. Surface adhesins, referred to as microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), bind to the host extracellular matrix and thus promote tissue colonization and infection (30). The major MSCRAMMs involved in S. aureus pathogenesis are particular surface proteins that are covalently bound to the cell wall peptidoglycan via a conserved LPXTG motif (22). Genomic analyses indicated that the S. aureus genome contains up to 21 such LPXTG surface proteins (38). In addition to their multiplicity, these proteins often have redundant functions, as exemplified by clumping factors A and B (ClfA and ClfB), which bind fibrinogen (1, 27), and fibronectin-binding proteins A and B (FnBPA and FnBPB), which bind fibronectin (21), fibrinogen (42), and elastin (37).It has been unambiguously demonstrated that in S. aureus ClfA and FnBPA are key pathogenicity factors, at least in infective endocarditis. This has been achieved by expressing these adhesins in bacteria lacking the rest of the S. aureus surface features (34). Using a variety of truncated and chimera constructs of these proteins, Que et al. observed that fibrinogen-binding domains were necessary and sufficient for colonization of damaged valves in experimental endocarditis, but not for persistence and invasion, whereas fibronectin-binding domains of FnBPA were unable to initiate infection but mediated aortic cell invasion and microbial persistence. Thus, the two adherence functions were necessary for progressive infection (35).Despite the great effort to establish whether there are specific genetic determinants that distinguish carriage and invasive infection strains, the answer was largely negative (8, 25). Genetic studies revealed that many S. aureus wild-type strains lack some of the genes coding for LPXTG motif proteins, but there is no overall difference between carriage and infection isolates (19). However, fnbA and clfA are nearly always present in carriage and clinical isolates, confirming their pivotal role. Moreover, sequence analysis of functional regions of the two proteins has shown that there is a high degree of conservation in sporadic and epidemic isolates of S. aureus (17). On the other hand, the presence of the genes does not imply that there is efficient expression of a protein on the cell surface. It is entirely possible that either the carriage isolates express FnBPA and ClfA at a lower level or the adherence to the host matrix is less efficient. To our knowledge, a comparison of the adherence phenotypes of infection and carriage isolates has never been conducted.Here we determined the levels of adherence to fibrinogen and fibronectin of 133 carriage isolates. We compared these isolates to 30 infective endocarditis and 30 blood culture isolates. In addition, we compared the infectivities of isolates displaying extreme adherence phenotypes in a rat model of experimental endocarditis.     

The involvement of an integrin-like protein and protein kinase C in amoebic adhesion to fibronectin and amoebic cytotoxicity

Adherence of a pathogen to the host cell is one of the critical steps in microbial infections. Naegleria fowleri, a causative agent of primary amoebic meningoencephalitis in humans, is expected to interact with extracellular components of the host, such as fibronectin, in a receptor-mediated mode. In this study, we investigated the interaction between N. fowleri and fibronectin to understand its cytopathology. In binding assays using immobilized fibronectin, the number of amoebae bound to fibronectin was increased compared to the controls, and was dependent on the amount of coated fibronectin present. A fibronectin binding protein of 60 kDa was found in extracts of N. fowleri. Western blot and immunolocalization assays using integrin ₅/FnR antibodies showed that a 60 kDa protein reacted with the antibodies in extracts of N. fowleri, which was localized on the surface of N. fowleri. Preincubation of N. fowleri with the integrin antibodies significantly inhibited amoebic binding to fibronectin and cytotoxicity to the CHO cells. Additionally, protein kinase C activity was detected in the extract of N. fowleri. When N. fowleri was pretreated with protein kinase C activator or inhibitor, the abilities of amoebic adhesion to fibronectin and cytotoxicity to the host cells were markedly affected compared to untreated amoebae. These results suggest that an amoebic integrin-like receptor and protein kinase C play important roles in amoebic cellular processes in response to fibronectin.     

Both complement- and fibrinogen-dependent mechanisms contribute to platelet aggregation mediated by Staphylococcus aureus clumping factor B

Staphylococcus aureus can stimulate activation and aggregation of platelets, which are thought to be factors in the development of infective endocarditis. Previous studies have identified clumping factor A (ClfA) and fibronectin binding proteins A and B (FnBPA and FnBPB) as potent platelet aggregators. These proteins are able to stimulate rapid platelet aggregation by either a fibrinogen- or a fibronectin-dependent process which also requires antibodies specific to each protein. Slower aggregation has been seen in other systems where specific fibrinogen binding ligands are absent and platelet aggregation is mediated by complement and specific antibodies. Bacteria expressing ClfB aggregate platelets with a longer lag time than ClfA or FnBPA and FnBPB. In order to investigate whether ClfB causes platelet aggregation in a complement- or fibrinogen-dependent manner, a non-fibrinogen-binding mutant of ClfB (ClfB Q235A) was constructed. Lactococcus lactis expressing ClfB Q235A was able to stimulate platelet aggregation in platelet-rich plasma without a significant increase in lag time. The requirements for platelet aggregation were investigated using gel-filtered platelets. Fibrinogen and specific anti-ClfB antibodies were found to be sufficient to allow platelet aggregation mediated by the wild-type ClfB protein. It seems that ClfB causes platelet aggregation by a fibrinogen-dependent mechanism. The non-fibrinogen-binding ClfB mutant was unable to stimulate platelet aggregation under these conditions. However, bacteria expressing ClfB Q235A caused platelet aggregation in a complement-dependent manner which required specific anti-ClfB antibodies.     

The opportunistic human pathogenic fungus Aspergillus fumigatus evades the host complement system

The opportunistic human pathogenic fungus Aspergillus fumigatus causes severe systemic infections and is a major cause of fungal infections in immunocompromised patients. A. fumigatus conidia activate the alternative pathway of the complement system. In order to assess the mechanisms by which A. fumigatus evades the activated complement system, we analyzed the binding of host complement regulators to A. fumigatus. The binding of factor H and factor H-like protein 1 (FHL-1) from human sera to A. fumigatus conidia was shown by adsorption assays and immunostaining. In addition, factor H-related protein 1 (FHR-1) bound to conidia. Adsorption assays with recombinant factor H mutants were used to localize the binding domains. One binding region was identified within N-terminal short consensus repeats (SCRs) 1 to 7 and a second one within C-terminal SCR 20. Plasminogen was identified as the fourth host regulatory molecule that binds to A. fumigatus conidia. In contrast to conidia, other developmental stages of A. fumigatus, like swollen conidia or hyphae, did not bind to factor H, FHR-1, FHL-1, and plasminogen, thus indicating the developmentally regulated expression of A. fumigatus surface ligands. Both factor H and plasminogen maintained regulating activity when they were bound to the conidial surface. Bound factor H acted as a cofactor to the factor I-mediated cleavage of C3b. Plasminogen showed proteolytic activity when activated to plasmin by urokinase-type plasminogen activator. These data show that A. fumigatus conidia bind to complement regulators, and these bound host regulators may contribute to evasion of a host complement attack.     

Moraxella catarrhalis Binds Plasminogen To Evade Host Innate Immunity

Several bacterial species recruit the complement regulators C4b-binding protein, factor H, and vitronectin, resulting in resistance against the bactericidal activity of human serum. It was recently demonstrated that bacteria also bind plasminogen, which is converted to plasmin that degrades C3b and C5. In this study, we found that a series of clinical isolates (n = 58) of the respiratory pathogen Moraxella catarrhalis, which is commonly isolated from preschool children and adults with chronic obstructive pulmonary disease (COPD), significantly binds human plasminogen. Ubiquitous surface protein A2 (UspA2) and hybrid UspA2 (UspA2H) were identified as the plasminogen-binding factors in the outer membrane proteome of Moraxella. Furthermore, expression of a series of truncated recombinant UspA2 and UspA2H proteins followed by a detailed analysis of protein-protein interactions suggested that the N-terminal head domains bound to the kringle domains of plasminogen. The binding affinity constant (K_D) values of full-length UspA2^30–539 (amino acids 30 to 539 of UspA2) and full-length UspA2H^50–720 for immobilized plasminogen were 4.8 × 10⁻⁸ M and 3.13 × 10⁻⁸ M, respectively, as measured by biolayer interferometry. Plasminogen bound to intact M. catarrhalis or to recombinant UspA2/UspA2H was readily accessible for a urokinase plasminogen activator that converted the zymogen into active plasmin, as verified by the specific substrate S-2251 and a degradation assay with fibrinogen. Importantly, plasmin bound at the bacterial surface also degraded C3b and C5, which consequently may contribute to reduced bacterial killing. Our findings suggest that binding of plasminogen to M. catarrhalis may lead to increased virulence and, hence, more efficient colonization of the host.