Conventional analyses distinguish between antimicrobial peptides by differences in amino acid sequence. Yet structural paradigms common to broader classes of these molecules have not been established. The current analyses examined the potential conservation of structural themes in antimicrobial peptides from evolutionarily diverse organisms. Using proteomics, an antimicrobial peptide signature was discovered to integrate stereospecific sequence patterns and a hallmark three-dimensional motif. This striking multidimensional signature is conserved among disulfide-containing antimicrobial peptides spanning biological kingdoms, and it transcends motifs previously limited to defined peptide subclasses. Experimental data validating this model enabled the identification of previously unrecognized antimicrobial activity in peptides of known identity. The multidimensional signature model provides a unifying structural theme in broad classes of antimicrobial peptides, will facilitate discovery of antimicrobial peptides as yet unknown, and offers insights into the evolution of molecular determinants in these and related host defense effector molecules.
The effect of copper deficiency on hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key enzyme regulating cholesterol biosynthesis, was investigated in the rat. Male weanling rats were fed synthetic diets containing copper levels of 6.5 ppm (adequate), 1.6 ppm (marginal) and 0.9 ppm (deficient) for 7 wks. At sacrifice plasma lipids, hepatic cholesterol, and hepatic HMG-CoA reductase, both total and active forms, were determined. Consistent with previous findings, plasma cholesterol and triglyceride levels were significantly elevated, 26% and 104% respectively, in copper deficient rats. Copper deficiency resulted in a significant decrease in hepatic total cholesterol levels. This change was due to decreased free cholesterol levels since cholesterol ester remained unchanged. Total HMg-CoA reductase was significantly elevated 2-fold with copper deficiency while the active form of the enzyme was not changed. This increase in reductase activity due to copper deficiency corresponds with the expected induction response to decreased hepatic cholesterol levels observed in this study. These data support the theory that copper deficiency results in a hyper-cholesterolemic state in the rat associated with increased hepatic cholesterol synthesis.
Different pathogens share similar medical settings and rely on similar virulence strategies to cause infections. We have previously applied 3-D computational modeling and bioinformatics to discover novel antigens that target more than one human pathogen. Active and passive immunization with the recombinant N-terminus of Candida albicans Hyr1 (rHyr1p-N) protect mice against lethal candidemia. Here we determine that Hyr1p shares homology with cell surface proteins of the multidrug resistant Gram negative bacterium, Acinetobacter baumannii including hemagglutinin (FhaB) and outer membrane protein A (OmpA). The A. baumannii OmpA binds to C. albicans Hyr1p, leading to a mixed species biofilm. Deletion of HYR1, or blocking of Hyr1p using polyclonal antibodies, significantly reduce A. baumannii binding to C. albicans hyphae. Furthermore, active vaccination with rHyr1p-N or passive immunization with polyclonal antibodies raised against specific peptide motifs of rHyr1p-N markedly improve survival of diabetic or neutropenic mice infected with A. baumannii bacteremia or pneumonia. Antibody raised against one particular peptide of the rHyr1p-N sequence (peptide 5) confers majority of the protection through blocking A. baumannii invasion of host cells and inducing death of the bacterium by a putative iron starvation mechanism. Anti-Hyr1 peptide 5 antibodies also mitigate A. baumannii /C. albicans mixed biofilm formation in vitro. Consistent with our bioinformatic analysis and structural modeling of Hyr1p, anti-Hyr1p peptide 5 antibodies bound to A. baumannii FhaB, OmpA, and an outer membrane siderophore binding protein. Our studies highlight the concept of cross-kingdom vaccine protection against high priority human pathogens such as A. baumannii and C. albicans that share similar ecological niches in immunocompromised patients.
ABSTRACT Candida albicans is usually a harmless human commensal. Because inflammatory responses are not normally induced by colonization, antimicrobial peptides are likely integral to first-line host defense against invasive candidiasis. Thus, C. albicans must have mechanisms to tolerate or circumvent molecular effectors of innate immunity and thereby colonize human tissues. Prior studies demonstrated that an antimicrobial peptide-resistant strain of C. albicans , 36082 R , is hypervirulent in animal models versus its susceptible counterpart (36082 S ). The current study aimed to identify a genetic basis for antimicrobial peptide resistance in C. albicans . Screening of a C. albicans genomic library identified SSD1 as capable of conferring peptide resistance to a susceptible surrogate, Saccharomyces cerevisiae . Sequencing confirmed that the predicted translation products of 36082 S and 36082 R SSD1 genes were identical. However, Northern analyses corroborated that SSD1 is expressed at higher levels in 36082 R than in 36082 S . In isogenic backgrounds, ssd1 Δ/ ssd1 Δ null mutants were significantly more susceptible to antimicrobial peptides than parental strains but had equivalent susceptibilities to nonpeptide stressors. Moreover, SSD1 complementation of ssd1 Δ/ ssd1 Δ mutants restored parental antimicrobial peptide resistance phenotypes, and overexpression of SSD1 conferred enhanced peptide resistance. Consistent with these in vitro findings, ssd1 null mutants were significantly less virulent in a murine model of disseminated candidiasis than were their parental or complemented strains. Collectively, these results indicate that SSD1 is integral to C. albicans resistance to host defense peptides, a phenotype that appears to enhance the virulence of this organism in vivo.
Mammalian platelets release platelet microbicidal proteins (PMPs) as components of their antimicrobial armamentarium. The present studies defined the structure of PMP-1 and examined its structure-activity relationships. Amino acid sequencing and mass spectroscopy demonstrated that distinct N-terminal polymorphism variants of PMP-1 isolated from nonstimulated or thrombin-stimulated platelets arise from a single PMP-1 propeptide. Sequence data (NH(2)-[S]D(1)DPKE(5)SEGDL(10)HCVCV(15)KTTSL(20) . . .) enabled cloning of PMP-1 from bone marrow and characterization of its full-length cDNA. PMP-1 is translated as a 106-amino-acid precursor and is processed to yield 73-residue (8,053 Da) and 72-residue (7,951-Da) variants. Searches with the BLAST program and sequence alignments demonstrated the homology of PMP-1 to members of the mammalian platelet factor 4 (PF-4) family of proteins. On the basis of phylogenetic relatedness, congruent sequence motifs, and predicted three-dimensional structures, PMP-1 shares the greatest homology with human PF-4 (hPF-4). By integration of its structural and antimicrobial properties, these results establish the identity of PMP-1 as a novel rabbit analogue of the microbicidal chemokine (kinocidin) hPF-4. These findings advance the hypothesis that stimuli in the setting of infection prompt platelets to release PF-4-class or related kinocidins, which have structures consistent with their likely multiple roles that bridge molecular and cellular mechanisms of antimicrobial host defense.
