Identification of molecular mechanisms used by Finegoldia magna to penetrate and colonize human skin

Finegoldia magna is a Gram‐positive anaerobic commensal of the human skin microbiota, but also known to act as an opportunistic pathogen. Two primary virulence factors of F. magna are the subtilisin‐like extracellular serine protease SufA and the adhesive protein FAF. This study examines the molecular mechanisms F. magna uses when colonizing or establishing an infection in the skin. FAF was found to be essential in the initial adherence of F. magna to human skin biopsies. In the upper layers of the epidermis FAF mediates adhesion through binding to galectin‐7 – a keratinocyte cell marker. Once the bacteria moved deeper into the skin to the basement membrane layer, SufA was found to degrade collagen IV which forms the backbone structure of the basement membrane. It also degraded collagen V, whereby F. magna could reach deeper dermal tissue sites. In the dermis, FAF interacts with collagen V and fibrillin, which presumably helps the bacteria to establish infection in this area. The findings of this study paint a clear picture of how F. magna interacts with human skin and explain how it is such a successful opportunistic pathogen in chronic wounds and ulcers.     

Constitutive and inflammation-dependent antimicrobial peptides produced by epithelium are differentially processed and inactivated by the commensal Finegoldia magna and the pathogen Streptococcus pyogenes

Epithelial linings serve as physical barriers and produce antimicrobial peptides (AMPs) to maintain host integrity. Examples are the bactericidal proteins midkine (MK) and BRAK/CXCL14 that are constitutively produced in the skin epidermal layer, where the anaerobic Gram-positive coccoid commensal Finegoldia magna resides. Consequently, this bacterium is likely to encounter both MK and BRAK/CXCL14, making these molecules possible threats to its habitat. In this study, we show that MK expression is upregulated during inflammation, concomitant with a strong downregulation of BRAK/CXCL14, resulting in changed antibacterial conditions. MK, BRAK/CXCL14, and the inflammation-dependent antimicrobial β-defensins human β-defensin (hBD)-2 and hBD-3 all showed bactericidal activity against both F. magna and the virulent pathogen Streptococcus pyogenes at similar concentrations. SufA, a released protease of F. magna, degraded MK and BRAK/CXCL14 but not hBD-2 nor hBD-3. Cleavage was seen at lysine and arginine residues, amino acids characteristic of AMPs. Intermediate SufA-degraded fragments of MK and BRAK/CXCL14 showed stronger bactericidal activity against S. pyogenes than F. magna, thus promoting survival of the latter. In contrast, the cysteine-protease SpeB of S. pyogenes rapidly degraded all AMPs investigated. The proteins FAF and SIC, released by F. magna and S. pyogenes, respectively, neutralized the antibacterial activity of MK and BRAK/CXCL14, protein FAF being the most efficient. Quantitation and colocalization by immunoelectron microscopy demonstrated significant levels and interactions of the molecules in in vivo and ex vivo samples. The findings reflect strategies used by a permanently residing commensal and a virulent pathogen, the latter operating during the limited time course of invasive disease.     

Mouse AMACO,a kidney and skin basement membrane associated molecule that mediates RGD-dependent cell attachment

The VWA domain-containing extracellular matrix protein AMACO has not been extensively characterized and its function remains unknown. It has been proposed as a potential cancer marker and carries a rare O-glucosylation and O-fucosylation on its first EGF-like domain. AMACO is a basement membrane associated protein, however its exact localization has not been determined. Here we show by immunogold electron microscopy of mouse kidney and skin that AMACO does not occur within the basement membrane but rather subjacent to the basement membrane at its stromal surface. In skin, AMACO often colocalizes with triple-helical domains of collagen VII containing anchoring fibrils as they emerge from the basal lamina. However, the immunogold patterns for AMACO and the C-terminal end of collagen VII show discrete differences, indicating that AMACO and collagen VII do not colocalize at anchoring plaques. In contrast, the localization pattern of AMACO partially overlaps with that for collagen XVIII. In addition, mouse AMACO was shown to support β1 integrin-mediated adhesion of a keratinocyte-like cell line, HaCaT, and a fibroblast cell line, Wi26, in an RGD-dependent manner, most likely using an RGD-motif near the C-terminus of AMACO. However, the loss of cell adhesion to the C-terminal part of the human AMACO, due to the unique absence of an RGD sequence in the human protein, suggests that cell adhesion is not AMACO's major function.     

Crystal structure and biological implications of a bacterial albumin binding module in complex with human serum albumin

Many bactericide species express surface proteins that interact with human serum albumin (HSA). Protein PAB from the anaerobic bacterium Finegoldia magna (formerly Peptostreptococcus magnus) represents one of these proteins. Protein PAB contains a domain of 53 amino acid residues known as the GA module. GA homologs are also found in protein G of group C and G streptococci. Here we report the crystal structure of HSA in complex with the GA module of protein PAB. The model of the complex was refined to a resolution of 2.7 A and reveals a novel binding epitope located in domain II of the albumin molecule. The GA module is composed of a left-handed three-helix bundle, and residues from the second helix and the loops surrounding it were found to be involved in HSA binding. Furthermore, the presence of HSA-bound fatty acids seems to influence HSA-GA complex formation. F. magna has a much more restricted host specificity compared with C and G streptococci, which is also reflected in the binding of different animal albumins by proteins PAB and G. The structure of the HSA-GA complex offers a molecular explanation to this unusually clear example of bacterial adaptation.     

Identification of a novel cell-adhesive protein spatiotemporally expressed in the basement membrane of mouse developing hair follicle

We used PCR-based cDNA subtraction to screen for genes up-regulated during mouse hair morphogenesis. One gene selected was predominantly expressed at the tip of developing hair follicles and encoded a protein characterized by the presence of twelve tandem repeats of approximately 120 amino acids and a novel N-terminal domain containing an Arg-Gly-Asp cell-adhesive motif. Immunohistochemistry demonstrated that the protein encoded by this gene, named QBRICK, was localized at the basement membrane zone of embryonic epidermis and hair follicles, in which it was more enriched at the tip rather than the stalk region. Cell adhesion assays showed that QBRICK was active in mediating cell-substratum adhesion through integrins containing alphav or alpha8 chain, but not integrin alpha5beta1. Immunohistochemistry showed that QBRICK colocalized with alphav-containing integrins in the interfollicular region, but with the alpha8-containing integrin at the tip region of developing hair follicles. These results, together, indicate that QBRICK is an adhesive ligand of basement membrane distinctively recognized by cells in the embryonic skin and hair follicles through different types of integrins directed to the Arg-Gly-Asp motif.     

Human keratinocytes cultured on collagen gels form an epidermis which synthesizes bullous pemphigoid antigens and alpha 2 beta 1 integrins and secretes laminin, type IV collagen, and heparan sulfate proteoglycan at the basal cell surface

Single cell suspensions of human keratinocytes when seeded onto floating three-dimensional gels constructed with type I collagen form a tissue resembling epidermis. These morphogenetic events occur in a serum-free environment in the absence of fibroblasts. Light and transmission electron microscopy show that cells form a basal layer plus suprabasilar cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasilar keratinocyte layers show morphologies which resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-fillagrin granules. The basal cell layer differs from skin in vivo in that there is no connection to a basement membrane via hemidesmosomes. Cells in the basal layers are polarized as evidenced by the secretion of type IV collagen, heparan sulfate proteoglycans, and laminin at the cell membrane interface with the collagen gel. These proteins are not organized into a cytological basement membrane. Bullous pemphigoid antigen, a protein component of hemidesmosomes, is synthesized by basal keratinocytes, but like the basement membrane proteins it is not incorporated into a definable cytological structure. Keratinocytes in the basal and suprabasilar layers also synthesize alpha 2 beta 1 integrins. The mechanisms of keratinocyte adhesion to the gel may be through the interactions of this cell surface receptor with laminin and type IV collagen synthesized by the cell and/or direct interactions between the receptor and type I collagen within the gel. This in vitro experimental system is a useful model for defining the molecular events which control the formation and turnover of basement membranes and the mechanisms by which keratinocytes adhere to type I collagen when sheets of keratinocytes are used clinically for wound coverage.     

The adhesion of coagulase negative staphylococci to human skin and its relevance to the bacterial flora of milk

Twenty-eight isolates of coagulase negative staphylococci were obtained from nipple swabs provided by one non-lactating woman and five nursing mothers. All but two of these isolates were shown by scanning electron microscopy to adhere to the surface of human skin. Experiments with frozen sections of human skin confirmed and extended these results by showing that isolates exhibited one of three patterns of adhesion, suggesting that there are three different adhesion receptors on epidermal cells. It is proposed that adhesion of staphylococci to the nipple and areolar epidermis provides a mechanism whereby large numbers of bacteria, nourished by residues of milk and saliva, are maintained on the surface of the skin.     

The adhesion of coagulase negative staphylococci to human skin and its relevance to the bacterial flora of milk

Twenty-eight isolates of coagulase negative staphylococci were obtained from nipple swabs provided by one non-lactating woman and five nursing mothers. All but two of these isolates were shown by scanning electron microscopy to adhere to the surface of human skin. Experiments with frozen sections of human skin confirmed and extended these results by showing that isolates exhibited one of three patterns of adhesion, suggesting that there are three different adhesion receptors on epidermal cells. It is proposed that adhesion of staphylococci to the nipple and areolar epidermis provides a mechanism whereby large numbers of bacteria, nourished by residues of milk and saliva, are maintained on the surface of the skin.     

Expression of MAEG, a novel basement membrane protein, in mouse hair follicle morphogenesis

We screened for genes specifically expressed in the mesenchymes of developing hair follicles using representational differential analysis; one gene identified was MAEG, which encodes a protein consisting of five EGF-like repeats, a linker segment containing a cell-adhesive Arg-Gly-Asp (RGD) motif, and a MAM domain. Immunohistochemistry showed that MAEG protein was localized at the basement membrane of embryonic skin and developing hair follicles, while MAEG expression diminished at the tip of the hair bud. A recombinant MAEG fragment containing the RGD motif was active in mediating adhesion of keratinocytes to the substratum in an RGD-dependent manner. One of the adhesion receptors recognizing the RGD motif was found to be the alpha8beta1 integrin, the expression of which was detected in the placode close to MAEG-positive mesenchymal cells, but later became restricted to the tip of the developing hair bud. Given its localized expression at the basement membrane in developing hair follicles and the RGD-dependent cell-adhesive activity, MAEG may play a role as a mediator regulating epithelial-mesenchymal interaction through binding to RGD-binding integrins including alpha8beta1 during hair follicle development.     

Anti-anaerobic activity of serum from patients treated with tigecycline for skin/soft tissue infections

To gain additional data concerning the anti-anaerobic activity of tigecycline in serum, we analyzed blood samples from six patients with a complicated skin/soft tissue infection who were receiving IV tigecycline 50 mg every 12 h. Venous blood samples were obtained after multiple doses of tigecycline at 1, 6 and 12 h after the initiation of a 1 h IV infusion. Sera from these samples were tested to determine serum inhibitory and bactericidal activity over time against 4 anaerobic bacteria (Bacteroides fragilis, Peptoniphilus asaccharolyticus, Prevotella bivia and Finegoldia magna). An analysis of serum titers found that tigecycline exhibited early (1 h) and prolonged (12 h) inhibitory activity against each study isolate. Moreover, it provided bactericidal activity for 12 h against these strains with the exception of F. magna. Tigecycline was found to exhibit antibacterial activity at serum concentrations below the MICs of the anaerobic bacteria tested. This finding further supports that the antimicrobial activity of tigecycline can be greater than that suggested by the free fraction of drug and that serum appears to enhance this antibacterial activity.     

DNA-based identification of a hepatic trematode in an elk calf

Liver fluke infection was identified as a probable cause of clinical disease in an approximately 6-mo-old elk (Cervus elaphus) in coastal Oregon. Clinical pathology and necropsy findings are described. The alcohol-fixed flukes that were submitted for identification were similar in size to Fasciola hepatica, but their shape resembled Fascioloides magna in that they lacked a distinctive anterior cone. A few structures consistent with the eggs of F. magna were observed in liver lesions, suggesting that at least some of the worms were sexually maturing. Due to difficulties in morphologic identification associated with improper fixation technique, DNA analysis was used to compare small subunit (SSU) and internal transcribed spacer 2 ribosomal RNA gene sequences of the recovered parasites with those of F. hepatica and F. magna, confirming these small, but sexually mature flukes were F. magna. This is the first publication of the SSU gene sequence for F. magna. Phylogenetic analysis showed that it is related to, but is an outlier, to the genus Fasciola. Due to the high mortality rate associated with this disease outbreak, the overall significance of trematodiasis in the herd is unclear.     

Type XIII collagen: a novel cell adhesion component present in a range of cell–matrix adhesions and in the intercalated discs between cardiac muscle cells

Recent analysis of type XIII collagen surprisingly showed that it is anchored to the plasma membranes of cultured cells via a transmembrane segment near its amino terminus. Here we demonstrate that type XIII collagen is concentrated in cultured skin fibroblasts and several other human mesenchymal cell lines in the focal adhesions at the ends of actin stress fibers, co-localizing with the known focal adhesion components talin and vinculin. This co-occurrence was also observed in rapidly forming adhesive structures of spreading and moving fibroblasts and in disrupting focal adhesions following microinjection of the Rho-inhibitor C3 transferase into the cells, suggesting that type XIII collagen is an integral focal adhesion component. Moreover, it appears to have an adhesion-related function since cell-surface expression of type XIII collagen in cells with weak basic adhesiveness resulted in improved cell adhesion on selected culture substrata. In tissues type XIII collagen was found in a range of integrin-mediated adherens junctions including the myotendinous junctions and costameres of skeletal muscle as well as many cell–basement membrane interfaces. Some cell–cell adhesions were found to contain type XIII collagen, most notably the intercalated discs in the heart. Taken together, the results strongly suggest that type XIII collagen has a cell adhesion-associated function in a wide array of cell–matrix junctions.     

Plasminogen activation in degradation and penetration of extracellular matrices and basement membranes by invasive bacteria

Methods to assess in vitro the role of plasminogen activation in enterobacterial degradation of extracellular matrices and their protein components as well as in penetration through basement membrane are described. Development of these methods was initiated after the findings that enterobacterial surface structures (fimbriae and the Pla surface protease) function in plasminogen activation as well as in laminin- and/or fibronectin-specific adhesion. Enterobacteria with these properties degrade radiolabeled laminin as well as metabolically labeled extracellular matrix from cultured endothelial or epithelial cells. Plasmin-coated bacteria also penetrate through the reconstituted basement membrane preparation Matrigel. The processes are dependent on plasminogen activation by the invasive bacteria. The results suggest a pathogenic similarity between enterobacteria and tumor cells in cellular metastasis through tissue barriers.     

Phosphorylation of the Fas associated factor FAF1 by protein kinase CK2 and identification of serines 289 and 291 as the in vitro phosphorylation sites

We previously identified the human Fas associated factor (FAF1) as one of the interacting partners of protein kinase CK2 beta subunit. Since FAF1 is a phosphoprotein we investigated whether it is a substrate for CK2. Here, we report the full length human FAF1 cDNA sequence, expression of FAF1 in Escherichia coli and purification and characterization of FAF1 as a substrate for CK2. FAF1 as well as an N-terminal 40 kDa degradation product serve as substrates for both the recombinant CK2 holoenzyme (km 100 microM) and the isolated catalytic alpha subunit (km 200 microM). Despite the high k(m) values, we obtained evidence that CK2 is the major cellular kinase responsible for FAF1 phosphorylation, using tissue extracts as kinase sources. By MALDI-MS we identified the two serine residues at positions 289 and 291 as the major in vitro CK2 phosphorylation sites. These data may help us elucidate the functions of FAF1 and the involvement of CK2 mediated phosphorylation in processes such as apoptotic signaling, ubiquitination, nuclear translocation and embryonic development.     

Human keratinocytes cultured on collagen gels form an epidermis which synthesizes bullous pemphigoid antigens and α2β1 integrins and secretes laminin, type IV collagen, and heparan sulfate proteoglycan at the basal cell surface

Single cell suspensions of human keratinocytes when seeded onto floating three-dimensional gels constructed with type I collagen form a tissue resembling epidermis. These morphogenetic events occur in a serum-free environment in the absence of fibroblasts. Light and transmission electron microscopy show that cells form a basal layer plus suprabasilar cell layers corresponding to the stratum spinosum, stratum granulosum, and stratum corneum. The suprabasilar keratinocyte layers show morphologies which resemble intact skin in which cells are connected by desmosomes and contain intermediate filaments and keratohyalin-fillagrin granules. The basal cell layer differs from skin in vivo in that there is no connection to a basement membrane via hemidesmosomes. Cells in the basal layers are polarized as evidenced by the secretion of type IV collagen, heparan sulfate proteoglycans, and laminin at the cell membrane interface with the collagen gel. These proteins are not organized into a cytological basement membrane. Bullous pemphigoid antigen, a protein component of hemidesmosomes, is synthesized by basal keratinocytes, but like the basement membrane proteins it is not incorporated into a definable cytological structure. Keratinocytes in the basal and suprabasilar layers also synthesize α2β1 integrins. The mechanisms of keratinocyte adhesion to the gel may be through the interactions of this cell surface receptor with laminin and type IV collagen synthesized by the cell and/or direct interactions between the receptor and type I collagen within the gel. This in vitro experimental system is a useful model for defining the molecular events which control the formation and turnover of basement membranes and the mechanisms by which keratinocytes adhere to type I collagen when sheets of keratinocytes are used clinically for wound coverage.     

Functional analysis of posttranslational cleavage products of the neuron-glia cell adhesion molecule, Ng-CAM

Neuron-glia cell adhesion molecule (Ng-CAM) mediates cell adhesion between neurons homophilically and between neurons and glia heterophilically; it also promotes neurite outgrowth. In the chick brain, Ng-CAM is detected as glycoproteins of 190 and 210 kD (Ng- CAM200) with posttranslational cleavage products of 135 kD (F135, which contains most of the extracellular region) and 80 kD (F80, which includes the transmembrane and the cytoplasmic domains). To examine the functions of each of these components, we have expressed Ng-CAM200, F135, and F80 in murine L cells, and F135 and F80 as GST fusion proteins in the pGEX vector in bacteria. Appropriately transfected L cells expressed each of these proteins on their surfaces; F135 was also found in the media of cells transfected with Ng-CAM200 and F135. In addition to binding homophilically, cells transfected with Ng-CAM200 and F135 bound heterophilically to untransfected L cells, suggesting that there is a ligand for Ng-CAM on fibroblasts that may be related to the glial ligand. Detailed studies using the transfected cells and the fusion proteins indicated that both the homophilic and the heterophilic binding activities of Ng-CAM are localized in the F135 fragment of the molecule. The results also indicated that proteolytic cleavage of Ng- CAM200 is not required either for its expression on the cell surface or for cell adhesion and that there is an "anchor" for F135 on L cells (and presumably on neurons). In contrast to the cell binding results, the F80 but not the F135 fusion protein enhanced the outgrowth of neurites from dorsal root ganglion cells; this activity was associated with the FnIII repeats of F80. The observations that a protein corresponding to F135 contains the cell aggregation sites whereas one corresponding to the F80 has the ability to promote neurite outgrowth suggest that proteolytic cleavage may be an important event in regulating these Ng-CAM activities during embryonic development and neural regeneration.     

Spatiotemporal expression of periostin during skin development and incisional wound healing: lessons for human fibrotic scar formation

Differentiation of fibroblasts to myofibroblasts and collagen fibrillogenesis are two processes essential for normal cutaneous development and repair, but their misregulation also underlies skin-associated fibrosis. Periostin is a matricellular protein normally expressed in adult skin, but its role in skin organogenesis, incisional wound healing and skin pathology has yet to be investigated in any depth. Using C57/BL6 mouse skin as model, we first investigated periostin protein and mRNA spatiotemporal expression and distribution during development and after incisional wounding. Secondarily we assessed whether periostin is expressed in human skin pathologies, including keloid and hypertrophic scars, psoriasis and atopic dermatitis. During development, periostin is expressed in the dermis, basement membrane and hair follicles from embryonic through neonatal stages and in the dermis and hair follicle only in adult. In situ hybridization demonstrated that dermal fibroblasts and basal keratinocytes express periostin mRNA. After incisional wounding, periostin becomes re-expressed in the basement membrane within the dermal-epidermal junction at the wound edge re-establishing the embryonic deposition pattern present in the adult. Analysis of periostin expression in human pathologies demonstrated that it is over-expressed in keloid and hypertrophic scars, atopic dermatitis, but is largely absent from sites of inflammation and inflammatory conditions such as psoriasis. Furthermore, in vitro we demonstrated that periostin is a transforming growth factor beta 1 inducible gene in human dermal fibroblasts. We conclude that periostin is an important ECM component during development, in wound healing and is strongly associated with pathological skin remodeling.     

Isolation of a hemidesmosome-rich fraction from a human squamous cell carcinoma cell line

Hemidesmosomes are cell-to-matrix adhesion complexes anchoring keratinocytes to basement membranes. For the first time, we present a method to prepare a fraction from human cultured cells that are highly enriched in hemidesmosomal proteins. Using DJM-1 cells derived from human squamous cell carcinoma, accumulation of hemidesmosomes was observed when these cells were cultured for more than 10 days in a commercial serum-free medium without supplemental calcium. Electron microscopy demonstrated that numerous electron-dense adhesion structures were present along the basal cell membranes of DJM-1 cells cultured under the aforementioned conditions. After removing cellular materials using an ammonia solution, hemidesmosomal proteins and deposited extracellular matrix were collected and separated by electrophoresis. There were eight major polypeptides, which were determined to be plectin, BP230, BP180, integrin α6 and β4 subunits, and laminin-332 by immunoblotting and mass spectrometry. Therefore, we designated this preparation as a hemidesmosome-rich fraction. This fraction contained laminin-332 exclusively in its unprocessed form, which may account for the promotion of laminin deposition, and minimal amounts of Lutheran blood group protein, a nonhemidesmosomal transmembrane protein. This hemidesmosome-rich fraction would be useful not only for biological research on hemidesmosomes but also for developing a serum test for patients with blistering skin diseases.     

Kindlin-1 is a phosphoprotein involved in regulation of polarity, proliferation, and motility of epidermal keratinocytes

A novel family of focal adhesion proteins, the kindlins, is involved in attachment of the actin cytoskeleton to the plasma membrane and in integrin-mediated cellular processes. Deficiency of kindlin-1, as a result of loss-of-function mutations in the KIND1 gene, causes Kindler syndrome, an autosomal recessive genodermatosis characterized by skin blistering, progressive skin atrophy, photosensitivity and, occasionally, carcinogenesis. Here we characterized authentic and recombinantly expressed kindlin-1 and show that it is localized in basal epidermal keratinocytes in a polar fashion, close to the cell surface facing the basement membrane, in the areas between the hemidesmosomes. We identified two forms of kindlin-1 in keratinocytes, with apparent molecular masses of 78 and 74 kDa, corresponding to phosphorylated and desphosphorylated forms of the protein. In kindlin-1-deficient skin, basal keratinocytes show multiple abnormalities: cell polarity is lost, proliferation is strongly reduced, and several cells undergo apoptosis. In vitro, deficiency of kindlin-1 in keratinocytes leads to strongly reduced cell proliferation, decreased adhesion, undirected motility, and intense protrusion activity of the plasma membrane. Taken together, these results show that kindlin-1 plays a role in keratinocyte adhesion, polarization, proliferation, and migration. It is involved in organization and anchorage of the actin cytoskeleton to integrin-associated signaling platforms.     

Expression of the Opc protein correlates with invasion of epithelial and endothelial cells by Neisseria meningitidis

Whereas capsulate strains of Neisseria meningitidis are dependent on pili for adhesion to human endothelial and epithelial cells, strains which lacked assembled pili and were partially capsule-deficient adhered to and invaded human endothelial and epithelial cells if they expressed the Opc protein. Bacteria expressing low or undetectable levels of Opc protein failed to adhere to or invade eukaryotic cells. In addition, the presence of OpaAC751 protein on the surface of bacteria did not increase bacterial interactions with host cells. Association of Opc-expressing bacteria was inhibited by antibodies against Opc. Invasion was dependent on the host-cell cytoskeletal activity and was inhibited by cytochalasin D. In some cells, infected at the apical surface, bacteria emerging from basal surface were detected by electron microscopy. Opc is found in diverse meningococci and may represent a common virulence factor which facilitates adherence and invasion by these bacteria.