Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2017 Jul 15.
Published in final edited form as: Anal Biochem. 2016 Apr 22;505:29–35. doi: 10.1016/j.ab.2016.04.001

Structure determination of lipopeptides from Mycobacterium avium subspecies paratuberculosis and identification of antigenic lipopeptide probes

Katsuhiko Mitachi 1, Lekh Nath Sharma Gautam 1, Jeffrey H Rice 2, Keiko Eda 2, Ashutosh Wadhwa 2, Eiichi Momotani 3, Joseph P Hlopak 2, Shigetoshi Eda 2, Michio Kurosu 1
PMCID: PMC4899129  NIHMSID: NIHMS780956  PMID: 27114041

Abstract

Mycobacterium avium subsp. paratuberculosis (MAP) causes chronic illnesses mostly in ruminants. MAP infection of intestinal tissue triggers a fatal inflammatory disorder, Johne’s disease (Paratuberculosis). Development of fast and reliable diagnostic methods for Johne’s disease in clinically suspected ruminants requires the discovery of MAP-specific antigens that induce immune responses. Despite a long-time interest in finding such antigens that can detect serum antibody responses with high sensitivity, the antigens currently used for a diagnosis of the MAP infections are the crude extracts from the whole cell. We performed the serum antibody response assay-guided purification of the ethanol extract from MAP isolated from an infected cow. As the results of extensive fractionations and in vitro assays, we identified that arachidil-D-Phe-N-Me-L-Val-L-Ile-L-Phe-L-Ala-OH (named lipopeptide IIβ, 3) exhibited the highest antibody binding activity in serum of a MAP-infected cattle compared to the other lipopeptides isolated from MAP. The absolute chemistry of 3 was determined unequivocally via our HPLC-amino acid data bases. α-Amino lipopeptide IIβ and its fluorescent probes were synthesized and evaluated in serum antibody binding activity assays. Lipopeptide IIβ-(2S)-NH2 (9) and its dansyl and FITC probes (10 and 11) exhibited antibody-mediate binding activity, and thus, such MAP specific lipopeptide probes can be potential biomarkers for the development of rapid and accurate diagnosis of Johne’s disease.

Keywords: Lipopeptide, New chiral reprivatizing agents, Mycobacterium avium subspecies paratuberculosis, Johne’s disease, Lipopeptide probes

Mycobacterium avium subsp. paratuberculosis (MAP) causes life-threatening chronic illnesses in animals.1–4 Unlike other Mycobacterium spp., MAP can adapt to gastrointestinal tract of ruminants, and its infections trigger a chronic inflammatory disorder, Johne’s disease (Paratuberculosis).5,6 Particularly, bovine Johne’s disease causes serious economic impact in the U.S. and other countries.7–10 Because neither practical treatment nor effective vaccine is available for bovine Johne’s disease, rapid and accurate diagnostic methods are very important to confirm the diseases.11–13 The enzyme-linked immunosorbent assay (ELISA) for detection of serum antibody is the most rapid and least costly test.14–16 However, antigens used for this assay are the crude mixtures derived from bacterial whole-cells that may cause false- positive and -negative results. Development of early diagnosis of MAP infections has been hampered by lack of studies on MAP-specific antigens.11 Eckstein et. al. reported that Para-LP-01 (Fig. 5) is a major lipopeptide, which was isolated as a mixture of fatty acids.17 Para-LP-01 is the only known lipopeptide isolated from MAP to date. Unfortunately, in our studies it was concluded that Para-LP-01 did not show strong antibody response and cell-mediated immune response binding activities at a practical concentration (10 μg/mL). Therefore, we aimed to identify a new and more sensitive MAP-specific diagnostic antigen for Johne’s disease ELISA tests. We previously reported that EtOH extract of MAP could be used for the detection of anti-MAP antibodies in serum and milk of MAP infected cattle, indicating that antigenic small molecules should exist in the EtOH extract.19 Such molecules could also be promising molecules to study humoral immunity in Johne’s disease.11,20

Figure 5.

Figure 5

Open in a new tab

Conformational analyses of lipopeptide IIβ; one of the most plausible conformers is shown.

Materials and methods

Chemical materials and methods

Difco Middlebrook 7H10 agar, Middlebrook 7H9 broth, Tryptic soy agar, Tryptic soy broth, MOPS, tris(hydroxymethyl)aminomethane, 2-mercaptoethanol, sucrose and triton-X 100 were purchased from Sigma-Aldrich. ADC enrichment was purchased from Fisher Scientific. Magnesium chloride and potassium chloride were obtained from VWR. All reagents and solvents were commercial grade and were used as received without further purification unless otherwise noted. Flash chromatography was performed with Whatman silica gel (Purasil 60 Å, 230–400 Mesh). Analytical thin-layer chromatography was performed with 0.25 mm coated commercial silica gel plates (EMD, Silica Gel 60F254) visualizing at 254 nm, or developed with ceric ammonium molybdate or anisaldehyde solutions by heating on a hot plate. 1H-NMR spectral data were obtained using 400, and 500 MHz instruments. 13C-NMR spectral data were obtained using 100 and125 MHz instruments. For all NMR spectra, δ values are given in ppm and J values in Hz.

Synthesis and chemical characterizations of lipopeptide IIβ (3) and its fluorescent probes, 11 and all new lipopeptide analogs studied in this article are summarized in supporting information.†

Serum samples

Four serum samples used in this study were kindly provided by Dr. Randy Capsel at the United States Department of Agriculture/National Veterinary Services Laboratory. Two Johne’s disease (JD)-negative serum samples (ID 15 and 22) were collected from female Holstein cattle (3.3 and 5.9 years old, respectively) in a Minnesota dairy farm where no Johne’s disease has been reported. Two JD-positive samples (ID 238 and 242) were collected from female Holstein cattle (4.0 and 5.0 years old, respectively) that were tested positive for Johne’s disease by a commercial enzyme-linked immunosorbent assay (ELISA) (IDEXX Laboratories, Westbrook, MA) and a standard fecal culture test.

Preparation of ethanol extract of Mycobacterium avium subsp. paratuberculosis (MAP)

MAP strain (Linda) used for the EVELISA antigen preparation were obtained from the Agricultural Research Service, U.S. Department of Agriculture (Ames, Iowa). MAP organisms were cultured in Middlebrook’s medium (Becton Dickinson, Cockeysville, MD) with the addition of 0.05% Tween 80 (Fisher Scientific, Fair Lawn, NJ) and 2 mg/l mycobactin J (Allied Monitor, Fayette, MO) at 37°C until used for experimentation. Ethanol extract of MAP was prepared as described previously. Briefly, bacilli of MAP were harvested from the liquid culture, suspended in 80% ethanol, and agitated by vortex to dislodge surface antigens. After removing MAP bacilli by centrifugation, ethanol extract was dried under vaccum.

Thin layer chromatography (TLC) of ethanol extract of Mycobacterium spp

MAP strains (K10 and Linda) were cultured and ethanol extract was prepared as described above. M. gordonae (TMC 1324), M. phlei (TMC 1548), M. smegmatis (TMC 1546), M. terrae (TMC 1450), M. avium subsp. avium (TMC 706, TMC 724), M. bovis BCG (TMC 1011), M. flavescens (TMC 1541), M. fortuitum (TMC 1529), M. intracellulae (TMC 1406), M. kansasii (TMC 1204), M. scroflaceum (TMC 1323), and M. szulgai (TMC 1328) by Dr. Pamera Small (the University of Tennessee Knoxville). These are cultured as described above for MAP except that the medium did not contain mycobactin J or the conditions provided by ATCC. Bacilli were harvested from liquid cultures at stationary phase and centrifuged at 5,000×g for 10 min. The pellet (~80 mg) was resuspended in 1 mL of 80% EtOH, and agitated by vortex at room temperature for 1 min., followed by centrifugation at 10,621 × g for 10 min. The resulting supernatant was filtered through a 0.22μm filter. The 1 mL of 80% EtOH extracted antigens was dried and further purified using a variation of the Folch Wash Method, resupended in 1 mL of CHCl3 : MeOH (2:1), and rocked for two hours. The Folch extraction was performed by the addition of 0.2 volumes of water, rocked for 1 h and centrifuged at 10,621 × g for 10 min. The CHCl3 layer was concentrated by drying overnight in fume hood. For one dimensional TLC, the chloroform fraction was dissolved in 1.6 mL of chloroform and 10 μl of the solution was loaded onto aluminum-backed silica-gel-60 plates (Merck), then developed with CHCl3-MeOH-H2O (150:10:1). Developed plates were sprayed to detect general components with Ce(SO4)2/((NH4)2MoO4 in 2 M sulfuric acid and charred. For two dimensional TLC, 10 μg dried weight of the chloroform layer dissolve in 10 μl of CHCl3 was loaded onto the silica gel and developed with CHCl3 : MeOH (94 : 6) for the first dimension and then with toluene : acetone (80 : 20) for the second dimension followed by staining with CuSO4 (3%), H3PO4 (15%) in H2O.

Detection of antibody binding to MAP antigens

Antibody binding to purified and chemically synthesized MAP antigens used in this study was conducted as described previously. MAP antigens were solubilized into dimethylsulfoxide (DMSO) at the concentration of 10 mg/ml and stored at −20°C until use. The stock solution of MAP antigen was diluted to desired concentration (e.g. 10 μg/mL) with absolute ethanol. The diluted MAP antigen solution was inoculated into a well of a 96-well plate (medium binding, Corning, NY) by evaporating ethanol, reacted with diluted (1:100) serum samples followed by horseradish peroxidase-labeled goat anti-bovine IgG (H+L) polyclonal antibody (diluted 1:500, Jackson ImmunoResearch Lab, Westgroup, PA). For dilution of serum sample and the secondary antibody, 10 mM phosphate buffered saline (pH 7.0) containing 0.05% Tween 20 and 10% SuperBlock (Thermo Scientific, Rockford, IL) was used. Binding of serum antibody was detected by using a substrate of horseradish peroxidase (2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) according to the manufacturer’s instructions.

Results and discussion

A number of bacterial species produce lipopeptides.21 Most of the antigenic lipids of Mycobacterium subspecies contain sugar moieties (e.g. 2,3-diacyl trehalose, phenolic glycolipids, lipooligosaccharides, and trehalose dimycolate).17,22 Interestingly, the lipid profile of MAP was quite different from that of the other Mycobacterium spp. (Fig. 1A). MAP is the closest species to M. avium subsp. avium, however, the lipid profile of MAP was significantly different from that of M. avium subsp. avium (lanes 5–6 vs 7–8 in Fig. 1A). As shown in Fig. 1B, presence of MAP-specific lipopeptides was confirmed by two-dimensional TLC; two major spots (indicated by circle) were found to be specific to MAP. The most abundant lipids found in MAP are lipopeptides (indicated by arrow in Fig. 1B). In our and other group’s analyses of MAP lipopeptides, it was revealed that they often contain the N-methyl α-amino acid(s).17 Marfey’s reagent (1-fluoro-2,4-dinitrophenyl-5-L-alanine amide, FDAA), which is commonly used for the chiral analysis of amino acids, reacts with primary amines and their derivatives are analysed by HPLC.23 Generally, FDAA showed the good enantioselectivity, but some amino acids (e.g. Tyr) and N-methyl amino acids showed poor reactivity against Marfey’s reagent.24 We have developed an unprecedented chiral derivatizing agent 5 (Fig. 3) that allows for the determination of the absolute configurations of a wide range of amino acids including secondary amines by 1H-NMR or HPLC.25 We have generated universal 1H-NMR and HPLC data bases of α-amino acids and their N-methyl derivatives in order to determine relative and absolute stereochemistry of the amino acid components of MAP lipopeptides. In the present work, we report a general method to determine the structure of lipopeptides and new MAP lipopeptides, named lipopeptide IIα-γ (2–4) and MAP lipopeptide probes 9–11 that showed the serum antibody response via ELISA.

Figure 1.

Figure 1

Open in a new tab

A. Comparison of lipids in EtOH extract of Mycobacterium spp. Spots specific to MAP are indicated by arrows. Lane 1. M. gordonae (TMC 1324), 2. M. phlei (TMC 1548), 3. M. smegmatis (TMC 1546), 4. M. terrae (TMC 1450), 5. MAP (K10), 6. MAP (Linda), 7. M. avium subsp. avium (TMC 706), 8. M. avium subsp. avium (TMC 724), 9. M. bovis BCG (TMC 1011), 10. M. flavescens (TMC 1541), 11. M. fortuitum (TMC 1529), 12. M. intracellulae (TMC 1406), 13. M. kansasii (TMC 1204), 14, M. scroflaceum (TMC 1323), 15. M. szulgai (TMC 1328). B. Two-dimentional TLC analysis of lipid components in EtOH extract of MAP and M. avium subsp. avium. Spots specific to MAP are indicated by circles. TLC conditions: A.CHCl3 : MeOH : H2O (150 : 10 : 1).; B. CHCl3 : MeOH (94 : 6), then toluene : acetone (80 : 20).; Stain: CuSO4 (3%), H3PO4 (15%) in H2O.

Figure 3.

Figure 3

Open in a new tab

Analyses of the diastereomers of α-amino acids via HPLC.

Isolations of antigenic lipopeptides

In an effort to identify antigenic molecules (elicitors) in the EtOH extract of MAP, we cultured MAP and obtained 50g of bacterial cell pellets. The MAP EtOH extract was subjected to a set of fractionation processes that were guided by the serum antibody response tests using an ELISA-based method. In the ELISA using sera prepared from a MAP-negative and -positive cattle, it was realized that the antigenic molecules are included in the fraction 4 (Fr4 in Fig. 2A). The Fr3 and Fr4 were determined to be lipopeptide-containing fractions based on a lipid-specific stain and 1H-NMR analyses. The molecule containing in the Fr3 was the most abundant lipopeptide (~0.2% isolation yield from 50g of cell pellets), named lipopeptide I (1). The Fr3 and purified 1 did not exhibit antibody response at a 10 μg/mL concentration.11 Lipopeptide I (1) was structurally identical to Para-LP-0110 except for that 1 was a mixture of saturated fatty acids (stearic acid (C18:0) : eicosanoic acid (C20:0): docosanoic acid (C22:0) = 1.0 : 2.3 : 1.38), whereas, Para-LP-01 was reported as a mixture of C16:0, C17:0, C18:0, C19:0, C20:0, C21:0, and C22:0. The antigenic lipopeptide fraction (Fr4 in Fig. 2A) showed very poor solubility in CDCl3, DMSO-d6, THF-d8, and D2O, but dissolved well in CD3OD, albeit, lipopeptide I dissolved in a wide range of NMR solvents. Due to poor solubility of Fr4 in conventional organic solvents for silica gel chromatography, further purification required applying reverse-phase HPLC. Under the HPLC conditions (solvent system: MeOH : 0.1% TFA in H2O = 95 : 5 to 100 : 0) Fr4 was purified and its chromatogram is illustrated in Fig. 2B. Among the fractionated samples, Fr4-4, Fr4-5, and Fr4-6 were isolated as their pure forms [named lipopeptide IIα-γ (2–4)] with quantities of 5.9, 13.3, 6.1 mg, respectively. All fractions (Fr4-1 to Fr4-7) obtained via HPLC purification were evaluated in ELISA for determining their serum antibody responses. At 10 μg/mL concentration, the HPLC fractions (Fr4-1~3 and Fr4-7 in Fig. 2B) did not show a significant activity against serum antibodies in a MAP-infected cattle compared with that of the negative control (DMSO). On the other hand, lipopeptide IIα-γ (Fr4-4, Fr4-5, and Fr4-6) exhibited serum antibody binding activities; serum antibody binding against these fractions were significantly higher than those against the other fractions.

Figure 2.

Figure 2

Open in a new tab

Isolation of new lipopeptides that exhibit serum antibody response activity.

Structure determinations of lipopeptide IIα, IIβ, and IIγ

Eckstein and co-workers determined the gross structure of Para-LP-01 via a combination of standard analytical methods including esterifications of the hydrolyzed amino acids with (R)- and (S)-2-butanol to determine their absolute stereochemistries.17 Esterifications of N-unprotected amino acids generally resulted in low conversions and their purification/analyses are time-consuming processes. Disadvantages of Marfey’s reagent for N-methyl amino acids and less nucleophilic amino acids are discussed above. In order to systematically determine structure of MAP lipopeptides including their absolute stereochemistry in a single chemical modification followed by HPLC analysis, we have generated universal HPLC data bases for a series of carbamates, which were synthesized via coupling reactions between natural and unnatural amino acids and chiral derivatizing agent (S)-5 and (R)-5.26,27 The HPLC data base of the carbamates 6 was realized as a powerful asset for determination of relative and absolute structures of minute quantities of α-amino acids and their N-methyl derivatives. As shown in Fig. 4B, all diastereomers derived from L- and D-series did not show problematic peak overlaps, and thus, the simultaneous determinations of relative/absolute configurations of α-amino acids were realized. Usefulness of the HPLC database of the α-amino acid-carbamates were demonstrated by using the crude mixture of amino acids derived from lipopeptide (1) via hydrolysis with 6N HCl for 6h at 105 °C. The carbamate formations of the hydrolyzed amino acids under iPr2NEt in acetone/H2O (3/1) furnished the corresponding products in quantitative yields (Fig. 3A). The generated carbamates showed excellent resolution on a C18-column using a mixture of solvents (AcOH and NaOAc in H2O-MeOH-MeCN) (Fig. 3B). Comparison of HPLC retention times of 6a–h with those of the database revealed that lipopeptide I (1) was determined to contain L-Ala, L-Phe, L-IIe, N-Me-L-Val, and D-Phe as observed for Para-LP-01. The fatty acid moiety of 1 was determined to exist as a mixture of C18-, C20-, and C22-saturated fatty acids via GC/MS analyses of the trimethylsilyl (TMS)-esters of the acid-hydrolysis product. NOESY experiments of 1 in CD3OD revealed that the N-methyl protons of N-Me-L-Val exhibited strong correlations with D-Phe and L-Ile residues, and the α-protons of the fatty acid moiety. Similarly, the N-Me-L-Val residue showed correlations with L-IIe and the partial structure of L-Phe-L-Ala-OMe was confirmed via NOESY correlations as illustrated in Fig. 4. Thus, the gross structures of lipopeptide I was unambiguously determined by single chemical transformations of the acid-hydrolysis product with (S)-5 followed by its HPLC analysis, fatty acid, and 2D-NMR analyses.

Figure 4.

Figure 4

Open in a new tab

NOESY data for lipopeptide I (1).

Amino acid analyses of the acid-hydrolysis products of lipopeptide IIα (2), IIβ (3), and IIγ (4) realized that their peptide portions also consist of L-Ala, L-Phe, L-IIe, N-Me-L-Val, and D-Phe (Fig 3B and 3C), however, 1H-NMR data for 2, 3, and 4 indicated that their C-terminal ends were not esterified with the methyl group. NOESY experiments and fatty acid analyses by GC-MS suggested the gross structures of lipopeptide IIα, IIβ, and IIγ as shown in Scheme 1. The pentapeptide moiety of 2, 3, and 4 were the same peptide sequence of D-Phe-L-(N-Me)Val-L-IIe-L-Phe-L-Ala-OH as observed in 1. Interestingly, their structures were differ in the lipid moiety; 2, 3, and 4 were the N-acyl pentapeptide of pure stearic acid (C18:0), arachidic acid (C20:0), and behenic acid (C22:0), respectively. In order to confirm relative and absolute stereochemistry of a series of lipopeptide II, 3 was chemically transformed to lipopeptide I-N-arachidic form. Treatment of 3 with CH3OH under a mild esterification condition (EDCI, NaHCO3, Glyceroacetonide-Oxyma in 5% H2O-CH3CN) furnished the corresponding methyl ester 7 in greater than 95% yield.28–30 The synthetic 7 was determined to be identical to the sample of natural 1 by 1H-NMR, 13C-NMR, [α]D, and TLC, except for the number of methylene protons and carbons in 1H-NMR and 13C-NMR, respectively. Consequently, gross structures of lipopeptide IIα (2), IIβ (3), and IIγ (4) were unequivocally determined.

Scheme 1.

Scheme 1

Open in a new tab

Structures of lipopeptide IIα, β, and γ, and methylation and amidation of lipopeptide IIβ (3).

Design and synthesis of the lipopeptide II probe

Lipopeptide I (1) and Para-LP-01 did not exhibit serum antibody binding activity at a 10 μg/mL concentration.18 Similarly, the C-terminal-capped derivatives 7 and 8 synthesized by methylation or amide-formation of lipopeptide IIβ (3) (see supporting information) quenched the serum antibody binding activity of 3,28 which were determined via ELISA (Scheme 1 and Fig. 6). Thus, we concluded that the C-terminal end of the lipopeptide requires being the free carboxylic acid to be an antigenic species. Based on the NOESY data and lowest energy conforms obtained via grid-Monte Calro conformational search algorithm,31 one of the most plausible conformers of lipopeptide IIβ (3) was speculated as shown in Fig. 5. It was hypothesized that functionalization of the 2α-position (indicated by arrow in Fig. 5) of the lipid chain may not change the overall conformation of the pentapeptide portion, retaining a specific serum antibody response activity. We designed (2S)-aminolipopeptide IIβ (9) and its fluorescent probes 10 and 11 (Scheme 2). The synthesis of the pentapeptide was conducted via water-mediated peptide-coupling conditions. The pentapeptide 12 was synthesized by using Glyceroacetonide-Oxyma, EDCI, and NaHCO3 in water without formation of racemization products.25 A simple aqueous NaHCO3 work-up could remove all reagents utilized in the reactions to afford coupling products in over 85% yield for 8 steps; sequential couplings of the building blocks and deprotections of the Boc group in solution phase furnished five gram quantities of the pentapepetide 12. The Boc protected (2S)-aminoarachidic acid 15 was synthesized via the Corey’s chiral phase transfer catalyst (PTC)32,33; asymmetric α-alkylation of the O’Donnell’s glycin ester 13 at 0 °C provided 14 in 90% yield with the selectivity of 9/1.34 Hydrolysis of the benzophenone-Shiff base and protection of the free amine with Boc2O gave rise to 15 in >95% overall yield. Coupling between 12 and 15 under Glyceroacetonide-Oxyma, EDCI, and NaHCO3 in a mixture of DMF and water (10/1) followed by global deprotections provided (2S)-aminolipopeptide IIβ (9) in >85% overall yield. The fluorescent probes, 10 and 11 were synthesized via coupling reactions with the corresponding isothiocyanates. The crude products were purified via reverse-phase HPLC to furnish 10 and 11 in 95–100% yields.

Figure 6.

Figure 6

Open in a new tab

Serum antibody binding activity of lipopeptide IIβ probes.

Scheme 2.

Scheme 2

Open in a new tab

Syntheses of 2α-aminolipopeptide IIβ and its fluorecent derivatives.

Specific serum antibody responses of lipid IIβ fluorescent probes

In vitro assays of 2α-amino lipopeptide IIβ (9) revealed that relative antibody binding activity of lipopeptide IIβ (3) was not significantly reduced by the introduction of the amino group at the 2α-position of the lipid moiety (Fig. 6). Each compound was screened in three times of more on different days. Gratifyingly, it was realized that the two fluorescent derivatives of 10 and 11 also exhibited serum antibody binding activity in ELISA.

Discussions and Conclusions

In conclusion, the HPLC database of the α-amino acid-carbamates derived from (S)-5 are demonstrated to be a convenient asset for structure determination of amino acids and their absolute configurations simultaneously. Amino acid analyses of lipopeptide I (1) and IIα–γ (2–4) can be performed via our HPLC database that allows determining their gross structures in combinations with 1H-NMR and GC-MS. The method using the chiral derivatizing agents 5 has a significant advantage over currently available methods for determination of the absolute configuration of free amino acids via 1H-NMR spectroscopy and/or HPLC.25

Lipopeptide I (1), a methyl ester, did not exhibit humoral immune against activity against serum antibodies in MAP-infected cattle at the concentrations tested. On the contrary, the free carboxylic acid, lipopeptide IIα–γ (2–4) showed serum antibody response activities in the same assay systems (the data only for 3 is shown in Fig. 6). Lipopeptide IIα–γ are isolated in a minute quantity, whereas, lipopeptide I contains ~0.2% in wet MAP cell pellets. Culturing a slow-growing MAP and purification of large quantity of lipopeptide I for the generation of lipopeptide IIβ require significant efforts and time. Thus, enough antigenic lipopeptide IIβ (3) is secured via a chemical synthesis; a gram quantity of 3 is achieved via water benign peptide-forming reactions in solution phase. Importantly, an antigenic lipopeptide IIβ (3) can be modified at the C2′-position of the lipid moiety. The free amino group of lipopeptide IIβ-(2S)-NH2 (9) will be applied onto polymer supports (e.g. poly-D-lysine coated well plates) for development of a reliable serum antibody ELISA. Lipopeptide IIβ-(2S)-NH-FITC (11) will be an ideal probe for a fluorescence polarization (FP) assay (excitation; 480 nm emission; 535 nm) of serum samples from MAP-infected cattle. Application of new lipopeptide probes described here for accurate diagnosis of Johne’s disease will be reported elsewhere.

Supplementary Material

supplement

Acknowledgments

We thank University of Tennessee internal grants for generous financial supports. Some synthetic intermediates were generated under the programs supported by National Institutes of Health (NIAID grants AI084411 and AI119796). We also thank Dr. Pamera Small (the University of Tennessee Knoxville) for generous donation of Mycobacterium spp. NMR and MS data were obtained on instruments supported by the NIH Shared Instrumentation Grants.

Footnotes

Supplementary Information

Additional supporting information may be found in the online version of this article at the publisher’s web-site.

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Notes and references

  • 1.Naser SA, Sagramsingh SR, Naser AS, Thanigachalam S. Mycobacterium avium subspecies paratuberculosis causes Crohn’s disease in some inflammatory bowel disease patients. World J Gastroenterol. 2014;20:7403–7415. doi: 10.3748/wjg.v20.i23.7403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Juste RA, Elguezabal N, Garrido JM, Pavon A, Geijo MV, Sevilla I, Cabriada JL, Tejada A, García-Campos F, Casado R, Ochotorena I, Izeta A, Greenstein RJ. On the prevalence of M. avium subspecies paratuberculosis DNA in the blood of healthy individuals and patients with inflammatory bowel disease. PLoS ONE. 2008;3:e2537. doi: 10.1371/journal.pone.0002537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Salem M, Heydel C, El-Sayed A, Ahmed SA, Zschöck M, Baljer G. Mycobacterium avium subspecies paratuberculosis: An insidious problem for the ruminant industry. Trop Anim Health Prod. 2013;45:351–366. doi: 10.1007/s11250-012-0274-2. [DOI] [PubMed] [Google Scholar]
  • 4.Stabel JR. Johne’s disease: a hidden threat. J Dairy Sci. 1998;81:283–288. doi: 10.3168/jds.S0022-0302(98)75577-8. [DOI] [PubMed] [Google Scholar]
  • 5.Weigoldt M, Meens J, Bange FC, Pich A, Gerlach GF, Goethe R. Metabolic adaptation of Mycobacterium avium subsp. paratuberculosis to the gut environment. Microbiol. 2013;159:380–391. doi: 10.1099/mic.0.062737-0. [DOI] [PubMed] [Google Scholar]
  • 6.Sweeney RW. Pathogenesis of paratuberculosis. Vet Clin North Am Food Anim Pract. 2011;27:537–456. doi: 10.1016/j.cvfa.2011.07.001. [DOI] [PubMed] [Google Scholar]
  • 7.Garcia AB, Shalloo L. Invited review: The economic impact and control of paratuberculosis in cattle. J Dairy Sci. 2015;98:5019–5039. doi: 10.3168/jds.2014-9241. [DOI] [PubMed] [Google Scholar]
  • 8.Pillars RB, Grooms DL, Gardiner JC, Kaneene JB. Association between risk-assessment scores and individual-cow Johne’s disease-test status over time on seven Michigan. Prevent Vet Med. 2011;98:10–18. doi: 10.1016/j.prevetmed.2010.10.001. [DOI] [PubMed] [Google Scholar]
  • 9.Hasonova L, Pavlik I. Paratuberculosis, a notifiable disease in Austria-current status, compulsory measures and first experiences. Vet Med. 2006;51:193–211. doi: 10.1016/j.prevetmed.2007.06.002. [DOI] [PubMed] [Google Scholar]
  • 10.McKenna SLB, Keefe GP, Tiwari A, VanLeeuwen J, Barkema HW. Johne’s disease in Canada Part II: Disease impacts, risk factors, and control programs for dairy producers. Can Vet J. 2006;47:1089–1099. [PMC free article] [PubMed] [Google Scholar]
  • 11.Thirunavukkarasu S, Plain KM, Eckstein TM, de Silva K, Whittington RJ. Cellular and humoral immunogenicity of Mycobacterium avium subsp. paratuberculosis specific lipopentapeptide antigens. Res Vet Sci. 2013;95:123–129. doi: 10.1016/j.rvsc.2013.03.002. [DOI] [PubMed] [Google Scholar]
  • 12.Gilardoni LR, Paolicchi FA, Mundo SL. Revista argentina de microbiología. Rev Argent Microbiol. 2012;44:201–215. [PubMed] [Google Scholar]
  • 13.Wadhwa A, Hickling GJ, Eda S. Opportunities for improved serodiagnosis of human tuberculosis, bovine tuberculosis, and paratuberculosis. Vet Med Int. 2012;2012:13. doi: 10.1155/2012/674238. Article ID 674238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Eda S, Bannantine JP, Waters WR, Mori Y, Whitlock RH, Scott MC, Speer CA. A highly sensitive and subspecies-specific surface antigen enzyme-linked immunosorbent assay for diagnosis of Johne’s Disease. Clin Vaccine Immunol. 2006;13:837–844. doi: 10.1128/CVI.00148-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Wadhwa A, Bannantine JP, Byrem TM, Stein TL, Saxton AM, Speer CA, Eda S. Use of ethanol extract of Mycobacterium bovis for detection of specific antibodies in sera of farmed red deer (Cervus elaphus) with bovine tuberculosis. Foodborne Pathog Dis. 2012;9:749–754. [Google Scholar]
  • 16.Gardner IA, Nielsen SS, Whittington RJ, Collins MT, Bakker D, Harris B, Sreevatsan S, Lombard JE, Sweeney R, Smith DR, Gavalchin J, Eda S. Consensus-based reporting standards for diagnostic test accuracy studies for paratuberculosis in ruminants. Prev Vet Med. 2011;101:18–34. doi: 10.1016/j.prevetmed.2011.04.002. [DOI] [PubMed] [Google Scholar]
  • 17.Eckstein TM, Chandrasekaran S, Mahapatra S, McNeil MR, Chatterjee D, Rithner CD, Ryan PW, Belisle JT, Inamine JM. A major cell wall lipopeptide of Mycobacterium avium subspecies paratuberculosis. J Biol Chem. 2006;281:5209–5215. doi: 10.1074/jbc.M512465200. [DOI] [PubMed] [Google Scholar]
  • 18.Non-specific bindings were observed in ELISA when >20 μg/mL of the lipopeptides were applied. Serum antibody response assays were carried out with the fixed concentration of 10 μg/mL of the lipopeptides.
  • 19.Momotani E, Romona NW, Yoshihara K, Momotani Y, Hori M, Ozaki H, Eda S, Ikegami M. Molecular pathogenesis of bovine paratuberculosis and human inflammatory bowel diseases. Vet Immunol Immunopathol. 2012;148:55–68. doi: 10.1016/j.vetimm.2012.03.005. [DOI] [PubMed] [Google Scholar]
  • 20.Meuwis MA, Fillet M, Chapelle JP, Malaise M, Louis E, Merville MP. New biomarkers of Crohn’s disease: serum biomarkers and development of diagnostic tools. Expert Rev Mol Diagn. 2008;8:327–337. doi: 10.1586/14737159.8.3.327. [DOI] [PubMed] [Google Scholar]
  • 21.Eisenstein BI. Lipopeptides, focusing on daptomycin, for the treatment of Gram-positive infections. Expert Opini Invest Drugs. 2004;13:1159–1169. doi: 10.1517/13543784.13.9.1159. [DOI] [PubMed] [Google Scholar]
  • 22.Lanéelle G, Daffé M. Mycobacterial cell wall and pathogenicity: a lipodologist’s view. Res Microbiol. 1991;142:433–437. doi: 10.1016/0923-2508(91)90116-r. [DOI] [PubMed] [Google Scholar]
  • 23.Bhushan R, Brückner H. Marfey’s reagent for chiral amino acid analysis: A review. Amino Acid. 2004;27:231–247. doi: 10.1007/s00726-004-0118-0. [DOI] [PubMed] [Google Scholar]
  • 24.Ngim KK, Zynger J, Downey B. Analysis of monoethanolamine by derivatization with Marfey’s reagent and HPLC. J Chromatgr Sci. 2007;45:126–130. doi: 10.1093/chromsci/45.3.126. [DOI] [PubMed] [Google Scholar]
  • 25.Kurosu M, Li K. New chiral derivatizing agents: Convenient determination of absolute configurations of free amino acids by 1H NMR. Org Lett. 2009;11:911–914. doi: 10.1021/ol8028719. [DOI] [PubMed] [Google Scholar]
  • 26.Kurosu M, Li K. (2,6-Dichloro-4-alkoxyphenyl)-(2, 4-dichlorophenyl)methyl trichloroacetimidates: protection of alcohols and carboxylic acids in solution or on polymer support. Synthesis. 2009;21:3633–3641. [Google Scholar]
  • 27.Wang Y, Aleiwi BA, Wang Q, Kurosu M. Selective esterifications of primary alcohols in a water-containing solvent. Org Lett. 2012;14:4910–4913. doi: 10.1021/ol3022337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Wang Q, Wang Y, Kurosu M. A new oxyma derivative for nonracemizable amide-forming reactions in water. Org Lett. 2012;14:3372–3375. doi: 10.1021/ol3013556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Aleiwi BA, Mitachi K, Kurosu M. Mild and convenient N-formylation protocol in water-containing solvents. Tetrahedron Lett. 2013;54:2077–2081. doi: 10.1016/j.tetlet.2013.02.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Katsuhiko M, Kurosu Y, Brandon HT, Kurosu M. Oxyma-based phosphates for racemization-free peptide segment couplings. J Petpt Sci. 2016 doi: 10.1002/psc.2859. in press. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Evans JS, Chan SI, Goddard WA. Prediction of polyelectrolyte polypeptide structures using Monte Carlo conformational search methods with implicit solvation modelling. Protein Sci. 1995;4:2019–2031. doi: 10.1002/pro.5560041007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Corey EJ, Xu F, Noe MC. A rational approach to catalytic enantioselective enolate alkylation using a structurally rigidified and defined chiral quaternary ammonium salt under phase transfer conditions. J Am Chem Soc. 1997;119:12414–12415. [Google Scholar]
  • 33.Lygo B, Wainwright PG. A new class of asymmetric phase-transfer catalysts derived from cinchona alkaloids-application in enantioselective synthesis of α-amino acids. Tetrahedron Lett. 1997;38:8595–8598. [Google Scholar]
  • 34.O’Donnell MJ. The enantioselective synthesis of α-amino acids by phase-transfer catalysis with achiral schiff base esters. Acc Chem Res. 2004;37:506–517. doi: 10.1021/ar0300625. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

supplement
NIHMS780956-supplement.pdf (1.7MB, pdf)

RESOURCES