Paul R. Hansen

Efficient protection against Mycobacterium tuberculosis by vaccination with a single subdominant epitope from the ESAT‐6 antigen

We have investigated the vaccine potential of two peptides derived from the 6‐kDa early secretory antigenic target (ESAT)‐6 antigen in the mouse model of tuberculosis. The peptides were both strongly immunogenic in B6CBAF1 (H‐2b,k) mice and primed recall responses of the same intensity after immunization. However, both tuberculosis infection and immunization with ESAT‐6 resulted in responses focused towards ESAT‐61 – 20. Multiple antigen peptide constructs as well as free peptides were emulsified with dimethyl dioctadecylammonium bromide / monophosphoryl lipid A / IL‐2 and tested as experimental vaccines in an i. v. and aerosol model of tuberculosis in mice. The peptide were highly immunogenic and induced cellular responses of the same magnitude. However, only vaccines based on the subdominant ESAT‐651 – 70 epitope promoted significant levels of protective immunity and the level of protection was equivalent to that achieved with ESAT‐6 and BCG. These findings demonstrate the potential of peptide‐based vaccines against tuberculosis and indicate that there is not direct correlation between the hierarchy of response to naturally processed peptides and their ability to induce protective immunity against Mycobacterium tuberculosis.

Use of peptide antibodies to probe for the mitoxantrone resistance-associated protein MXR/BCRP/ABCP/ABCG2

Recent studies have characterized the ABC half-transporter associated with mitoxantrone resistance in human cancer cell lines. Encoded by the ABCG2 gene, overexpression confers resistance to camptothecins, as well as to mitoxantrone. We developed four polyclonal antibodies against peptides corresponding to four different epitopes on the mitoxantrone resistance-associated protein, ABCG2. Three epitopes localized on the cytoplasmic region of ABCG2 gave rise to high-affinity antibodies, which were demonstrated to be specific for ABCG2. Western blot analysis of cells with high levels of ABCG2 showed a single major band of the expected 72-kDa molecular size of ABCG2 under denaturing conditions. Immunoblot analysis performed under non-reducing conditions and after treatment with cross-linking reagents demonstrated a molecular weight shift from 72 kDa to several bands of 180 kDa and higher molecular weight, suggesting detection of dimerization products of ABCG2. Evidence of N-linked glycosylation was also obtained using tunicamycin and N-glycosidase F. Finally, both by light, fluorescence and electron microscopic immunohistochemical staining, we demonstrate cytoplasmic and predominantly plasma membrane localization of ABCG2 in cell lines with high levels of expression. Plasma membrane staining was observed on the surface of the chorionic villi in placenta. These results support the hypothesis that ABCG2 is an ABC half-transporter that forms dimers in the plasma membrane, functioning as an ATP-dependent outward pump for substrate transport.

Sinapis phylogeny and evolution of glucosinolates and specific nitrile degrading enzymes

Levels of sinalbin (4-hydroxybenzylglucosinolate) and 28 other glucosinolates were determined in leaves and roots of 20 species that were either phylogenetically close to Sinapis alba, Sinapis arvensis, or Sinapis pubescens (tribe Brassiceae, Brassicaceae), or were expected to contain arylalkyl nitrilase activity. Comparison with a molecular phylogenetic tree based on ITS DNA sequences identified two separate occurrences of sinalbin. The first in a group of species related to S. alba (including members of the genera Coincya and Kremeriella); and the second in S. arvensis, nested among sinalbin deficient species. Significant 4-hydroxyphenylacetonitrile degrading enzyme activity was found in both S. alba and S. arvensis, but in S. alba the major product was the corresponding carboxylic acid, while in S. arvensis the major product was the amide. Both investigated enzyme activities, nitrilase and nitrile hydratase, were specific, accepting only certain arylacetonitriles such as 4-hydroxy and 4-methoxyphenylacetonitrile. Only the S. alba enzyme required an oxygen in para position of the substrate, as found in sinalbin. Indole-3-acetonitrile, arylcyanides, and arylpropionitriles were poor substrates. The nitrilase activity of S. alba was quantitatively comparable to that reported in the monocot Sorghum bicolor (believed to be involved in cyanogenic glycoside metabolism). Glucosinolates derived from methionine were found in all Sinapis clades. Glucosinolate patterns suggested a complex evolution of glucosinolates in the investigated species, with several apparent examples of abrupt changes in glucosinolate profiles including chain length variation and appearance of glucosinolates derived from branched-chain amino acids. NMR data for desulfated homosinalbin, 9-methylsulphonylnonylglucosinolate, 3-methylpentylglucosinolate and related glucosinolates are reported, and a facultative connection between sinalbin and specific nitrilases is suggested.

Antimicrobial Activities of Twenty Lysine-Peptoid Hybrids against Clinically Relevant Bacteria and Fungi

Background: This paper describes the antimicrobial activities of 20 lysine-peptoid hybrids against a selection of clinically relevant bacteria and fungi. Methods: Minimal inhibitory concentrations were determined against methicillin-susceptible Staphylococcus aureus (ATCC 29213), methicillin-resistant S. aureus (ATCC 33591), vancomycin-intermediate S. aureus (ATCC 700699 MU50), vancomycin-resistant Enterococcus faecium (ATCC 700221), Pseudomonas aeruginosa (ATCC 27853), Salmonella typhimurium (clinical isolate), Klebsiella pneumoniae (clinical isolate), amphotericin-B-resistant C. albicans (ATCC 200955) and Cryptococcus neoformans (clinical isolate). Results: The lysine-peptoid hybrids proved to be active against all strains tested, except K. pneumoniae. For each susceptible strain, we identified at least 4 lysine-peptoid hybrids showing excellent activity. The most active compounds displayed minimal inhibitory concentrations ranging from ≤1.6 to 6.25 µM. Conclusion: This study demonstrates that lysine-peptoid hybrids show activity against drug-resistant pathogens.

Antigenic Profiling of a Chlamydia trachomatis Gene-Expression Library

The obligate intracellular bacterium Chlamydia trachomatis is the causative agent of sexually transmitted chlamydia infections. A panel of 116 recombinant C. trachomatis proteins was evaluated comparatively to characterize both cell-mediated and humoral immune responses in patients with confirmed C. trachomatis genital infection. The antigens identified were categorized as being recognized exclusively by T cells (CT004, CT043, CT184, CT509, and CT611), B cells (CT082, CT089, CT322, CT396, and CT681), or both T cells and B cells (CT110 and CT443). This grouping of C. trachomatis antigens was correlated to their predicted cellular localization. The comparative evaluation presented here indicates that T cell antigens are located in all bacterial compartments, whereas antibody targets are mainly localized to the outer membrane (P = .0013). Overall, we have identified 5 T cell antigens, 5 B cell antigens, and 2 T/B cell antigens that are potential components for a future chlamydia vaccine.

The chaperone and potential mannan-binding lectin (MBL) co-receptor calreticulin interacts with MBL through the binding site for MBL-associated serine proteases.

The chaperone calreticulin has been suggested to function as a C1q and collectin receptor. The interaction of calreticulin with mannan‐binding lectin (MBL) was investigated by solid‐phase binding assays. Calreticulin showed saturable and time‐dependent binding to recombinant MBL, provided that MBL was immobilized on a solid surface or bound to mannan on a surface. The binding was non‐covalent and biphasic with an initial salt‐sensitive phase followed by a more stable salt‐insensitive interaction. For plasma‐derived MBL, known to be complexed with MBL‐associated serine proteases (MASPs), no binding was observed. Interaction of calreticulin with recombinant MBL was fully inhibited by recombinant MASP‐2, MASP‐3 and MAp19, but not by the MASP‐2 D105G and MAp19 Y59A variants characterized by defective MBL binding ability. Furthermore, MBL point mutants with impaired MASP binding showed no interaction with calreticulin. Comparative analysis of MBL with complement component C1q, its counterpart of the classical pathway, revealed that they display similar binding characteristics for calreticulin, providing further indication that calreticulin is a common co‐receptor/chaperone for both proteins. In conclusion, the potential MBL co‐receptor calreticulin binds to MBL at the MASP binding site and the interaction may involve a conformational change in MBL.

Complex metabolism of aromatic glucosinolates in Pieris rapae caterpillars involving nitrile formation, hydroxylation, demethylation, sulfation, and host plant dependent carboxylic acid formation.

We investigated the metabolism of two chain elongated phenolic glucosinolates and the corresponding O-methyl derivatives upon ingestion by caterpillars of the butterfly Pieris rapae (L.). The glucosinolates (GSLs) were 4-hydroxyphenethylGSL, (R)-2-hydroxy-2-(4-hydroxyphenyl)ethylGSL, 4-methoxyphenethylGSL, and (R)-2-hydroxy-2-(4-methoxyphenyl)ethylGSL, variously occurring in foliage of two Arabis species: Arabis hirsuta (L.) Scop. and Arabis soyeri Reut. & Huet subsp. subcoriacea (Gren. ex Nyman) Breitstr. (Brassicaceae). Frass from caterpillars reared on each Arabis species contained two sulfated nitriles (4-sulfates of 3-(4-hydroxyphenyl)propanenitrile and 3-hydroxy-3-(4-hydroxyphenyl)propanenitrile) as apparent GSL metabolites. Comparison of glucosinolate levels in foliage and levels of sulfated nitriles in frass, and experiments with isolated GSLs spiked to crucifer foliage and ingested by P. rapae, demonstrated that phenolic GSLs and the corresponding O-methyl derivatives were metabolised to sulfated nitriles, and that metabolites lacking a beta-hydroxy group were partially hydroxylated in this position during metabolism in P. rapae. In contrast, an induction experiment did not show increased levels of beta-hydroxylated GSLs in A. soyeri plants upon caterpillar feeding. Frass contents of other putative GSL metabolites from the interaction with the two Arabis species differed significantly; caterpillars reared on A. hirsuta excreted significant amounts of four carboxylic acids (3-(4-hydroxyphenyl)propanoic acid, 3-hydroxy-3-(4-hydroxyphenyl)propanoic acid, and the corresponding 4-sulfates), which were low or absent when the caterpillars were reared on A. soyeri. The excreted carboxylic acids could be formed by hydrolysis of nitriles to carboxylic acids in caterpillar guts by an ingested nitrilase enzyme from A. hirsuta foliage; this hypothesis was supported by demonstration of 3-(4-hydroxyphenyl)propanenitrile hydrolysing nitrilase activity (E.C. 3.5.5.x) in a crude A. hirsuta extract. Some hypothetic metabolites, glycine conjugates of phenolic carboxylic acids, were not detected. Conditions for group separation and HPLC isolation of intact GSLs and sulfated metabolites were optimised, NMR spectroscopic data of the compounds are reported, and evolutionary and ecological implications are discussed.

Structure–activity study of the antibacterial peptide fallaxin

Fallaxin is a 25‐mer antibacterial peptide amide, which was recently isolated from the West Indian mountain chicken frog Leptodactylus fallax. Fallaxin has been shown to inhibit the growth of several Gram‐negative bacteria including Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Here, we report a structure–activity study of fallaxin based on 65 analogs, including a complete alanine scan and a full set of N‐ and C‐terminal truncated analogs. The fallaxin analogs were tested for hemolytic activity and antibacterial activity against methicillin‐resistant Staphylococcus aureus (MRSA), vancomycin‐intermediate resistant S. aureus, (VISA), methicillin‐susceptible S. aureus (MSSA), E. coli, K. pneumoniae, and P. aeruginosa. We identified several analogs, which showed improved antibacterial activity compared to fallaxin. Our best candidate was FA12, which displayed MIC values of 3.12, 25, 25, and 50 μM against E. coli, K. pneumoniae, MSSA, and VISA, respectively. Furthermore, we correlated the antibacterial activity with various structural parameters such as charge, hydrophobicity 〈H〉, mean hydrophobic moment 〈μH〉, and α‐helicity. We were able to group the active and inactive analogs according to mean hydrophobicity 〈H〉 and mean hydrophobic moment 〈μH〉. Far‐UV CD‐spectroscopy experiments on fallaxin and several analogs in buffer, in TFE, and in membrane mimetic environments (small unilamellar vesicles) indicated that a coiled‐coil conformation could be an important structural trait for antibacterial activity. This study provides data that support fallaxin analogs as promising lead structures in the development of new antibacterial agents.

Rational Design of Alpha-Helical Antimicrobial Peptides: Do's and Don'ts.

Antimicrobial peptides (AMPs) are promising candidates for battling multiresistant bacteria. Despite extensive research, structure–activity relationships of AMPs are not fully understood, and there is a lack of structural data relating to AMPs in lipids. Here we present the NMR structure of anoplin (GLLKRIKTLL‐NH2) in a micellar environment. A vast library of substitutions was designed and tested for antimicrobial and hemolytic activity, as well as for changes in structure and lipid interactions. This showed that improvement of antimicrobial activity without concomitant introduction of strong hemolytic activity can be achieved through subtle increases in the hydrophobicity of the hydrophobic face or through subtle increases in the polarity of the hydrophilic face of the helix, or—most efficiently—a combination of both. A set of guidelines based on the results is given, for assistance in how to modify cationic α‐helical AMPs in order to control activity and selectivity. The guidelines are finally tested on a different peptide.

Second-generation nanofiltered plasma-derived mannan-binding lectin product: process and characteristics.

Background and Objectives  Mannan‐binding lectin (MBL) is an important component of the innate immune defence; it binds to carbohydrate structures on pathogenic micro‐organisms resulting in complement activation and opsonization. Individuals with low MBL levels are at risk of recurrent and severe infections. Substitution therapy with plasma‐derived MBL is a promising treatment of diseases associated with MBL deficiency. A first‐generation MBL product has been shown to be safe and well tolerated, and patients have benefited from MBL treatment. Following is a description of the development of a nanofiltered second‐generation MBL product from Cohn fraction III, with the use of a new affinity matrix for MBL purification and the characteristics of this improved product.