Antimicrobial Peptides: Mechanisms of Action and Resistance.

More than 40 antimicrobial peptides and proteins (AMPs) are expressed in the oral cavity. These AMPs have been organized into 6 functional groups, 1 of which, cationic AMPs, has received extensive attention in recent years for their promise as potential antibiotics. The goal of this review is to describe recent advances in our understanding of the diverse mechanisms of action of cationic AMPs and the bacterial resistance against these peptides. The recently developed peptide GL13K is used as an example to illustrate many of the discussed concepts. Cationic AMPs typically exhibit an amphipathic conformation, which allows increased interaction with negatively charged bacterial membranes. Peptides undergo changes in conformation and aggregation state in the presence of membranes; conversely, lipid conformation and packing can adapt to the presence of peptides. As a consequence, a single peptide can act through several mechanisms depending on the peptide's structure, the peptide:lipid ratio, and the properties of the lipid membrane. Accumulating evidence shows that in addition to acting at the cell membrane, AMPs may act on the cell wall, inhibit protein folding or enzyme activity, or act intracellularly. Therefore, once a peptide has reached the cell wall, cell membrane, or its internal target, the difference in mechanism of action on gram-negative and gram-positive bacteria may be less pronounced than formerly assumed. While AMPs should not cause widespread resistance due to their preferential attack on the cell membrane, in cases where specific protein targets are involved, the possibility exists for genetic mutations and bacterial resistance. Indeed, the potential clinical use of AMPs has raised the concern that resistance to therapeutic AMPs could be associated with resistance to endogenous host-defense peptides. Current evidence suggests that this is a rare event that can be overcome by subtle structural modifications of an AMP.

Antimicrobial properties and dentin bonding strength of magnesium phosphate cements.

The main objective of this work was to assess the antimicrobial properties and the dentin-bonding strength of novel magnesium phosphate cements (MPC). Three formulations of MPC, consisting of magnesium oxide and a phosphate salt, NH4H2PO4, NaH2PO4 or a mixture of both, were evaluated. As a result of the setting reaction, MPC transformed into either struvite (MgNH4PO4·6H2O) when NH4H2PO4 was used or an amorphous magnesium sodium phosphate when NaH2PO4 was used. The MPC had appropriate setting times for hard tissue applications, high early compressive strengths and higher strength of bonding to dentin than commercial mineral trioxide aggregate cement. Bacteriological studies were performed with fresh and aged cements against three bacterial strains, Escherichia coli, Pseudomonas aeruginosa (planktonic and in biofilm) and Aggregatibacter actinomycetemcomitans. These bacteria have been associated with infected implants, as well as other frequent hard tissue related infections. Extracts of different compositions of MPC had bactericidal or bacteriostatic properties against the three bacterial strains tested. This was associated mainly with a synergistic effect between the high osmolarity and alkaline pH of the MPC. These intrinsic antimicrobial properties make MPC preferential candidates for applications in dentistry, such as root fillers, pulp capping agents and cavity liners.

Parotid secretory granules: crossroads of secretory pathways and protein storage.

Saliva plays an important role in digestion, host defense, and lubrication. The parotid gland contributes a variety of secretory proteins-including amylase, proline-rich proteins, and parotid secretory protein (PSP)-to these functions. The regulated secretion of salivary proteins ensures the availability of the correct mix of salivary proteins when needed. In addition, the major salivary glands are targets for gene therapy protocols aimed at targeting therapeutic proteins either to the oral cavity or to circulation. To be successful, such protocols must be based on a solid understanding of protein trafficking in salivary gland cells. In this paper, model systems available to study the secretion of salivary proteins are reviewed. Parotid secretory proteins are stored in large dense-core secretory granules that undergo stimulated secretion in response to extracellular stimulation. Secretory proteins that are not stored in large secretory granules are secreted by either the minor regulated secretory pathway, constitutive secretory pathways (apical or basolateral), or the constitutive-like secretory pathway. It is proposed that the maturing secretory granules act as a distribution center for secretory proteins in salivary acinar cells. Protein distribution or sorting is thought to involve their selective retention during secretory granule maturation. Unlike regulated secretory proteins in other cell types, salivary proteins do not exhibit calcium-induced aggregation. Instead, sulfated proteoglycans play a role in the storage of secretory proteins in parotid acinar cells. This work suggests that unique sorting and retention mechanisms are responsible for the distribution of secretory proteins to different secretory pathways from the maturing secretory granules in parotid acinar cells.

Design and validation of anti-inflammatory peptides from human parotid secretory protein.

Parotid secretory protein (PSP) and palate-lung-nasal epithelium clone (PLUNC) are novel secretory proteins that are expressed in the oral cavity and upper airways. Both proteins are related to bactericidal/permeability increasing protein (BPI). Cationic peptides derived from BPI exhibit anti-inflammatory activity. To test if PSP (C20orf70 gene product) also contains anti-inflammatory peptides, we designed 3 cationic peptides based on the predicted structure of PSP and known active regions of BPI. Each peptide inhibited the lipopolysaccharide (LPS)-stimulated secretion of TNFalpha from RAW 264.7 macrophage cells. At 200 microg/mL, the peptide GK-7 exhibited inhibition similar to that achieved with 10 microg/mL of polymyxin B. PSP peptides directly inhibited the binding of LPS to LPS-binding protein. The cationic peptide Substance P had no inhibitory effect in these assays, confirming the specificity of the PSP peptides. These findings suggest that PSP peptides can serve as templates for the design of novel anti-inflammatory peptides.

Expression and anti-bacterial activity of human parotid secretory protein (PSP).

Parotid secretory protein (PSP) is an abundant protein in mouse and rat parotid glands. A related sequence (C20orf70) was identified on human chromosome 20. The goal of this study was to determine if PSP is expressed in the human parotid gland. The cDNA for human PSP was amplified from a human parotid cDNA sample. A peptide antibody, raised to the C-terminal peptide of PSP, identified the protein in human parotid tissue by immunofluorescence microscopy. Immunoaffinity chromatography suggested that PSP was expressed in human saliva. PSP is related to bactericidal/permeability-increasing protein (BPI). To test if PSP exhibits anti-bacterial activity, epitope-tagged PSP was expressed in rat GH4C1 cells. The secretion medium exhibited bacteristatic or bactericidal effects on Pseudomonas aeruginosa in a colony-forming assay when compared with secretion medium from GH4C1 cells that did not express PSP. These results suggest that PSP is expressed in the human parotid gland and saliva, where it functions as a BPI-like anti-bacterial protein.

A sulfated proteoglycan is necessary for storage of exocrine secretory proteins in the rat parotid gland.

Sulfated proteoglycans have been proposed to play a role in the sorting and storage of secretory proteins in exocrine secretory granules. Rat parotid acinar cells expressed a 40- to 60-kDa proteoglycan that was stored in secretory granules. Treatment of the tissue with the proteoglycan synthesis inhibitor paranitrophenyl xyloside resulted in the complete abrogation of the sulfated proteoglycan. Pulse-chase experiments in the presence of the xyloside analog showed a significant reduction in the stimulated secretion and granule storage of the newly synthesized regulated secretory proteins amylase and parotid secretory protein. Inhibition of proteoglycan sulfation by chlorate did not affect the sorting of these proteins. The effect of proteoglycan synthesis inhibition on protein sorting was completely reversed upon treatment with a weak acid. These results suggest that the sulfated proteoglycan is necessary for sorting and storage of regulated secretory proteins in the exocrine parotid gland. Preliminary evidence suggests that the mechanism involves the modulation of granule pH by the proteoglycan rather than a direct interaction with other granule components.

Oligomerization of green fluorescent protein in the secretory pathway of endocrine cells.

Green fluorescent protein (GFP) is used extensively as a reporter protein to monitor cellular processes, including intracellular protein trafficking and secretion. In general, this approach depends on GFP acting as a passive reporter protein. However, it was recently noted that GFP oligomerizes in the secretory pathway of endocrine cells. To characterize this oligomerization and its potential role in GFP transport, cytosolic and secretory forms of enhanced GFP (EGFP) were expressed in GH4C1 and AtT-20 endocrine cells. Biochemical analysis showed that cytosolic EGFP existed as a 27 kDa monomer, whereas secretory forms of EGFP formed disulphide-linked oligomers. EGFP contains two cysteine residues (Cys(49) and Cys(71)), which could play a role in this oligomerization. Site-directed mutagenesis of Cys(49) and Cys(71) showed that both cysteine residues were involved in disulphide interactions. Substitution of either cysteine residue resulted in a reduction or loss of oligomers, although dimers of the secretory form of EGFP remained. Mutation of these residues did not adversely affect the fluorescence of EGFP. EGFP oligomers were stored in secretory granules and secreted by the regulated secretory pathway in endocrine AtT-20 cells. Similarly, the dimeric mutant forms of EGFP were still secreted via the regulated secretory pathway, indicating that the higher-order oligomers were not necessary for sorting in AtT-20 cells. These results suggest that the oligomerization of EGFP must be considered when the protein is used as a reporter molecule in the secretory pathway.

Comparative sorting of neuroendocrine secretory proteins: a search for common ground in a mosaic of sorting models and mechanisms.

Endocrine, neuroendocrine and exocrine cells store regulated secretory proteins in secretory granules, while constitutive and constitutive-like secretory proteins are secreted directly without storage. Sorting of secretory proteins takes place in the trans-Golgi network (sorting for entry) or immature secretory granules (sorting by retention). The relative contribution of these sorting steps and the sorting signals and mechanisms involved in each step has been the subject of intense studies and debate in recent years. New evidence now suggests that: (1) two proteins with structurally similar sorting signals can use different sorting mechanisms; (2) one protein with multiple sorting signals can be sorted differently in different cell types; and (3) one cell type can recognize different sorting signals and use different sorting mechanisms. The latter finding suggests that sorting must be a regulated event. While the current image of sorting is complex, recent findings are pointing to common features that form a mosaic of related sorting mechanisms.

Trafficking of non-regulated secretory proteins in insulin secreting (INS-1) cells.

AIMS/HYPOTHESIS: Sorting of proinsulin to the regulated secretory pathway of pancreatic beta cells and retention of insulin in dense-core granules of this pathway is remarkably efficient. To monitor the specificity of these events, the secretion of two exogenous secretory proteins not known to carry information for sorting or retention in the regulated pathway was investigated in INS-1 cells. METHODS: SEGFP, a fusion protein consisting of a signal peptide N-terminal to EGFP (mutant green fluorescent protein with enhanced fluorescence) and secreted alkaline phosphatase (SEAP) were expressed in INS-1 cells by transfection and by infection with recombinant adenovirus, respectively. Secretion of SEGFP was monitored by quantitative western blotting and that of SEAP by its activity. RESULTS: Secreted alkaline phosphatase showed high basal secretion (6.6% total) but only modest (3.6-fold) stimulation of secretion by secretagogues, in keeping with secretion largely through the constitutive pathway. By contrast SEGFP had a secretory pattern similar to insulin, with low basal secretion (0.8% total) and 16-fold stimulation by secretagogues. Granular localization of SEGFP was confirmed by high resolution electron microscopy immunocytochemistry. Pulse-chase experiments indicated retention of SEGFP in granules at least 24 h after synthesis. The secretory SEGFP, but not cytosolic EGFP, formed disulphide-linked oligomers. This could be implicated in its regulated secretion. CONCLUSION/INTERPRETATION: These data indicate that in INS-1 cells SEGFP, but not SEAP, is unexpectedly handled as a regulated secretory protein and stored along with insulin in granules. This raises questions about the specificity and mechanism of the sorting of proteins to granules in INS-1 cells or their retention therein or both.

Aggregation chaperones enhance aggregation and storage of secretory proteins in endocrine cells.

Calcium-induced aggregation has been proposed to play a role in the sorting and storage of secretory proteins in secretory granules of endocrine cells. The regulation of this process is not known. Hexahistidine epitope tags were used to create aggregation chaperones that enhance the calcium-induced aggregation of secretory granule proteins in vitro. Indeed, 100% recovery of the aggregating target protein was achieved without any modification of the target protein. The aggregation chaperone is not trapped in the aggregates. Co-expression of His(6)-tagged secreted alkaline phosphatase and the regulated secretory protein chromogranin A resulted in an increased chromogranin storage in secretory granules, and stimulated secretion of chromogranin A increased 50%. However, secretion of secreted alkaline phosphatase was not affected by the hexahistidine epitope tag. Thus, calcium-induced aggregation is not a passive process; rather, aggregation and sorting of secretory proteins can be regulated by aggregation chaperones in the secretory pathway of endocrine cells.

N- and C-terminal domains direct cell type-specific sorting of chromogranin A to secretory granules.

Chromogranins are a family of regulated secretory proteins that are stored in secretory granules in endocrine and neuroendocrine cells and released in response to extracellular stimulation (regulated secretion). A conserved N-terminal disulfide bond is necessary for sorting of chromogranins in neuroendocrine PC12 cells. Surprisingly, this disulfide bond is not necessary for sorting of chromogranins in endocrine GH4C1 cells. To investigate the sorting mechanism in GH4C1 cells, we made several mutant forms removing highly conserved N- and C-terminal regions of bovine chromogranin A. Removing the conserved N-terminal disulfide bond and the conserved C-terminal dimerization and tetramerization domain did not affect the sorting of chromogranin A to the regulated secretory pathway. In contrast, removing the C-terminal 90 amino acids of chromogranin A caused rerouting to the constitutive secretory pathway and impaired aggregation properties as compared with wild-type chromogranin A. Since this mutant was sorted to the regulated secretory pathway in PC12 cells, these results demonstrate that chromogranins contain independent N- and C-terminal sorting domains that function in a cell type-specific manner. Moreover, this is the first evidence that low pH/calcium-induced aggregation is necessary for sorting of a chromogranin to the regulated secretory pathway of endocrine cells.

Polarized secretion of the regulated secretory protein chromogranin A.

Bovine chromogranin A (CgA), together with secreted alkaline phosphatase (SEAP) as an external control for apical secretion were expressed in MDCK cells to test if CgA contains sorting signals for polarized secretion. CgA, SEAP, and the endogenous apical marker GP80 were secreted 75-80% apically. Basolateral secretion of SEAP was inhibited 40% by ammonium chloride. Sulfate labeling and digestion with chondroitinase ABC revealed a 120 kDa proteoglycan-CgA and 75 kDa CgA. Inhibition of proteoglycan synthesis did not affect apical secretion of CgA. As CgA is not N-glycosylated, we used tunicamycin to test if cellular N-glycosylation is required for apical sorting. Tunicamycin reversed the polarity of secretion of CgA to the basolateral side. These results suggest that CgA contains dominant apical and recessive basolateral sorting information.

Disruption of disulfide bonds exhibits differential effects on trafficking of regulated secretory proteins.

For several secretory proteins, it has been hypothesized that disulfide-bonded loop structures are required for sorting to secretory granules. To explore this hypothesis, we employed dithiothreitol (DTT) treatment in live pancreatic islets, as well as in PC-12 and GH(4)C(1) cells. In islets, disulfide reduction in the distal secretory pathway did not increase constitutive or constitutive-like secretion of proinsulin (or insulin). In PC-12 cells, DTT treatment caused a dramatic increase in unstimulated secretion of newly synthesized chromogranin B (CgB), presumably as a consequence of reducing the single conserved chromogranin disulfide bond (E. Chanat, U. Weiss, W. B. Huttner, and S. A. Tooze. EMBO J. 12: 2159-2168, 1993). However, in GH(4)C(1) cells that also synthesize CgB endogenously, DTT treatment reduced newly synthesized prolactin and blocked its export, whereas newly synthesized CgB was routed normally to secretory granules. Moreover, on transient expression in GH(4)C(1) cells, CgA and a CgA mutant lacking the conserved disulfide bond showed comparable multimeric aggregation properties and targeting to secretory granules, as measured by stimulated secretion assays. Thus the conformational perturbation of regulated secretory proteins caused by disulfide disruption leads to consequences in protein trafficking that are both protein and cell type dependent.

Sorting of a constitutive secretory protein to the regulated secretory pathway of exocrine cells.

Exocrine cells secrete granule proteins by regulated or constitutive-like secretory pathways. It is thought that all secretory proteins can enter immature secretory granules in exocrine cells. To test this hypothesis, we expressed the constitutive secretory protein secreted alkaline phosphatase (SEAP) in the exocrine cell line AR42J and compared its secretion to that of amylase, an endogenous regulated secretory protein. Secretion of SEAP and amylase were stimulated about 1.5-fold by substance P and 2-fold by barium chloride. In dexamethasone-treated cells, SEAP and amylase secretion were stimulated about 1.8-fold by substance P, 5-fold by barium chloride, and 4-fold by cholecystokinin-8. Cycloheximide reduced basal secretion of SEAP and amylase by 50%, increasing cholecystokinin-stimulated secretion to about 10-fold. Sodium butyrate induced expression of SEAP 2-fold but had no effect on stimulated secretion. These results suggest that SEAP is stored in secretory granules in AR42J cells.

Differential storage of prolactin, granins (Chromogranin B and secretogranin II), and constitutive secretory markers in rat pituitary GH4C1 cells.

The rat pituitary cell line GH4C1 secretes granins (chromogranin B and secretogranin II) and prolactin by the regulated secretory pathway. The intracellular storage of prolactin is preferentially induced by hormone treatment with estradiol, insulin, and epidermal growth factor. The goal of this study was to determine the effect of hormone treatment on storage of granins and constitutive secretory markers. The granins were efficiently stored in both hormone-treated and -untreated cells (17% of total secreted in 4 h). Secreted alkaline phosphatase (SEAP), a truncated membrane protein that would not be expected to enter secretory granules, and glycosaminoglycan, a marker for the constitutive secretory pathway, exhibited 70 80% secretion under both conditions. In comparison, the relative prolactin secretion was 31 and 68% from hormone-treated and -untreated cells, respectively. Phorbol ester and KCl stimulated prolactin secretion 2.3-fold from untreated cells and 5. 5-fold from hormone-treated cells. In contrast, SEAP secretion was stimulated 1.5-fold from both treated and untreated cells, consistent with secretion by the constitutive secretory pathway. Stimulated secretion of granins was detected from both hormone-treated and -untreated cells. These results suggest that granin and prolactin storage are differentially regulated and that the constitutive secretory pathway is not affected by hormone treatment.

Aggregation and concentration-dependent sorting of exocrine regulated secretory proteins.

Sorting between the regulated and the constitutive secretory pathway in exocrine cells is thought to involve aggregation of regulated secretory proteins. This study demonstrates that, unlike endocrine secretory proteins, exocrine secretory proteins, including amylase, do not undergo homotypic aggregation under the conditions found in the sorting organelles. Also, unlike other exocrine proteins, amylase does not aggregate with chondroitin sulfate. Since amylase exhibits heterotypic aggregation, the role of protein concentration in amylase sorting was tested in AR42J cells. Secretion was stimulated with substance P and cholecystokinin from both untreated and dexamethasone-treated cells, with more efficient stimulation from dexamethasone-treated cells. These results indicate that amylase sorting is enhanced when its expression is stimulated by dexamethasone treatment.

An N-terminal hydrophobic peak is the sorting signal of regulated secretory proteins.

Endocrine and exocrine cells each contain a regulated and constitutive secretory pathway. The presence of two distinct secretory pathways in the same cell type requires a sorting step to direct secretory proteins to the correct pathway. It is thought that regulated secretory proteins contain a specific sorting signal. However, this signal has not been identified. Amino acid sequence comparisons have not revealed any significant similarity between different regulated secretory proteins, suggesting that the sorting signal does not consist of a conserved primary sequence. In the present report, we have analyzed the predicted secondary structures of regulated secretory proteins and identified an N-terminal hydrophobic peak (NHP) which is located approximately from amino acids 9-26, overlaps with a predicted alpha-helix and contains charged amino acid residues. This signal is present in regulated secretory proteins that exhibit an N-terminal sorting sequence, but it is absent from constitutively secreted proteins and proteins where the sorting sequence is not located near the N-terminus. It appears that the NHP is both necessary and sufficient for sorting of many secretory proteins to the regulated secretory pathway.

Pancreastatin stimulates secretion from neonatal rat atrial cells.

Atrial myocardial cells store atrial natriuretic factor and secrete the peptide in response to extracellular stimulation. Ventricular myocardial cells also exhibit stimulated secretion of atrial natriuretic factor but with little or no intracellular storage. The stimulated secretion of other proteins from these cells is poorly characterized. In the present study the secretion of sulfated macromolecules from Na2(35)SO4 labeled atrial and ventricular myocardial cells was quantitated. Both cell types secreted a chondroitin sulfate proteoglycan, as evidenced by glycanase digestion. Endothelin-1, a known stimulator of ANF secretion, stimulated secretion from atrial cells about 70% but had no effect on ventricular cell secretion. The differential stimulation of atrial and ventricular cell secretion was not due to the absence of a regulated secretory pathway in the latter cell type since phorbol ester stimulated secretion from both cell types. This result indicates that differences exist in the early steps of signal transduction between atrial and ventricular cells. Pancreastatin, a regulatory peptide derived from chromogranin A, stimulated atrial cell secretion 90%. This result suggests that chromogranin A, which has been identified in atrial secretory granules, may play an autoregulatory role in atrial secretion.

Regulated, but not constitutive, secretory proteins bind porcine chymotrypsinogen.

Endocrine and exocrine cells exhibit both a constitutive and a regulated secretory pathway. In the latter pathway, secretory proteins are stored at a high concentration in secretory granules and are released by exocytosis in response to appropriate external stimuli. Sorting between the two secretory pathways is believed to take place in the trans-Golgi tubular network. To account for experimental data, it has been proposed that sorting receptors exist which bind a variety of regulated secretory proteins, including foreign secretory proteins introduced into the cells by transfection. In support of the sorting receptor hypothesis Chung et al. (Chung, K.-N., Walter, P., Aponte, G. W., and Moore, H.-P.H. (1989) Science 243, 192-197) isolated a group of 25-kDa canine pancreatic "hormone-binding proteins" that bound regulated but not constitutive secretory proteins. To determine if similar proteins are present in other species and tissues, we have screened porcine pancreas, parathyroid, adrenal medulla, and pituitary glands. A 31-kDa protein, similar to that identified by Chung et al. (1989), which binds to regulated but not to constitutive secretory proteins was identified in porcine pancreas. This protein was not detected in the parathyroid, adrenal medulla, or pituitary glands, however, which argues against it serving as a general sorting receptor. NH2-terminal sequencing, immunoreactivity, and proteolytic activity data indicate that the porcine 31-kDa protein is similar if not identical to porcine chymotrypsinogen A or B.