Arie V. Nieuw Amerongen

Antimicrobial Peptides: Properties and Applicability

Abstract All organisms need protection against microorganisms, e. g. bacteria, viruses and fungi. For many years, attention has been focused on adaptive immunity as the main antimicrobial defense system. However, the adaptive immune system, with its network of humoral and cellular responses is only found in higher animals, while innate immunity is encountered in all living creatures. The turning point in the appreciation of the innate immunity was the discovery of antimicrobial peptides in the early eighties. In general these peptides act by disrupting the structural integrity of the microbial membranes. It has become clear that membraneactive peptides and proteins play a crucial role in both the innate and the adaptive immune system as antimicrobial agents. This review is focused on the functional and structural features of the naturally occurring antimicrobial peptides, and discusses their potential as therapeutics.

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, &agr;-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 (&mgr;g/min, controlling for salivary flow rate) of MUC7, lactoferrin, &agr;-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.

Stress and secretory immunity.

Publisher Summary This chapter focuses on the relationship between stress and saliva secretory immunity in humans. Salivary gland function is largely under autonomic control; the parasympathetic nerves mainly govern salivary fluid secretion, whereas the sympathetic nerves regulate protein secretion. The primary salivary centers in the brain stem receive inhibitory and excitatory inputs from neural structures in the forebrain and brain stem. As well as governing typical salivary functions, these structures are also involved in generating bodily changes associated with stress. It is, therefore, reasonable to assume that salivary changes during stress are an integral part of a centrally coordinated stress response that encompasses many other bodily functions. The autonomic receptors in the salivary glands can be divided into two main groups: the classic autonomic receptor types, which respond to either noradrenaline or acetylcholine, and the nonadrenergic–noncholinergic (NANC) receptors that respond to other autonomic messenger substances, such as peptides, nitric oxide, and purines. Differential activation of these receptor types can cause additive, synergistic, or antagonistic intracellular responses, ultimately resulting in a protein release that is capable of being differentially regulated among glands.

Psychological stress as a determinant of protein levels and salivary-induced aggregation of Streptococcus gordonii in human whole saliva

Several pathologies of the oral cavity have been associated with stress, so we investigated salivary-induced aggregation during psychological stress. In addition, salivary total protein, alpha-amylase, and secretory immunoglobulin A (s-IgA) were assessed. In this longitudinal study, 28 dental students provided unstimulated whole saliva during 10 minutes before an academic examination and subsequently 2 weeks and 6 weeks later in a nonstress situation. The effect of whole saliva on the aggregation of Streptococcus gordonii (HG 222) was determined spectrophotometrically. The results show a significant stress-mediated increase of salivary total protein concentration, alpha-amylase activity, amylase/protein ratio, alpha-amylase output, s-IgA concentration, and s-IgA output. There was also a trend for increased total protein output, whereas salivary flow rate was unchanged. The aggregation of S. gordonii in whole saliva collected before examination was 13.1%, whereas the aggregation in whole saliva collected during nonstress was 23.3%. This reduction was statistically significant (p <.01). Furthermore, the decrease in bacterial aggregation was related to the increase in state-anxiety (p <.05). The reduction in aggregation of S. gordonii under stress was not correlated with changes in salivary flow rate, s-IgA concentration, total protein concentration, or alpha-amylase activity. These results suggest that acute psychological stress exerts its influence on both salivary composition and salivary function. Reduced bacterial aggregation may be a contributing factor in the often reported relationship between stress and impaired oral health.

Lactoferrampin: a novel antimicrobial peptide in the N1-domain of bovine lactoferrin.

The antimicrobial activity of bovine lactoferrin is attributed to lactoferricin, situated in the N1-domain. Based on common features of antimicrobial peptides, a second putative antimicrobial domain was identified in the N1-domain of lactoferrin, designated lactoferrampin. This novel peptide exhibited candidacidal activity, which was substantially higher than the activity of lactoferrin. Furthermore, lactoferrampin was active against Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa, but not against the fermenting bacteria Actinomyces naeslundii, Porphyromonas gingivalis, Streptococcus mutans and Streptococcus sanguis. Notably, lactoferrampin is located in the N1-domain in close proximity to lactoferricin, which plays a crucial role in membrane-mediated activities of lactoferrin.

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 byDMBT1 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. mutansand 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.

Candidacidal effects of two antimicrobial peptides: histatin 5 causes small membrane defects, but LL-37 causes massive disruption of the cell membrane

The effects of antimicrobial peptides on artificial membranes have been well-documented; however, reports on the ultrastructural effects on the membranes of micro-organisms are relatively scarce. We compared the effects of histatin 5 and LL-37, two antimicrobial peptides present in human saliva, on the functional and morphological properties of the Candida albicans cell membrane. Fluorescence microscopy and immunogold transmission electron microscopy revealed that LL-37 remained associated with the cell wall and cell membrane, whereas histatin 5 transmigrated over the membrane and accumulated intracellularly. Freeze-fracture electron microscopy revealed that LL-37 severely affected the membrane morphology, resulting in the disintegration of the membrane bilayer into discrete vesicles, and an instantaneous efflux of small molecules such as ATP as well as larger molecules such as proteins with molecular masses up to 40 kDa. The effects of histatin 5 on the membrane morphology were less pronounced, but still resulted in the efflux of nucleotides. As the morphological defects induced by histatin 5 are much smaller than those induced by LL-37, but the efflux of nucleotides is similar at comparable candidacidal concentrations, we suggest that the loss of nucleotides plays an important role in the killing process.

The salivary lipocalin Von Ebner's gland protein is a cysteine proteinase inhibitor

The lipocalins make up a heterogeneous superfamily of proteins. Although showing almost no sequence homology, they share very similar secondary and tertiary structures. Their ability to bind hydrophobic ligands is well established, but the physiological function of most lipocalins remains unclear. The lipocalin from the human Von Ebners Gland of the tongue (VEGh) contains three sequence motifs corresponding with the papain-binding domains of cystatins, a family of naturally occurring cysteine proteinase inhibitors. We found that VEGh inhibited papain activity to a similar extent as salivary cystatin S. Furthermore, synthetic peptides derived from VEGh and cystatin C, comprising these three motifs, inhibited papain, too. We conclude that VEGh is a physiological inhibitor of cysteine proteinases and therefore can play a role in the control of inflammatory processes in oral and ocular tissues.

A critical comparison of the hemolytic and fungicidal activities of cationic antimicrobial peptides

The hemolytic and fungicidal activity of a number of cationic antimicrobial peptides was investigated. Histatins and magainins were inactive against human erythrocytes and Candida albicans cells in phosphate buffered saline, but displayed strong activity against both cell types when tested in 1 mM potassium phosphate buffer supplemented with 287 mM glucose. The HC50/IC50 ratio, indicative of the therapeutic index, was about 30 for all peptides tested. PGLa was most hemolytic (HC50=0.6 μM) and had the lowest therapeutic index (HC50/IC50=0.5). Susceptibility to hemolysis was shown to increase with storage duration of the erythrocytes and also significant differences were found between blood collected from different individuals. In this report, a sensitive assay is proposed for the testing of the hemolytic activities of cationic peptides. This assay detects subtle differences between peptides and allows the comparison between the hemolytic and fungicidal potency of cationic peptides.

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

Abstract 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.