Title: An era of the mycobacterial cell wall lipid biomarkers

Authors: Houdini H.T. Wu^1,2,3, Oona Y-C. Lee^1,3, David E. Minnikin¹, Gurdyal S. Besra¹

Affiliations: ¹Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, United Kingdom B15 2TT; ^b UK Health Security Agency, Public Health Birmingham laboratory, Heartlands Hospital, Birmingham, United Kingdom B5 9SS; ³Coventry Road Medical Centre, Heartlands Hospital, Birmingham, United Kingdom B5 9SS

Abstract: This paper is dedicated to the memory of Professor David Ernest Minnikin (1939–2021) for his ground-breaking work in microbial chemotaxonomy, mycobacterial tuberculosis cell envelope, developing the value of mycobacterial cell wall lipids biomarkers in ancient tuberculosis and leprosy, as well as their evolution and beyond.

Mycobacterium tuberculosis and Mycobacterium leprae produce unusual cell envelope lipids at their outer membrane. Deployment of these mycobacterial cell wall components is established as specific lipids biomarkers to enable the diagnosis of ancient tuberculosis and leprosy in bone samples from archaeological skeletons. Accessible biomarkers include mycolic, mycocerosic and mycolipenic acids and the phthiocerol family. From over two decades of work, profiles of mycolic acids have enabled the diagnosis of the oldest known human tuberculosis case in skeletons up to 9ka from Atlit-Yam, Israel (Hershkovitz et al. 2008 PLoS ONE, 3, e3426) and up to 1030CE in Argentina (Luna et al. 2020 Tuberculosis 125, 101995) supported with mycocerosic, mycolipenic acids and phthiocerol data. Detection of pristine mycolipenic acid and mycocerosic acids, accompanied by clear phthiocerol components has complimented tuberculosis diagnosis in a 17ka Bison antiquus from Natural Trap Cave, Wyoming (Lee et al. 2012 PLoS ONE, 7: e41923). Mycolic acid traces have helped characterise medieval leprosy (Taylor et al. 2013 PLoS ONE 8: e62406; Schuenemann et al. 2013 Science341, 179). In cases of mixed leprosy and tuberculosis infections, the relative proportions of the two diseases can be estimated by analysis of mycolic, mycolipenic and mycocerosate profiles (Lee et al. 2012 in: Proc. 12^th Ann. BABAO Conf.;eds. PD Mitchell & J Buckberry. BAR International Series 2380, Archaeopress, Oxford, 63-69).

An era of the mycobacterial cell wall lipid biomarkers is shown to be an invaluable tool in the diagnosis of ancient tuberculosis and leprosy, as well as demonstrating the presence of members of the Mycobacterium tuberculosis complex. These should be used in combination with palaeopathological findings, ancient DNA studies and genomic analyses, wherever applicable to provide insight into the evolution of tuberculosis.

Keywords: Tuberculosis; Leprosy; Mycobacterial Cell Wall Lipid Biomarkers