Design of a specific peptide against phenolic glycolipid-1 from Mycobacterium leprae and its implications in leprosy bacilli entry.

BACKGROUND: Mycobacterium leprae, the causative agent of Hansen's disease, causes neural damage through the specific interaction between the external phenolic glycolipid-1 (PGL-1) and laminin subunit alpha-2 (LAMA2) from Schwann cells. OBJECTIVE: To design a LAMA2-based peptide that targets PGL-1 from M. leprae. METHODS: We retrieved the protein sequence of human LAMA2 and designed a specific peptide using the Antimicrobial Peptide Database and physicochemical parameters for antimycobacterial peptide-lipid interactions. We used the AlphaFold2 server to predict its three-dimensional structure, AUTODOCK-VINA for docking, and GROMACS programs for molecular dynamics simulations. FINDINGS: We analysed 52 candidate peptides from LAMA2, and subsequent screening resulted in a single 60-mer peptide. The mapped peptide comprises four ß-sheets and a random coiled region. This peptide exhibits a 45% hydrophobic ratio, in which one-third covers the same surface. Molecular dynamics simulations show that our predicted peptide is stable in aqueous solution and remains stable upon interaction with PGL-1 binding. In addition, we found that PGL-1 has a preference for one of the two faces of the predicted peptide, which could act as the preferential binding site of PGL-1. MAIN CONCLUSIONS: Our LAMA2-based peptide targeting PGL-1 might have the potential to specifically block this key molecule, suggesting that the preferential region of the peptide is involved in the initial contact during the attachment of leprosy bacilli to Schwann cells.

The Intergenic Recombinant HLA-B∗46:01 Has a Distinctive Peptidome that Includes KIR2DL3 Ligands.

HLA-B∗46:01 was formed by an intergenic mini-conversion, between HLA-B∗15:01 and HLA-C∗01:02, in Southeast Asia during the last 50,000 years, and it has since become the most common HLA-B allele in the region. A functional effect of the mini-conversion was introduction of the C1 epitope into HLA-B∗46:01, making it an exceptional HLA-B allotype that is recognized by the C1-specific natural killer (NK) cell receptor KIR2DL3. High-resolution mass spectrometry showed that HLA-B∗46:01 has a low-diversity peptidome that is distinct from those of its parents. A minority (21%) of HLA-B∗46:01 peptides, with common C-terminal characteristics, form ligands for KIR2DL3. The HLA-B∗46:01 peptidome is predicted to be enriched for peptide antigens derived from Mycobacterium leprae. Overall, the results indicate that the distinctive peptidome and functions of HLA-B∗46:01 provide carriers with resistance to leprosy, which drove its rapid rise in frequency in Southeast Asia.

Bacillus amyloliquefaciens Q-426 as a potential biocontrol agent against Fusarium oxysporum f. sp. spinaciae.

In recent years, Bacillus species have received considerable attention for the biological control of many fungal diseases. In this study, Bacillus amyloliquefaciens Q-426 was tested for its potential use against a variety of plant pathogens. Our screen for genes involved in the biosynthesis of antifungal agents revealed that the fen and bmy gene clusters are present in the Q-426 genome. Lipopeptides such as bacillomycin D, fengycin A, and fengycin B were purified from the bacterial culture broth and subsequently identified by ESI-mass spectrometry. The minimal inhibitory concentration of fengycin A against Fusarium oxysporum f. sp. spinaciae W.C. Snyder & H.N. Hansen O-27 was determined to be 31.25 µg ml(-1) . However, exposure of fungal cells to 50 µg ml(-1) of fengycin A did not allow permeation of fluorescein diacetate into the cytoplasm through the cell membrane. Moreover, leakage of intracellular inorganic cations, nucleic acid and protein were also not detected, indicating that the fungal cell membrane is not the primary target of action for fengycin A. Profound morphological changes were observed in the F. oxysporum strain and spore germination was completely inhibited, suggesting that 50 µg ml(-1) of fengycin A acts, at least, as a fungistatic agent.

[Antistress cross-protection of UV irradiated yeast cells with participation of extracellular peptide factors].

Antistress effect of extracellular peptides on UV irradiated yeast of different phylogenetic groups was studied. Yeast from different ecotopes and taxonomic groups exposed to UV radiation of a lethal intensity showed a protective effect and reactivating effect with participation of extracellular peptides. The highest protective activity was found in peptide reactivation factors (RFs) of bakery yeast-Saccharomyces cerevisiae, Kluyveromyces fragilis, and Candida utilis; the highest reactivating activity was exhibited by factors of the above-mentioned cultures and Debariomyces hansenii. Cross-protective and reactivating effects of RFs of yeast belonging to different taxonomic groups were demonstrated. Cross-protection increased two to three times after preexposure of reactivation factors to UV light (activation) in contrast to their reactivating effect.

Purification and characterization of an alkaline protease from the marine yeast Aureobasidium pullulans for bioactive peptide production from different sources.

The extracellular alkaline protease in the supernatant of cell culture of the marine yeast Aureobasidium pullulans 10 was purified to homogeneity with a 2.1-fold increase in specific protease activity as compared to that in the supernatant by ammonium sulfate fractionation, gel filtration chromatography (Sephadex G-75), and anion-exchange chromatography (DEAE Sepharose Fast Flow). According to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis data, the molecular mass of the purified enzyme was estimated to be 32.0 kDa. The optimal pH and temperature of the purified enzyme were 9.0 and 45 degrees C, respectively. The enzyme was activated by Cu(2+) (at a concentration of 1.0 mM) and Mn(2+) and inhibited by Hg(2+), Fe(2+), Fe(3+), Zn(2+), and Co(2+). The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride, but weakly inhibited by EDTA, 1-10-phenanthroline, and iodoacetic acid. The K(m) and V(max) values of the purified enzyme for casein were 0.25 mg/ml and 0.0286 micromol/min/mg of protein, respectively. After digestion of shrimp protein, spirulina (Arthospira platensis) protein, proteins of marine yeast strains N3C (Yarrowia lipolytica) and YA03a (Hanseniaspora uvarum), milk protein, and casein with the purified alkaline protease, angiotensin I converting enzyme (ACE) inhibitory activities of the resulting peptides reached 85.3%, 12.1%, 29.8%, 22.8%, 14.1%, and 15.5%, respectively, while the antioxidant activities of these were 52.1%. 54.6%, 25.1%, 35%, 12.5%, and 24.2%, respectively, indicating that ACE inhibitory activity of the resulting peptides from the shrimp protein and antioxidant activity of those produced from the spirulina protein were the highest, respectively. These results suggest that the bioactive peptides produced by digestion of the shrimp protein with the purified alkaline protease have potential applications in the food and pharmaceutical industries.

Computer-assisted prediction of HLA-DR binding and experimental analysis for human promiscuous Th1-cell peptides in the 24 kDa secreted lipoprotein (LppX) of Mycobacterium tuberculosis.

The secreted 24 kDa lipoprotein (LppX) is an antigen that is specific for Mycobacterium tuberculosis complex and M. leprae. The present study was carried out to identify the promiscuous T helper 1 (Th1)-cell epitopes of the M. tuberculosis LppX (MT24, Rv2945c) antigen by using 15 overlapping synthetic peptides (25 mers overlapping by 10 residues) covering the sequence of the complete protein. The analysis of Rv2945c sequence for binding to 51 alleles of nine serologically defined HLA-DR molecules, by using a virtual matrix-based prediction program (propred), showed that eight of the 15 peptides of Rv2945c were predicted to bind promiscuously to >/=10 alleles from more than or equal to three serologically defined HLA-DR molecules. The Th1-cell reactivity of all the peptides was assessed in antigen-induced proliferation and interferon-gamma (IFN-gamma)-secretion assays with peripheral blood mononuclear cells (PBMCs) from 37 bacille Calmette-Guérin (BCG)-vaccinated healthy subjects. The results showed that 17 of the 37 donors, which represented an HLA-DR-heterogeneous group, responded to one or more peptides of Rv2945c in the Th1-cell assays. Although each peptide stimulated PBMCs from one or more donors in the above assays, the best positive responses (12/17 (71%) responders) were observed with the peptide p14 (aa 196-220). This suggested a highly promiscuous presentation of p14 to Th1 cells. In addition, the sequence of p14 is completely identical among the LppX of M. tuberculosis, M. bovis and M. leprae, which further supports the usefulness of Rv2945c and p14 in the subunit vaccine design against both tuberculosis and leprosy.

Identification of an I-Ad restricted peptide on the 65-kilodalton heat shock protein of Mycobacterium avium.

The 65 kilodalton heat shock protein (Hsp65) from mycobacterial species elicits immune responses and in some cases protective immunity. Here we have used a DNA sublibrary approach to identify antigenic fragments of Mycobacterium avium Hsp65 and a synthetic peptide approach to delineate CD4+ T cell determinants. A panel of Hsp65 reactive CD4+ T cell clones was established from lymph node cells obtained from BALB/c mice immunized with recombinant Hsp65. The clones were tested for proliferative reactivity against the products of the DNA sublibrary of the hsp65 gene. A T cell epitope, restricted by the I-Ad molecule, was identified within the C-terminal region of Hsp65 and the minimal epitope (amino acid residues 489-503) delineated using overlapping peptides spanning the C-terminal fragment. Additionally, the CD4+ T cell clone recognizing this epitope also responded to native Hsp65 present in M. avium lysates by both proliferation and cytokine production, indicating that the epitope was present and processed similarly both in the native and the recombinant forms of Hsp65. This sequence identified in BALB/c mice (Hsp65 489-503) is identical in other mycobacteria, notably M. tuberculosis, M. bovis and M. leprae, suggesting the epitope may have wider application in murine models of other mycobacterial infections.

Solution structure of the A loop of 23S ribosomal RNA.

The A loop is an essential RNA component of the ribosome peptidyltransferase center that directly interacts with aminoacyl (A)-site tRNA. The A loop is highly conserved and contains a ubiquitous 2'-O-methyl ribose modification at position U2552. Here, we present the solution structure of a modified and unmodified A-loop RNA to define both the A-loop fold and the structural impact of the U2552 modification. Solution data reveal that the A-loop RNA has a compact structure that includes a noncanonical base pair between C2556 and U2552. NMR evidence is presented that the N3 position of C2556 has a shifted pKa and that protonation at C2556-N3 changes the C-U pair geometry. Our data indicate that U2552 methylation modifies the A-loop fold, in particular the dynamics and position of residues C2556 and U2555. We compare our structural data with the structure of the A loop observed in a recent 50S crystal structure [Ban, N., Nissen, P., Hansen, J., Moore, P. B. & Steitz, T. A. (2000) Science 289, 905--920; Nissen, P., Hansen, J., Ban, N., Moore, P. B. & Steitz, T. A. (2000) Science 289, 920--930]. The solution and crystal structures of the A loop are dramatically different, suggesting that a structural rearrangement of the A loop must occur on docking into the peptidyltransferase center. Possible roles of this docking event, the shifted pKa of C2556 and the U2552 2'-O-methylation in the mechanism of translation, are discussed.

Evidence for human CD4+ T cells in the CD1-restricted repertoire: derivation of mycobacteria-reactive T cells from leprosy lesions.

Both the CD4-CD8- (double negative) and CD4-CD8+ T cell lineages have been shown to contain T cells which recognize microbial lipid and glycolipid Ags in the context of human CD1 molecules. To determine whether T cells expressing the CD4 coreceptor could recognize Ag in the context of CD1, we derived CD4+ T cell lines from the lesions of leprosy patients. We identified three CD4+ Mycobacterium leprae-reactive, CD1-restricted T cell lines: two CD1b restricted and one CD1c restricted. These T cell lines recognize mycobacterial Ags, one of which has not been previously described for CD1-restricted T cells. The response of CD4+ CD1-restricted T cells, unlike MHC class II-restricted T cells, was not inhibited by anti-CD4 mAb, suggesting that the CD4 coreceptor does not impact positive or negative selection of CD1-restricted T cells. The CD4+ CD1-restricted T cell lines produced IFN-gamma and GM-CSF, the Th1 pattern of cytokines required for cell-mediated immunity against intracellular pathogens, but no detectable IL-4. The existence of CD4+ CD1-restricted T cells that produce a Th1 cytokine pattern suggests a contributory role in immunity to mycobacterial infection.

Structural features of MHC class I molecules that might facilitate alternative pathways of presentation.

Comparisons of the structures of different mouse MHC class I molecules define how polymorphic residues determine the unique structural motif and atomic anchoring of their bound peptides. Here, Ted Hansen and colleagues speculate that quantitative differences in how class I molecules interact with peptide, beta2-microglobulin and molecular chaperones that facilitate peptide loading might determine their relative participation in different pathways of antigen presentation.

A DR17-restricted T cell epitope from a secreted Mycobacterium tuberculosis antigen only binds to DR17 molecules at neutral pH.

The assembly of peptide-major histocompatability class II complexes in vitro is accelerated at low pH, comparable to that found in the intracellular compartments of metabolically active antigen-presenting cells (APC). Mycobacteria such as Mycobacterium tuberculosis reside in phagosomes with only mildly acidic pH. Therefore, we investigated the pH dependency of peptide-HLA-DR binding for several T cell epitopes of mycobacterial proteins, focussing particularly on well-defined, immunodominant HLA-DR17(3)-restricted T cell epitopes: peptide (p) 3-13 from the cytoplasmic 65-kDa heat shock protein of M. tuberculosis/M. leprae, and peptide 56-65 from the secreted 30/31-kDa protein from M. tuberculosis/M. leprae. p3-13 bound to purified, cell-free DR17 under both acidic and neutral conditions. Four other, unrelated DR17-binding peptides showed the same pH-dependent binding characteristics as p3-13. p56-65, however, only bound to purified DR17 at pH 7 but not at all at pH 4.5. These DR17 peptide binding data were confirmed in cell-bound DR17, in T cell stimulation assays in which fixed APC were peptide-pulsed at acidic or neutral pH before addition of peptide-specific DR17-restricted T cells. As far as we are aware, p56-65 is the only human T cell epitope binding to HLA exclusively at neutral pH. The binding characteristics of p56-65 may reflect dominant processing in alternative, less acidic vacuolar compartments specifically related to the generation of epitopes from (secreted) mycobacterial proteins. The observation that p56-65 is an immunodominant epitope for anti-mycobacterial T cells suggests the relevance of such novel processing compartments in T cell-mediated immunity.

Recognition of Mycobacterium leprae recombinant 18,000 MW epitopes by IgG subclasses in leprosy.

IgG subclasses are known to be differentially regulated by cytokines (elaborated by activated T cells), which act as growth factors and immunoglobulin switch factors on B cells. In leprosy, we have previously shown that IgG subclass antibodies to a purified recombinant antigen of Mycobacterium leprae (18,000 MW) are restricted to IgG1 and IgG3 across the disease spectrum. The only significant difference observed was that lepromatous patients with low to undetectable T-cell responses showed a strong correlation between IgG1 and IgG3 (P < 0.001) antibodies while tuberculoid patients who showed strong T-cell responses did not show such a correlation. To examine if these differences were related to T-cell-mediated class switching in tuberculoid leprosy patients, we have studied epitope recognition by IgG1 and IgG3 using a panel of synthetic peptides spanning the 18,000 MW molecule in an enzyme-linked immunosorbent assay (ELISA). In lepromatous patients there was little similarity in peptide recognition by IgG1 and IgG3, with IgG1 recognition being restricted to a single dominant carboxy-terminal peptide while the IgG3 antibodies recognized a diverse set of peptides in the N-terminal half of the 18,000 MW molecule. In tuberculoid patients both IgG1 and IgG3 antibody showed recognition of similar peptides in the N-terminal half of the 18,000 MW molecule. Our results therefore support the hypothesis that immunoglobulin class switching is occurring in tuberculoid but not in lepromatous patients.