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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 1  |  Issue : 1  |  Page : 37-41

A novel small molecule immunoassay to detect the mycobacterial siderophore carboxymycobactin


1 Division of Pulmonology, University of Cape Town, Groote Schuur Hospital, Observatory, Cape Town, South Africa
2 Department of Pathogen Molecular Biology, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, WC1E 7HT, UK

Date of Web Publication24-Jul-2017

Correspondence Address:
Ruth McNerney
Division of Pulmonology, University of Cape Town, Groote Schuur Hospital, Observatory, Cape Town
South Africa
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_20_17

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  Abstract 

Background: To diagnose tuberculosis (TB), it is necessary to demonstrate the presence of Mycobacterium tuberculosis in a clinical specimen such as sputum. A simple, low-cost rapid test for blood or urine is urgently needed but remains elusive. Tests for host response-derived biomarkers or secreted bacterial compounds have so far failed to provide sufficient sensitivity or specificity and alternative approaches are needed. Carboxymycobactins are amphiphilic siderophores unique to the mycobacteria that have the ability to transverse mammalian cell walls. They have insufficient mass to induce an antibody response and there are currently no simple sensitive tests for their detection. Methods: We report the development of an enzyme-linked immunosorbent assay (ELISA) for carboxymycobactin. Polyclonal antibodies were raised in rabbits following conjugation to a carrier protein, bovine serum albumin allowing development of a sensitive indirect double antibody ELISA. A second keyhole limpet hemocyanin conjugate was manufactured to adhere the hapten to the wells of the microplate. We established a limit of detection for the assay of 1 pg/ml. Carboxymycobactin was detected in culture supernatant from mycobacteria including clinical isolates of M. tuberculosis, but not from cultures of Rhodococcus, Nocardia, Streptomyces, Bacillus cereus, Klebsiella oxytoca, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Sample concentration was achieved following extraction with chloroform. Results: We have demonstrated for the first time the direct detection of carboxymycobactin in culture supernatant by immunoassay. Conclusions: We recommend testing samples from humans and animals to establish the potential utility of carboxymycobactin as a diagnostic marker of active mycobacterial disease.

Keywords: Diagnosis, enzyme-linked immunosorbent assay, mycobacteria, mycobactin, polyclonal antibody, tuberculosis


How to cite this article:
McNerney R, Moyo M. A novel small molecule immunoassay to detect the mycobacterial siderophore carboxymycobactin. Biomed Biotechnol Res J 2017;1:37-41

How to cite this URL:
McNerney R, Moyo M. A novel small molecule immunoassay to detect the mycobacterial siderophore carboxymycobactin. Biomed Biotechnol Res J [serial online] 2017 [cited 2021 Dec 6];1:37-41. Available from: https://www.bmbtrj.org/text.asp?2017/1/1/37/211407




  Introduction Top


Tuberculosis (TB) has superseded HIV as the world's most deadly infectious disease.[1] Diagnosis currently depends on the detection of M. tuberculosis (MTB) in specimens such as sputum by microscopy, culture, or amplification of nucleic acids. Global case detection rates are poor, and the World Health Organization and Stop TB Partnership have prioritized the development of a rapid simple test that is affordable in low-income countries where TB is endemic.[2] Approaches to diagnosing TB are illustrated in [Figure 1]. Current tests for pulmonary TB require expectorated sputum, and the diagnosis of extra-pulmonary disease necessitates invasive sampling from the site of infection. The complexities of the immune response to MTB infection render the detection of host-derived biomarkers ineffective for the diagnosis of active disease. Novel markers to differentiate TB disease from latent infection are needed that may be detected using simple, low-cost, rapid technology. Efforts to develop blood- or urine-based tests have focused on macromolecules such as proteins or lipid-based molecules, but with the exception of lipoarabinomannan in the urine of severely immunosuppressed patients,[3] specific TB markers have not been found in sufficient quantities to permit their detection with easy-to-use rapid tests.[4] Contributing factors toward their failure may include low copy number, localization due to inability to traverse cell wall/membrane barriers, or their inherent instability within biological systems. Alternative markers are needed that more readily diffuse from the site of disease. Attempts to harness the simplicity of detecting low molecular weight volatile compounds released from samples or in patient breath have also failed to provide a solution, in part due to their lack of specificity.[5] In a novel approach, we proposed to explore small compounds (>1000 Da) secreted exclusively by mycobacteria that are amphiphilic in nature.
Figure 1: Current and new approaches under investigation for diagnosing tuberculosis. Current diagnosis requires detection of the bacteria in clinical samples, this is achieved by microscopy, culture, or by detecting Mycobacterium tuberculosis DNA. Detection of macromolecules and small volatile molecules in low.cost simple tests for tuberculosis has not yet proved successful

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Carboxymycobactins are secreted mycobacterial siderophores involved in the extracellular sequestration of iron.[6],[7] Also known as exomycobactins or water-soluble mycobactins, they are believed to play a role in virulence and survival in macrophages.[8],[9],[10] These mycobacterial siderophores have also been shown to have a protective effect for mammalian cells due to their ability to bind reactive radicals.[11],[12] As shown in [Figure 2], they have a core structure similar to that of the cell bound mycobactins, both being derived from a salicylate precursor.[13],[14] With molecular masses ranging from 650 to 800 Da, they are able to freely diffuse through the cell wall.[15] Carboxymycobactins have an extremely high affinity for iron,[16] and when complexed with Fe (III), they are chloroform soluble and may be extracted and purified using chromatographic methodologies.[17] They have been reported to occur in bacilli of the MTB complex and in Mycobacterium smegmatis, Mycobacterium intracellulare, Mycobacterium scrofulaceum, Mycobacterium triviale, Mycobacterium xenopi, and Mycobacterium neoaurum but not in Mycobacterium vaccae, and some strains of Mycobacterium avium and M. paratuberculosis were found to require supplementation with mycobactin.[14],[15],[18] They are upregulated during growth in iron-depleted media and their apparent uniqueness to mycobacteria and their ability to transverse cell walls suggest that they may have value as a diagnostic predictor of mycobacterial disease. However, current methods for their detection are complex and not suited for diagnostic use. Immunoassay, the detection of compounds using antibodies, provides rapid low-cost methods of diagnosis, and we therefore determined to develop an immunoassay for the detection of these mycobacterial siderophores. Carboxymycobactin has a high affinity for iron, and the ferric (Iron III) complex [Figure 2]b was used throughout this study.
Figure 2: Structure of carboxymycobactins. (a) n = 2-9; R1=C or CH3; R2=CH3 or C2H5; R3=CH3 or C2H5 (b) Ferric carboxymycobactin

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  Methods Top


To render carboxymycobactin immunogenic, it was conjugated to a carrier protein by the mixed anhydride method, linking through the carboxyl group of the hapten and amino groups in the albumin.[19],[20] Ferric carboxymycobactin MS purified from the supernatant of iron-depleted cultures of M. smegmatis was conjugated to bovine serum albumin (BSA). The BSA (Fraction V, Sigma-Aldrich Co. Ltd, UK) was dissolved in de-ionized water with dioxane (Fluka Sigma-Aldrich Co. Ltd, UK) and the pH was adjusted to 9.5 with 1 M sodium hydroxide. Carboxymycobactin MS was dissolved in dioxane and cooled to 12°C. Tri n-butylamine (Fluka Sigma-Aldrich Co. Ltd, UK) was added and left for 20 min at 12°C. Iso-butylchloroformate (Fluka Sigma-Aldrich Co. Ltd, UK) was added and the mixture was left for a further 15 min at 12°C. The two solutions were mixed and the pH was adjusted to 8.0 with sodium hydroxide and left stirring for 4 h at 4°C prior to dialysis, lyophilization, and purification on a Sephadex PD-10 column (Amersham Pharmacia Biotech, UK).

To facilitate binding carboxymycobactin to the solid-phase microwell as required for the immunoassay, a second conjugate was prepared with keyhole limpet hemocyanin (KHL). To minimize the chances of cross reaction, a different method of conjugation was adopted than that used for the immunogen and a carbodiimide-linking reagent was used.[19],[20] KHL (Sigma-Aldrich Co. Ltd, UK) was dissolved in 0.1 M sodium chloride. The carboxymycobactin MS was dissolved in ethanol and added to 0.1 M phosphate buffer pH 7.0. After mixing, N-(3-Dimethylamionopropyl) N-ethyl-carbodiimide hydrochloride (Fluka, Sigma-Aldrich Co. Ltd, UK) was added. The conjugate was purified by dialysis prior to lyophilization.

Antibodies against the BSA-carboxymycobactin conjugate were raised in New Zealand White rabbits using Imject® Alum (Pierce Chemical Company, USA) as an adjuvant, in accordance with the UK Home Office requirements. The sera were tested for the presence of antibodies against the carboxymycobactin hapten by exposure to KLH-carboxymycobactin bound in the wells of the microtiter plate. Following washing, immobilized antibody was detected by alkaline phosphatase-labeled anti-rabbit IgG and the substrate p-nitrophenyl phosphate (Sigma-Aldrich Co. Ltd, Poole, UK) with optical densities (OD) recorded at 405 nm. Investigation of pre- and post-inoculation sera demonstrated binding of postinoculation sera to the carboxymycobactin conjugate but not to KLH alone, indicating successful production of antibodies against the siderophore target. A double antibody competition enzyme-linked immunosorbent assay (ELISA) was established using sera diluted at 1:10,000.


  Results Top


During the ELISA, carboxymycobactin free in solution competed with that bound to the well and thus a reduced OD indicated the presence of carboxymycobactin in the sample. As presented in [Figure 3], standard curves were constructed with serial dilutions of ferric carboxymycobactin MS.
Figure 3: A standard curve for quantitation of carboxymycobactin by a double antibody competitive enzyme-linked immunosorbent assay, indicating a range of detection of 1 pg to 10 ng (n = 3)

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To investigate carboxymycobactin secreted by bacterial cultures, bacteria were inoculated in Sauton's broth[21] prepared with and without the addition of 50 mM ferric ammonium citrate and incubated at 37°C. Samples of supernatant were filtered (0.2 μm) prior to testing. As shown in [Table 1], M. smegmatis mc155 and M. bovis Bacillus Calmette–Guérin (BCG) produced carboxymycobactin more readily in broth lacking the iron supplement. Further evidence that the ELISA was detecting the carboxymycobactin target and not a non-specific protein was provided by successful extraction with chloroform. Samples were extracted by shaking with chloroform which was removed to a clean tube and dried by evaporation before reconstitution in ethanol and assay buffer prior to retesting by the ELISA. The positive samples remained strongly positive following chloroform extraction.
Table 1: Carboxymycobactin production by Mycobacterium smegmatis mc2 and Mycobacterium bovis Bacillus Calmette–Guérin grown in Sauton's broth with and without iron supplementation

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Investigation was undertaken of other bacteria belonging to the Actinomycetes family, including additional species of mycobacteria and clinical isolates of MTB. Bacteria were incubated at 37°C in Sauton's medium without iron supplement. Growth of bacteria was assessed by visual observation of the turbidity of the broth and recorded as heavy growth, moderate growth, light growth, or no observable growth. As shown in [Table 2], carboxymycobactin was secreted by MTB H37Rv and M. terrae and clinical isolates of MTB and M. fortuitum. Modest amounts were observed in cultures of M. xenopi and M. avium. The level of carboxymycobactin produced appeared to be related to the amount of bacterial growth observed. However, carboxymycobactin production may be associated with stationary rather than log-phase growth, and further investigation of this phenomenon was outside the scope of this study. In keeping with previous reports, carboxymycobactin production was not observed from the nonmycobacterial species such as Rhodococcus, Nocardia, and Streptomyces demonstrating high specificity for the mycobacterial siderophore. Similarly, carboxymycobactin was not observed in supernatants from cultures of Bacillus cereus, Klebsiella oxytoca, Staphylococcus aureus, Escherichia coli DH5α, and Pseudomonas aeruginosa grown in Sauton's media in the absence of ferric ammonium citrate.
Table 2: Carboxymycobactin production by Actinomycetes bacteria grown in Sauton's broth without iron supplementation

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  Discussion Top


We have demonstrated production of polyclonal antibodies against carboxymycobactin following conjugation to a carrier protein. This is the first report of antibodies for these mycobacterial siderophores. Siderophore-conjugate vaccines are of interest as potential therapeutic agents. However, it is the potential of carboxymycobactin as a diagnostic marker of mycobacterial infection that most warrants further attention.

The antibodies were duly incorporated in an ELISA and were used to investigate bacterial culture supernatant. The immunoassay methodology is faster and simpler to perform than the traditional chromatographic methods of detection and permitted direct sampling of culture supernatants. However, should it prove necessary, the solubility of carboxymycobactins in solvents such as chloroform provides a means to concentrate samples and increases the sensitivity of detection. Carboxymycobactin secretion was observed with both fast-growing environmental mycobacterial species that are rarely pathogenic such as M. smegmatis and in the slow-growing highly virulent MTB. The modest production observed in cultures of M. avium may reflect previous reports of the requirement of some strains for supplementation with mycobactin.[14] Specificity for the mycobacteria was observed, but it should be noted that growth conditions and sampling times were not optimized for each of the bacteria, and further investigations are required to validate these findings. The levels of carboxymycobactin in patient samples have not been investigated. Evidence from other bacterial pathogens, including E. coli 0111 and Klebsiella pneumoniae, suggests that measurable levels of siderophores may be detected during infection.[22],[23] MTB exposure to iron-limited environments is likely to be extensive during active disease, and the ability of these molecules to pass through mammalian cell walls might facilitate their entry into circulating body fluids, offering convenient sampling for diagnostic purposes. However, this remains speculation as the presence of carboxymycobactin within infected tissues or body fluids has yet to be demonstrated. To this end, further studies are required to investigate samples from MTB-infected humans or animals.

This study was funded by the UK Department for International Development.

Financial support and sponsorship

This study was funded by the UK Department for International Development.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2]


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