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Three anti-microbial peptides were compared with respect to their killing activity against Candida albicans and their ability to disturb its cellular and internal membranes. Histatin 5 is an anti-fungal peptide occurring naturally in human saliva, while dhvar4 and dhvar5 are variants of its active domain, with increased anti-microbial activity. dhvar4has increased amphipathicity compared with histatin 5, whereas dhvar5has amphipathicity comparable with that of histatin 5. All three peptides caused depolarization of the cytoplasmic and/or mitochondrial membrane, indicating membranolytic activity. For the variant peptides both depolarization and killing occurred at a faster rate. With FITC-labelled peptides, no association with the cytoplasmic membrane was observed, contradicting the formation of permanent transmembrane multimeric peptide pores. Instead, the peptides were internalized and act on internal membranes, as demonstrated with mitochondrion- and vacuole-specific markers. In comparison with histatin 5, the variant peptides showed a more destructive effect on mitochondria. Entry of the peptides and subsequent killing were dependent on the metabolic state of the cells. Blocking of the mitochondrial activity led to complete protection against histatin 5 activity, whereas that of dhvar4 was hardly affected and that of dhvar5 was affected only intermediately.

Concentrations and output of lactoferrin and of low-Mr mucin MG2 were determined in saliva of subjects suffering from Actinobacillus actinomycetemcomitans-associated periodontal disease and healthy subjects. Periodontal patients were clinically examined and a microbiological sample was taken from the deepest bleeding pockets in each quadrant. The number of viable A. actinomycetemcomitans was determined in the sampled sites of each patient. The MG2 output in the diseased subjects (13.6 microg protein/min) was decreased at least by a factor three compared to periodontal healthy subjects (44.3 microg protein/min). On the other hand, output of lactoferrin was not significantly different in healthy (9.5 microg/min) and diseased subjects (7.6 microg/min). Western analyses demonstrated a higher iron-saturation of lactoferrin in diseased subjects in comparison with the healthy subjects. Lactoferrin degrading enzymes, probably derived from microbial sources, could be detected in saliva of the periodontally diseased subjects, but not in saliva of healthy subjects. The combination of iron-saturation and degradation of lactoferrin suggests that anti-microbial properties of lactoferrin are diminished in periodontitis patients. Moreover, the low concentration of mucin MG2 suggests a decline in mucin defence and consequently a higher susceptibility for oral infection. A negative correlation (r= -0.4, p < 0.05) between the number of subgingival A. actinomycetemcomitans and lactoferrin in saliva suggested that low concentrations of lactoferrin favour the growth of the bacterium. These data indicate that a decline in the salivary defence system might increase the risk for oral infection by A. actinomycetemcomitans.

There are several reasons for hyposalivation, each affecting the salivary composition in different ways. The aim of this study was to analyze and compare lactoferrin, amylase and mucin MUC5B in stimulated whole saliva collected from subjects with hyposalivation of different origins and to relate the results to the presence of some microbial species associated with oral disorders. Albumin was determined as a marker of serum leakage. The characteristic feature for subjects with radiation-induced hyposalivation was a large increase in lactoferrin, probably due to leakage through inflamed mucosal tissues, while it was a high albumin content for the group with primary Sjögren's syndrome, probably due to disruption of the fragile mucosa. The saliva composition in subjects with hyposalivation of unknown origin or due to medicines was close to that in the healthy controls. All three hyposalivation groups tended to display a decrease in the concentrations of MUC5B and amylase. None of the microbial species analyzed (streptococci, mutans streptococci, Lactobacillus spp., Fusobacterium nucleatum, Prevotella intermedia/Prevotella nigrescens, Candida albicans, Staphylococcus aureus and enterics) correlated with concentration of MUC5B in saliva. The RT group, having the highest concentration of lactoferrin, had the lowest median number of F. nucleatum and was the only group in which median number of P. intermedia/P. nigrescens was zero.

Salivary agglutinin is a 300-400 kDa salivary glycoprotein that binds to antigen B polypeptides of oral streptococci, thereby playing a role in their colonization and the development of caries. A mass spectrum was recorded of a trypsin digest of agglutinin. A dominant peak of 1460 Da was sequenced by quadrupole time-of-flight (Q-TOF) tandem MS. The sequence showed 100% identity with part of the scavenger receptor cysteine-rich ('SRCR') domain found in gp-340/DMBT1 (deleted in malignant brain tumours-1). The mass spectrum revealed 11 peaks with an identical mass as a computer-simulated trypsin digest of gp-340. gp-340 is a 340 kDa glycoprotein isolated from bronchoalveolar lavage fluid that binds specifically to lung surfactant protein-D. DMBT1 is a candidate tumour suppressor gene. A search in the human genome revealed only one copy of this gene. The molecular mass, as judged from SDS/PAGE and the amino acid composition of agglutinin, was found to be nearly identical with that of gp-340. It was shown by Western blotting that monoclonal antibodies against gp-340 reacted with salivary agglutinin, and monoclonals against agglutinin reacted with gp-340. It was demonstrated that gp-340 and agglutinin bound in a similar way to Streptococcus mutans and surfactant protein-D. Histochemically, the distribution of gp-340 in the submandibular salivary glands was identical with the agglutinin distribution, as shown in a previous paper [Takano, Bogert, Malamud, Lally and Hand (1991) Anat. Rec. 230, 307-318]. We conclude that agglutinin is identical with gp-340, and that this molecule interacts with S. mutans and surfactant protein-D.

No abstract

Periodontitis is associated with the presence of certain Gram-negative bacteria in the oral cavity, among these Actinobacillus actinomycetemcomitans. In order to determine which types of salivary components interact with A. actinomycetemcomitans two strains (HG 1175 and FDC Y4) were incubated with whole saliva and individual glandular secretions, viz. parotid, submandibular, and sublingual saliva. Immunochemical analysis by immunoblotting of bacteria-bound salivary proteins showed that IgA, the low-molecular mucin MG2, parotid agglutinin, and a 300 kDa sublingual and submandibular glycoprotein, were bound to the bacterial strains tested. In addition, adherence of A. actinomycetemcomitans to salivary proteins in a solid-phase was studied. After electrophoresis and transfer of salivary proteins to nitrocellulose membranes A. actinomycetemcomitans adhered only to MG2. In this assay periodate treatment, mild acid hydrolysis or neuraminidase digestion of the saliva glycoproteins abolished binding of two clinical isolates (HG 1175 and NY 664), suggesting that sialic acid residues on MG2 are involved in the binding. In contrast, adherence of the smooth laboratory strain Y4 was not affected by removal of sialic acid residues or even periodate treatment of MG2.

The antimicrobial activity of bovine lactoferrin (bLF) is attributed to lactoferricin, which is situated in the N1-domain of bLF. Recently, another antimicrobial domain consisting of residues 268-284, designated lactoferrampin (LFampin), has been identified in the N1-domain of bLF, which exhibited antimicrobial activity against Candida albicans and several bacteria. In the present study, the candidacidal activity of a series of peptides spanning this antimicrobial domain was investigated in relation to the charge and the capacity to form a helical conformation in hydrophobic environments. C-Terminal truncation of LFampin resulted in a drastic decrease in candidacidal activity. Positively charged residues clustered at the C-terminal side of the LFampin domain appeared to be crucial for the candidacidal activity. The ability to adopt helical conformations did not change when LFampin was truncated at the C-terminal side. N-Terminally truncated LFampin peptides, truncated up to the sequence 270-284, were more reluctant to adopt a helical conformation. Therefore, we conclude that the C-terminal part of LFampin 265-284, which is the most active peptide, is crucial for its candidacidal activity, due to the presence of clustered positive charges, and that the N-terminal part is essential for activity as it facilitates helix formation.