Did Saint Leonard suffer from Madura foot at the time of death? Infectious disease diagnosis by paleo-proteotyping

Abstract

Leonard was one of the most renowned saints during the Western Middle Ages. His biography, written in the 11th century, reports that he was a noble man converted to Christianity by Saint Remi, in 496, therefore at the same time as Clovis, one of the oldest kings of France.1Vita et miracula sancti Leonardi, Confessoris: initium desideratur. French National Library, ML5134.Google Scholar Leonardo obtained from his sovereign the right to visit and possibly release the prisoners he deemed worthy of release, at any time. And it is because he freed a large number of prisoners that he became the patron saint of prisoners. Clovis offers him to be elevated to the dignity of bishop, but he refuses, and enters the Saint-Mesmin abbey of Micy near Orléans, in central France. Then he became a hermit in the forests of Limousin, further south, where a large number of disciples came to join him. Clovis asks him to pray so that his wife gives birth to a male to whom the throne can be passed. and it was indeed a boy to whom she gave birth; to thank him, the sovereign offered him land in Noblat, where he founded an abbey, around which a village (Saint-Léonard-de-Noblat) was established. Always very close to the community of prisoners, Leonardo was joined by those who escaped - sometimes with miraculous help - and worked alongside him to drain the swamps or domesticate the woods and fields. The causes of his death in 559 have always remained unknown. Relics of Saint Leonard are known since the 11th c., held in Saint-Leonard-de-Noblat (next to Limoges, France), and presented to the veneration of devotees each 7 years during ceremonies under the auspices of UNESCO (“Ostensions limousines”) since 1094 to cure victims of “Burden Disease”. On the occasion of such a collective presentation, we subjected the entire skeleton to a complete anthropological and paleopathological analysis. Male sex was confirmed by the pelvis examination, with an estimated age at death of less than 40 years-old. All the inferior long bones (both femurs, tibias and fibulas) were affected by mild to severe chronic osteomyelitis, confirmed by X-ray examination (Fig. 1).2Bury D.C. Rogers T.S. Dickman M.M. Osteomyelitis: diagnosis and treatment.Am Fam Physician. 2021; 104: 395-402PubMed Google Scholar To pinpoint the pathogen at the origin of such an infection, a small 0.5 cm3 sample of pathological bone (left femur) was subjected to proteotyping analysis by tandem mass spectrometry. Dried material (≈50 mg) was mixed with 100 µL lithium dodecyl sulfate (LDS) 1X lysis buffer (Thermo) supplemented with silica and glass beads, and subjected to bead-beating using a Precellys Evolution instrument (Bertin Technologies). The extracted proteins were denatured for 5 min at 99 °C, subjected to electrophoresis on a NuPAGE 4–12% Bis-Tris gel for 5 min, and then treated and proteolyzed in-gel with trypsin. The resulting peptides were resolved for 30 min by reverse phase chromatography on a Vanquish Neo UHPLC and characterized by tandem mass spectrometry with an Orbitrap Exploris 480 instrument (Thermo). MS/MS spectra were interpreted to identify the taxonomical origin of the peptides as previously described.3Grenga L. Pible O. Miotello G. Culotta K. Ruat S. Roncato M.A. et al.Taxonomical and functional changes in Covid-19 faecal microbiome could be related to SARS-CoV-2 faecal load.Environ Microbiol. 2022; 24: 4299-4316Crossref PubMed Scopus (14) Google Scholar Interpretation parameters were 3-ppm mass tolerance on the parent peptide ion, 0.02-Da tolerance on the secondary fragment ions, carboxyamidomethylation of cysteines as fixed modification, and oxidation of methionine and proline residues as variable modification. Taxon-Spectrum Matches and taxon-specific peptide sequences were counted at all possible taxonomical ranks.4Pible O. Allain F. Jouffret V. Culotta K. Miotello G. Armengaud J. et al.Estimating relative biomasses of organisms in microbiota using “phylopeptidomics”.Microbiome. 2020; 830Crossref PubMed Scopus (25) Google Scholar Although the quantity of protein material was low, 9103 MS/MS spectra could be recorded and one third (3068) could be assigned to peptide sequences. As expected, due to its ancient origin, a large ratio of MS/MS spectra correspond to peptides encompassing oxidized prolines (35%). Most of this signal belongs to human proteins (328 Homo sapiens species-specific peptides). Among the microbial signals, two bacteria were unambiguously detected: Actinomadura (20 specific peptides) and Cupriavidus metallidurans (13 specific peptides). This last species is described as heavy metal stress tolerant while the Actinomadura genus has been identified as responsible of Mycetoma (Madura foot) pathogenesis. In order to pinpoint which Actinomadura species is present in the sample, a pan-proteomics analysis was performed with a database encompassing all presently sequenced organisms belonging to this genus (i.e. 58 full-length genomes, totaling 736,894 proteins sequences). The results indicate the presence of a strain closely related to Actinomadura bangladeshensis and sharing some sequences with a relative strain not yet taxonomically characterized (Actinomadura sp. BRA 177). Noteworthy, peptides assigned to Actinomadura containing proline residues are oxidized, as expected due to the advanced age of this biological material. Such Actinomadura bacteria are the causing agent of mycetoma5Zijlstra E.E. van de Sande W.W. Fahal A.H. Mycetoma: a long journey from neglect.PLoS Negl Trop Dis. 2016; 10e0004244Crossref PubMed Scopus (37) Google Scholar a chronic, deforming subcutaneous infection of the foot, due to bacteria or fungal inoculation by minor trauma, which, if left untreated with antibiotics, spreads to the soft tissues and bones. Incubation ranges from a few weeks to several years. The disease is characterized by soft, painless formations associated with discharges of white granules. Here, the pathogen causing the observed osteomyelitis was directly identified by tandem mass spectrometry. Today, mycetoma is present in tropical and subtropical environments, and recognized as a neglected tropical disease,5Zijlstra E.E. van de Sande W.W. Fahal A.H. Mycetoma: a long journey from neglect.PLoS Negl Trop Dis. 2016; 10e0004244Crossref PubMed Scopus (37) Google Scholar but the results described here show that this disease was probably widespread in Europe before the era of antibiotic-based medicine. Not applicable (no involvement of contemporary patients/animals). None for any of both authors. Both authors wish to thank the radiology department of Saint-Léonard-de-Noblat community hospital (France), Alexandre Mazin and other members of the Confrérie Saint-Léonard de Noblat, as well as Jean-Charles Gaillard for his help with mass spectrometry measurements.

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