Synergistic effects of low doses of histatin 5 and its analogues on amphotericin B anti-mycotic activity.

The increase in the use of antifungal agents for prophylaxis and therapy has led to the development of antifungal drug resistance. Drug combinations may prevent or delay resistance development. The aim of the present study was to investigate whether naturally and designed cationic antifungal peptides act synergistically with commonly used antimycotics. No enhanced activity was found upon addition of dhvar4, a designed analogue of the human salivary peptide histatin 5, or PGLa to fluconazole or 5-flucytosine, respectively. In contrast, strong synergism of amphotericin B with the peptides was found against several Aspergillus, Candida, and Cryptococcus strains, and against an amphotericin B-resistant C. albicans laboratory mutant in the standardised broth microdilution assays according to the NCCLS standard method M27-T. Amphotericin B showed synergism with dhvar5, another designed analogue of histatin 5, and with magainin 2 against all seven tested strains. Combinations of amphotericin B with histatin 5, dhvar4, and PGLa showed synergism against four of the seven strains. The growth inhibitory activity of amphotericin B was enhanced by sub-MIC concentrations of peptide, but its haemolytic activity remained unaffected, suggesting that its cytotoxicity to host cells was not increased and that peptides may be suitable candidates for combination therapy.

Amphotericin B- and fluconazole-resistant Candida spp., Aspergillus fumigatus, and other newly emerging pathogenic fungi are susceptible to basic antifungal peptides.

The present study shows that a number of basic antifungal peptides, including human salivary histatin 5, a designed histatin analog designated dhvar4, and a peptide from frog skin, PGLa, are active against amphotericin B-resistant Candida albicans, Candida krusei, and Aspergillus fumigatus strains and against a fluconazole-resistant Candida glabrata isolate.

Phase variation in Helicobacter pylori lipopolysaccharide.

Helicobacter pylori NCTC 11637 lipopolysaccharide (LPS) expresses the human blood group antigen Lewis x (Le(x)) in a polymeric form. Le(x) is beta-D-galactose-(1-4)-[alpha-L-fucose-(1-3)]-beta-D-acetylglucosamine. Schematically the LPS structure is (Le(x))n-core-lipid A. In this report, we show that Le(x) expression is not a stable trait but that LPS displays a high frequency (0.2 to 0.5%) of phase variation, resulting in the presence of several LPS variants in one bacterial cell population. One type of phase variation implied the loss of alpha1,3-linked fucose, resulting in variants that expressed nonsubstituted polylactosamines (also called the i antigen), i.e., Le(x) minus fucose; LPS: (lactosamine)n-core-lipid A. The switch of Le(x) to i antigen was reversible. A second group of variants arose by loss of polymeric main chain which resulted in expression of monomeric Le(y); LPS: (Le(y))-core-lipid A. A third group of variants arose by acquisition of alpha1,2-linked fucose which hence expressed Le(x) plus Le(y); LPS: (Le(y))(Le(x))n-core-lipid A. The second and third group of variants switched back to the parental phenotype [(Le(x))-core-lipid A] in lower frequencies. Part of the variation can be ascribed to altered expression levels of glycosyltransferase levels as assessed by assaying the activities of galactosyl-, fucosyl-, and N-acetylglucosaminyltransferases. Clearly phase variation increases the heterogeneity of H. pylori, and this process may be involved in generating the very closely related yet genetically slightly different strains that have been isolated from one patient.

Synthetic histatin analogues with broad-spectrum antimicrobial activity.

Histatins are salivary histidine-rich cationic peptides, ranging from 7 to 38 amino acid residues in length, that exert a potent killing effect in vitro on Candida albicans. Starting from the C-terminal fungicidal domain of histatin 5 (residues 11-24, called dh-5) a number of substitution analogues were chemically synthesized to study the effect of amphipathicity of the peptide in helix conformation on candidacidal activity. Single substitutions in dh-5 at several positions did not have any effect on fungicidal activity. However, multi-site substituted analogues (dhvar1 and dhvar2) exhibited a 6-fold increased activity over dh-5. In addition, dhvar1 and dhvar2 inhibited the growth of the second most common yeast found in clinical isolates, Torulopsis glabrata, of oral- and non-oral pathogens such as Prevotella intermedia and Streptococcus mutans, and of a methicillin-resistant Staphylococcus aureus. In their broad-spectrum activity, dhvar1 and dhvar2 were comparable to magainins (PGLa and magainin 2), antimicrobial peptides of amphibian origin. Both the fungicidal and the haemolytic activities of dhvar1, dhvar2 and magainins increased at decreasing ionic strength.

Typing of Helicobacter pylori with monoclonal antibodies against Lewis antigens in lipopolysaccharide.

Recently, it has been shown that the lipopolysaccharide (LPS) O antigen of Helicobacter pylori contains Lewis x (Lex), Lewis y (Ley), or both Lex and Ley antigens. We applied a serotyping method for H. pylori by an enzyme-linked immunosorbent assay with monoclonal antibodies (MAbs) specific for these antigens and the related fucosylated H type 1 (H1) antigen. The selected MAbs recognized the Lex and/or Ley structures in the LPS of H. pylori. The agreement between the results of biochemical compositional analysis and the serological data validated our serotyping system. A total of 152 strains from different geographic origins (The Netherlands, Canada, Poland, Italy, and People's Republic of China) were examined for typeability based on the presence of Lewis antigens. One hundred twenty-nine (84.9%) strains were typeable, and 12 different serotyping patterns were observed; 80.9% of the strains contained Lex and/or Le(y) antigens, and 18.4% reacted with the MAb against the related H1 antigen either alone or in combination with the Lex and/or Ley antigen. Our results show that the Lex and Ley antigens are frequently encountered in the LPS of H. pylori strains from various geographic origins. This typing method is an easy-to-perform technique, which can be used for strain differentiation in epidemiological studies of H. pylori infections.

Potential role of molecular mimicry between Helicobacter pylori lipopolysaccharide and host Lewis blood group antigens in autoimmunity.

Helicobacter pylori is involved in gastritis, gastric and duodenal ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. Earlier studies already suggested a role for autoimmune phenomena in H. pylori-linked disease. We now report that lipopolysaccharides (LPS) of H. pylori express Lewis y, Lewis x, and H type I blood group structures similar to those commonly occurring in gastric mucosa. Immunization of mice and rabbits with H. pylori cells or purified LPS induced an anti-Lewis x or y or anti-H type I response, yielding antibodies that bound human and murine gastric glandular tissue, granulocytes, adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma cells. Experimental oral infections in mice or natural infection in humans yielded anti-Lewis antibodies also. The beta chain of gastric (H+,K+)-ATPase, the parietal cell proton pump involved in acid secretion, contained Lewis y epitopes; gastric mucin contained Lewis x and y antigenic determinants. Growth in mice of a hybridoma that secretes H. pylori-induced anti-Lewis y monoclonal antibodies resulted in histopathological evidence of gastritis, which indicates a direct pathogenic role for anti-Lewis antibodies. In conclusion, our observations demonstrate that molecular mimicry between H. pylori LPS and the host, based on Lewis antigens, and provide understanding of an autoimmune mechanism for H. pylori-associated type B gastritis.