DMBT1 functions as pattern-recognition molecule for poly-sulfated and poly-phosphorylated ligands.

Deleted in malignant brain tumors 1 (DMBT1) is a secreted glycoprotein displaying a broad bacterial-binding spectrum. Recent functional and genetic studies linked DMBT1 to the suppression of LPS-induced TLR4-mediated NF-kappaB activation and to the pathogenesis of Crohn's disease. Here, we aimed at unraveling the molecular basis of its function in mucosal protection and of its broad pathogen-binding specificity. We report that DMBT1 directly interacts with dextran sulfate sodium (DSS) and carrageenan, a structurally similar sulfated polysaccharide, which is used as a texturizer and thickener in human dietary products. However, binding of DMBT1 does not reduce the cytotoxic effects of these agents to intestinal epithelial cells in vitro. DSS and carrageenan compete for DMBT1-mediated bacterial aggregation via interaction with its bacterial-recognition motif. Competition and ELISA studies identify poly-sulfated and poly-phosphorylated structures as ligands for this recognition motif, such as heparansulfate, LPS, and lipoteichoic acid. Dose-response studies in Dmbt1(-/-) and Dmbt1(+/+) mice utilizing the DSS-induced colitis model demonstrate a differential response only to low but not to high DSS doses. We propose that DMBT1 functions as pattern-recognition molecule for poly-sulfated and poly-phosphorylated ligands providing a molecular basis for its broad bacterial-binding specificity and its inhibitory effects on LPS-induced TLR4-mediated NF-kappaB activation.

Bactericidal activity of LFchimera is stronger and less sensitive to ionic strength than its constituent lactoferricin and lactoferrampin peptides.

The innate immunity factor lactoferrin harbours two antimicrobial moieties, lactoferricin and lactoferrampin, situated in close proximity in the N1 domain of the molecule. Most likely they cooperate in many of the beneficial activities of lactoferrin. To investigate whether chimerization of both peptides forms a functional unit we designed a chimerical structure containing lactoferricin amino acids 17-30 and lactoferrampin amino acids 265-284. The bactericidal activity of this LFchimera was found to be drastically stronger than that of the constituent peptides, as was demonstrated by the need for lower dose, shorter incubation time and less ionic strength dependency. Likewise, strongly enhanced interaction with negatively charged model membranes was found for the LFchimera relative to the constituent peptides. Thus, chimerization of the two antimicrobial peptides resembling their structural orientation in the native molecule strikingly improves their biological activity.

A common binding motif for various bacteria of the bacteria-binding peptide SRCRP2 of DMBT1/gp-340/salivary agglutinin.

Abstract Salivary agglutinin (DMBT1SAG) is identical to lung glycoprotein-340 and encoded by deleted in malignant brain tumors-1. It is a member of the scavenger receptor cysteine-rich (SRCR) superfamily, proteins that have one or more SRCR domains. Salivary agglutinin plays a role in oral innate immunity by the binding and agglutination of oral streptococci. S. mutans has been shown to bind to a 16-mer peptide (QGRVEVLYRGSWGTVC) located within the SRCR domains. Within this peptide, designated SRCR Peptide 2, residues VEVL and W were critical for binding. The aim of this study was to investigate binding of DMBT1SAG to other bacteria. Therefore, interaction between a series of bacteria and DMBT1(SAG), SRCR peptide 2 and its alanine substitution variants was studied in adhesion and agglutination assays. For different bacteria there was a highly significant correlation between adhesion to DMBT1SAG and adhesion to SRCR peptide 2 suggesting that SRCR peptide 2 is the major bacteria binding site. An alanine substitution scan showed that 8 amino acids were involved in binding (xRVEVLYxxSWxxxx). The binding motifs varied for different species were found, but the residues VxVxY and W were always present. In conclusion, a common binding motif (RVEVLYxxxSW) within the SRCR domains is responsible for the broad bacteria-binding spectrum of DMBT1SAG.

Salivary agglutinin/glycoprotein-340/DMBT1: a single molecule with variable composition and with different functions in infection, inflammation and cancer.

Salivary agglutinin (SAG), lung glycoprotein-340 (gp-340) and Deleted in Malignant Brain Tumours 1 (DMBT1) are three names for identical proteins encoded by the dmbt1 gene. DMBT1/SAG/gp-340 belongs to the scavenger receptor cysteine-rich (SRCR) superfamily of proteins, a superfamily of secreted or membrane-bound proteins with SRCR domains that are highly conserved down to sponges, the most ancient metazoa. On the one hand, DMBT1 may represent an innate defence factor acting as a pattern recognition molecule. It interacts with a broad range of pathogens, including cariogenic streptococci and Helicobacter pylori, influenza viruses and HIV, but also with mucosal defence proteins, such as IgA, surfactant proteins and MUC5B. Stimulation of alveolar macrophage migration, suppression of neutrophil oxidative burst and activation of the complement cascade point further to an important role in the regulation of inflammatory responses. On the other hand, DMBT1 has been demonstrated to play a role in epithelial and stem cell differentiation. Inactivation of the gene coding for this protein may lead to disturbed differentiation, possibly resulting in tumour formation. These data strongly point to a role for DMBT1 as a molecule linking innate immune processes with regenerative processes.

Antimicrobial peptides: review of their application in musculoskeletal infections.

Antimicrobial resistance is expected to increase the burden of osteomyelitis drastically. The rise in resistant bacterial strains is driving researchers to find new treatment options. As a potential new antibiotic class, antimicrobial peptides (AMPs) combine several attractive intrinsic properties. Their minimal propensity for inducing antimicrobial resistance could be of particular clinical significance. AMPs act as an essential part of the innate immune system and have been identified in virtually all forms of life. These short, positively charged peptides have a combined pore-forming and intracellular killing effect on a broad range of microorganisms. Their reported spectrum of action includes resistant bacterial strains, viruses, and fungi. Moreover, immunomodulating, antitumoric, and angiogenic mechanisms have been reported. We have designed degradable and nondegradable drug-release systems for local treatment with AMPs. In animal models of osteomyelitis, these systems reduced bone infection caused by both resistant and nonresistant strains. The systemic application of several peptides for experimental detection and treatment of bone and soft-tissue infection is also discussed in this review. Radioactive-labeled peptides have accurately discriminated sterile inflammation from active infection in imaging studies. Successful preclinical studies of AMPs indicate that clinical evaluation of these powerful antibiotic agents is in order.

Effect of amino acid substitutions on the candidacidal activity of LFampin 265-284.

LFampin 265-284, derived from bovine lactoferrin, has broad-spectrum antimicrobial activity against the yeast Candida albicans and several Gram-positive and Gram-negative bacteria. A glycine substitution scan was used to identify residues that are important for its candidacidal activity. Each single substitution of a positively charged residue led to considerable reduction in candidacidal activity, for each residue to a different extent. Substitution within the helix-facilitating N-terminal sequence DLIW had less severe effect; substitution of Ile and Trp led to a somewhat reduced potency. No substantial effects were found on the propensity to adopt a helical structure or to bind to C. albicans cells.

Bacteria binding by DMBT1/SAG/gp-340 is confined to the VEVLXXXXW motif in its scavenger receptor cysteine-rich domains.

The scavenger receptor cysteine-rich (SRCR) proteins form an archaic group of metazoan proteins characterized by the presence of SRCR domains. These proteins are classified in group A and B based on the number of conserved cysteine residues in their SRCR domains, i.e. six for group A and eight for group B. The protein DMBT1 (deleted in malignant brain tumors 1), which is identical to salivary agglutinin and lung gp-340, belongs to the group B SRCR proteins and is considered to be involved in tumor suppression and host defense by pathogen binding. In a previous study we used nonoverlapping synthetic peptides covering the SRCR consensus sequence to identify a 16-amino acid bacteria-binding protein loop (peptide SRCRP2; QGRVEVLYRGSWGTVC) within the SRCR domains. In this study, using overlapping peptides, we pinpointed the minimal bacteria-binding site on SRCRP2, and thus DMBT1, to an 11-amino acid motif (DMBT1 pathogen-binding site 1 or DMBT1pbs1; GRVEVLYRGSW). An alanine substitution scan revealed that VEVL and Trp are critical residues in this motif. Bacteria binding by DMBT1pbs1 was different from the bacteria binding by the macrophage receptor MARCO in which an RXR motif was critical. In addition, the homologous consensus sequences of a number of SRCR proteins were synthesized and tested for bacteria binding. Only consensus sequences of DMBT1 orthologues bound bacteria by this motif.

A peptide domain of bovine milk lactoferrin inhibits the interaction between streptococcal surface protein antigen and a salivary agglutinin peptide domain.

The peptide domain of salivary agglutinin responsible for its interaction with cell surface protein antigen (PAc) of Streptococcus mutans or bovine lactoferrin was found in the same peptide, scavenger receptor cysteine-rich domain peptide 2 (SRCRP2). Inhibition studies suggest that PAc and lactoferrin, of which residues 480 to 492 seem important, competitively bind to the SRCRP2 domain of salivary agglutinin.

Binding of salivary agglutinin to IgA.

SAG (salivary agglutinin), which is identical to gp-340 (glycoprotein-340) from the lung, is encoded by DMBT1 (deleted in malignant brain tumours 1). It is a member of the SRCR (scavenger receptor cysteine-rich) superfamily and contains 14 SRCR domains, 13 of which are highly similar. SAG in saliva is partially complexed with IgA, which may be necessary for bacterial binding. The goal of the present study was to characterize the binding of purified SAG to IgA. SAG binds to a variety of proteins, including serum and secretory IgA, alkaline phosphatase-conjugated IgGs originating from rabbit, goat, swine and mouse, and lactoferrin and albumin. Binding of IgA to SAG is calcium dependent and is inhibited by 0.5 M KCl, suggesting that electrostatic interactions are involved. Binding of IgA was destroyed after reduction of SAG, suggesting that the protein moiety is involved in binding. To pinpoint further the binding domain for IgA on SAG, a number of consensus-based peptides of the SRCR domains and SRCR interspersed domains were designed and synthesized. ELISA binding studies with IgA indicated that only one of the peptides tested, comprising amino acids 18-33 (QGRVEVLYRGSWGTVC) of the 109-amino-acid SRCR domain, exhibited binding to IgA. This domain is identical to the domain of SAG that is involved in binding to bacteria. Despite this similar binding site, IgA did not inhibit binding of Streptococcus mutans to SAG or peptide. These results show that the binding of IgA to SAG is specifically mediated by a peptide sequence on the SRCR domains.

Reactive oxygen species play no role in the candidacidal activity of the salivary antimicrobial peptide histatin 5.

The mechanism of action of antimicrobial peptides is still a matter of debate. The formation of ROS (reactive oxygen species) has been suggested to be the crucial step in the fungicidal mechanism of a number of antimicrobial peptides, including histatin 5 and lactoferrin-derived peptides. In the present study we have investigated the effects of histatin 5 and of a more amphipathic synthetic derivative, dhvar4, on the generation of ROS in the yeast Candida albicans, using dihydroethidium as an indicator for ROS. With both peptides, a substantial enhancement of fluorescence was observed. However, TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl), a cell-permeant ROS scavenger, did not have an inhibitory effect on killing or on the enhancement of fluorescence. Furthermore, antimycin and azide, which have been reported to induce ROS in vitro, were not able to enhance the dihydroethidium fluorescence, while chlorhexidine, a non-specific antiseptic agent, enhanced dihydroethidium fluorescence to the same extent as did the peptides. Fluorescence microscopy showed the fluorescence enhancement to be a consequence of the release of unbound preformed ethidium from the mitochondrial matrix within the cell. It is concluded that ROS do not play a role in the histatin 5-mediated killing of C. albicans.

Prevention and treatment of salivary gland hypofunction related to head and neck radiation therapy and chemotherapy.

Radiation therapy and chemotherapy for malignant tumors in the head and neck region are inevitably associated with injury to oral tissues, including the salivary glands. This often results in salivary gland hypofunction. Until now there has been no effective method of preventing damage caused by cancer therapies. Although not yet supported by sufficient evidence, there are some clinical trials indicating a potential benefit from radical scavengers and saliva stimulants. Other developments are in gene transfer to regain salivary gland function and stem cell transplantation to regenerate a diseased salivary gland. While irradiation on salivary gland tissue is irreversible to a large extent, hyposalivation associated with chemotherapy is usually less severe and reversible. In case of significant residual secretory capacity, supportive care is indicated. These patients are advised to stimulate their salivary glands by mechanical or gustatory stimuli. Alternatively, salivary flow can be stimulated by cholinergic stimulation (eg, pilocarpine or cevimeline). In the case of little or no residual capacity, palliative treatment is the only option. In such patients, nocturnal oral dryness can be alleviated by spraying oral surfaces with water or by applying a saliva substitute, particularly a substitute with gellike properties. During the day, the application of mouthwashes and saliva substitutes is indicated if moistening of oral surfaces with water is not sufficient. Recent developments are focusing on bioactive saliva substitutes and mouthwashes containing antimicrobial peptides to protect oral tissues against microbial colonization and to suppress or cure mucosal and gingival inflammation.

Comparison of lacrimal and salivary gland involvement in Sjögren's syndrome.

OBJECTIVES: To determine the performance of different tear and salivary tests applied in Sjögren's syndrome (SS) and to disclose how these tests relate to common serologic tests in SS. DESIGN: In addition to the routine ocular and oral tests for diagnosing SS (Schirmer test, rose bengal score, unstimulated whole saliva flow, and parotid sialography), tear breakup time and flow rate of glandular saliva (parotid and submandibular-sublingual [SM/SL]) were evaluated in patients referred for diagnosis of SS. Patients were categorized into primary SS, secondary SS, and non-SS groups according to the revised European classification criteria for SS. SETTING: Referral center. PATIENTS: Referred sample of 80 consecutive patients. MAIN OUTCOME MEASURE: Correlation between ocular and salivary measures. RESULTS: Breakup time performed insufficiently in diagnosing SS, as opposed to the rose bengal score. In patients with primary and secondary SS, a clear correlation was noted between tear and saliva quality and secretion rate, and between the rose bengal score and parotid sialography. Increased rose bengal scores also correlated significantly with hyperglobulinemia and presence of SS-B antibodies in serum, with duration of subjective eye dryness, and with decreased tear-gland function. With regard to the oral tests, whole, parotid, and SM/SL salivary flow decreased significantly with increasing duration of oral complaints, with the stimulated SM/SL flow rate showing the strongest decrease and being more specific in diagnosing SS. Also, parotid sialography was more specific in excluding patients without SS than the commonly applied diagnostic criterion of secretion of unstimulated whole saliva. CONCLUSIONS: The rose bengal score remains the eye test of choice, as it has the highest specificity for SS. Hyperglobulinemia and especially positive serologic findings for SS-B may warrant close monitoring of the eyes, since these serum findings appear to relate to the severity of ocular surface damage. Parotid sialography and stimulated secretion of SM/SL saliva are more specific in diagnosing SS than unstimulated secretion of whole saliva.

Stress as a determinant of saliva-mediated adherence and coadherence of oral and nonoral microorganisms.

OBJECTIVE: The mucosal secretory proteins, such as the salivary proteins, play a key role in the acquisition and regulation of the mucosal microflora. Most notably, some microorganisms utilize the host's secretory proteins to adhere to the mucosa; a first step in colonization and infection. The secretory proteins also influence colonization by affecting the binding among microorganisms, a process denoted as coadherence. Previously we reported that acute stressors cause specific changes in saliva composition. The present study investigated to what extent these changes influence saliva-mediated microbial adherence and coadherence (ex vivo). METHODS: Thirty-two male undergraduates provided unstimulated saliva before and during a control condition and two stressors: A memory test and a surgery video presentation. We used saliva-coated microplates to test the adherence of bacteria for which the oral cavity is either a natural reservoir (eg, viridans streptococci) or a portal of entry (eg, Helicobacter pylori). We also tested the saliva-mediated co-adherence between Streptococcus gordonii and the yeast Candida albicans. Correlation analyses were performed to determine the relationships between changes in microbial adherence and the concentrations of potential salivary ligands, viz. cystatin S, the mucins MUC5B and MUC7, S-IgA, lactoferrin, alpha-amylase, and total salivary protein. RESULTS: During the memory test, saliva-mediated adhesion of Streptococcus sanguis, Streptococcus gordonii, and H. pylori increased, whereas the coadherence of C. albicans with S. gordonii decreased. During the surgical video presentation the saliva-mediated adherence of H. pylori, S. sanguis, and Streptococcus mitis increased. These changes were independent of salivary flow rate, but correlated with specific changes in salivary protein composition. CONCLUSION: The results show that even moderate stressors, by altering the activity of the mucosal secretory glands, may affect microbial colonization processes such as adherence and coadherence. This study hereby presents a mechanism by which stress may affect the mucosal microflora and susceptibility to infectious disease.

Internalisation and degradation of histatin 5 by Candida albicans.

Histatins, salivary antimicrobial peptides, are susceptible to proteolytic degradation, often ascribed to host proteinases. In this study, we addressed the question whether proteolytic activity from microbial sources can contribute to this degradation. Candida albicans, an opportunistic yeast that is susceptible to the histatins, was used as target organism. The most potent histatin (histatin 5: sequence: DSHAKRHHGYKRKFHEKHHSHRGY), two histatin 5 fragments (dh-5: sequence: KRKFHEKHHSHRGY; P-113: sequence: AKRHHGYKRKFH) and an all-D isomer of the latter (P-113D) were used as model peptides. All L-peptides were susceptible to degradation by C. albicans. Cleavage was established at Lys5 and His19 of histatin 5, Lys11, Arg12, Phe14, Glu16, Lys17, His18 and Ser20 of dh-5 and Ala4 and Lys11 of P-113. In addition, it was found that secreted C. albicans enzymes are not involved in the degradation process and that blocking cell entry of the peptides greatly impedes degradation. Moreover, P-113D, which is biologically as active as P-113, was hardly susceptible to proteolysis. These data imply that proteolysis occurs mainly intracellularly and is not used as a protective mechanism against histatin activity. Together, our results suggest that, besides host proteinases, microbial enzymes play an important role in histatin degradation.

Innate secretory immunity in response to laboratory stressors that evoke distinct patterns of cardiac autonomic activity.

OBJECTIVE: Most infections begin at mucosal surfaces. These surfaces are covered by the secretory proteins of the exocrine glands (eg, the salivary, respiratory, and gastrointestinal glands), which provide a first line of innate defense. The release of these secretory proteins is under neuroendocrine control and thus, in theory, sensitive to modulation by psychosocial stress. This was empirically tested by measuring the salivary secretion of cystatin S, lactoferrin, alpha-amylase, the mucins MUC5B and MUC7, and total salivary protein in response to stressors known to evoke distinct patterns of cardiac autonomic activity. METHODS: Thirty-two undergraduate volunteers were each subjected to two laboratory stressors and a control condition. Stressors were an active coping memory test and a passive coping video presentation showing surgical procedures. In the control condition participants viewed a didactic video presentation. RESULTS: The stressors evoked the expected distinct patterns of cardiac autonomic activity. The memory test produced a strong increase in sympathetic activity (evidenced by a shortened preejection period), and a decrease in cardiac parasympathetic activity (evidenced by a decrease in heart rate variability). This active coping response was associated with an enhanced secretion ( microg/min, controlling for salivary flow rate) of MUC7, lactoferrin, alpha-amylase, and total salivary protein. Conversely, the surgical video produced an increase in cardiac vagal tone and a modest increase in sympathetic activity. This passive coping response was associated with an enhanced secretion of all proteins studied. These secretory responses were generally larger than the secretory responses during the active coping memory test. Correlation analyses indicated that for both stressors autonomic and cardiovascular reactivity was positively associated with an enhanced and prolonged secretory activity. CONCLUSIONS: Stress-induced modulation of innate secretory immunity may be a contributing factor in the observed relationship between stress and susceptibility to infectious diseases. We further propose a more differentiated approach to acute stress by distinguishing among stressors with distinct autonomic nervous system effects.

Identification of the bacteria-binding peptide domain on salivary agglutinin (gp-340/DMBT1), a member of the scavenger receptor cysteine-rich superfamily.

Salivary agglutinin is encoded by DMBT1 and identical to gp-340, a member of the scavenger receptor cysteine-rich (SRCR) superfamily. Salivary agglutinin/DMBT1 is known for its Streptococcus mutans agglutinating properties. This 300-400 kDa glycoprotein is composed of conserved peptide motifs: 14 SRCR domains that are separated by SRCR-interspersed domains (SIDs), 2 CUB (C1r/C1s Uegf Bmp1) domains, and a zona pellucida domain. We have searched for the peptide domains of agglutinin/DMBT1 responsible for bacteria binding. Digestion with endoproteinase Lys-C resulted in a protein fragment containing exclusively SRCR and SID domains that binds to S. mutans. To define more closely the S. mutans-binding domain, consensus-based peptides of the SRCR domains and SIDs were designed and synthesized. Only one of the SRCR peptides, designated SRCRP2, and none of the SID peptides bound to S. mutans. Strikingly, this peptide was also able to induce agglutination of S. mutans and a number of other bacteria. The repeated presence of this peptide in the native molecule endows agglutinin/DMBT1 with a general bacterial binding feature with a multivalent character. Moreover, our studies demonstrate for the first time that the polymorphic SRCR domains of salivary agglutinin/DMBT1 mediate ligand interactions.