Sensitive lipid biomarker detection for tuberculosis in late Neanderthal skeletons from Subalyuk Cave, Hungary.

Skeletal remains of two Neanderthal individuals, a 25-35 year-old woman and a 3-4 year-old child, were discovered in a Subalyuk Cave in North-Eastern Hungary. Radiocarbon dating of the female and child remains revealed an age of 39,732-39,076 and 36,117-35,387 cal BP, respectively. Paleopathological studies of these Neanderthal remains revealed probable evidence of skeletal mycobacterial infection, including in the sacrum of the adult specimen and the endocranial surface of the child's skull. Application of PCR amplification to the juvenile cranium and a vertebra gave a positive result (IS6110) for tuberculosis, backed up by spoligotyping. Lipid biomarker analyses of the same two specimens revealed definitive signals for C32 mycoserosates, a very characteristic component of the Mycobacterium tuberculosis complex (MTBC). A vertebra from the adult provided weak evidence for mycocerosate biomarkers. The correlation of probable skeletal lesions with characteristic amplified DNA fragments and a proven lipid biomarker points to the presence of tuberculosis in these Neanderthals. In particular, the closely similar biomarker profiles, for two distinct juvenile cranial and vertebral bones, strengthen this diagnosis.

Professor David Minnikin Memorial Lecture: An era of the mycobacterial cell wall lipid biomarkers.

This paper is dedicated to the memory of Professor David Ernest Minnikin (1939-2021). David was one of the key scientists who pioneered the field of Mycobacterium tuberculosis cell envelope research for over half a century. From the classification, identification, and extraction of the unusual lipids of the mycobacterial cell wall, to exploiting them as characteristic lipid biomarkers for sensitive detection, his ideas enlightened a whole world of possibilities within the tuberculosis (TB) field. In addition, his definition of the intricate models now forms a key milestone in our understanding of the M. tuberculosis cell envelope and has resolved many unanswered questions on the evolution of M. tuberculosis.

A re-investigation of the mycolic acids of Mycobacterium avium.

Mycolic acid methyl esters were extracted from Mycobacterium avium by a mild saponification protocol, designed to preserve labile components. The resulting mixture of α-, keto- and wax ester mycolates was accompanied by some degraded ω-carboxymycolic acid dimethyl esters, whose overall structures were found to support previous studies. Chromatography of the mono-carboxylic mycolates gave an inseparable mixture of keto- and wax ester mycolates and separate α-mycolates. Reduction of the ketomycolate components allowed isolation and characterisation of intact wax ester mycolates for the first time. Minor α- and ω-carboxymycolates were detected in which methyl branches were located on either the proximal or distal sides of trans-alkene groups.

The role of hydrophobicity in tuberculosis evolution and pathogenicity.

The evolution of tubercle bacilli parallels a route from environmental Mycobacterium kansasii, through intermediate "Mycobacterium canettii", to the modern Mycobacterium tuberculosis complex. Cell envelope outer membrane lipids change systematically from hydrophilic lipooligosaccharides and phenolic glycolipids to hydrophobic phthiocerol dimycocerosates, di- and pentaacyl trehaloses and sulfoglycolipids. Such lipid changes point to a hydrophobic phenotype for M. tuberculosis sensu stricto. Using Congo Red staining and hexadecane-aqueous buffer partitioning, the hydrophobicity of rough morphology M. tuberculosis and Mycobacterium bovis strains was greater than smooth "M. canettii" and M. kansasii. Killed mycobacteria maintained differential hydrophobicity but defatted cells were similar, indicating that outer membrane lipids govern overall hydrophobicity. A rough M. tuberculosis H37Rv ΔpapA1 sulfoglycolipid-deficient mutant had significantly diminished Congo Red uptake though hexadecane-aqueous buffer partitioning was similar to H37Rv. An M. kansasii, ΔMKAN27435 partially lipooligosaccharide-deficient mutant absorbed marginally more Congo Red dye than the parent strain but was comparable in partition experiments. In evolving from ancestral mycobacteria, related to "M. canettii" and M. kansasii, modern M. tuberculosis probably became more hydrophobic by increasing the proportion of less polar lipids in the outer membrane. Importantly, such a change would enhance the capability for aerosol transmission, affecting virulence and pathogenicity.

Identification of a Desaturase Involved in Mycolic Acid Biosynthesis in Mycobacterium smegmatis.

Mycolic acids are unique long chain fatty acids found in the cell walls of mycobacteria including the tubercle bacillus, Mycobacterium tuberculosis. The introduction of double bonds in mycolic acids remains poorly understood, however, genes encoding two potential aerobic desaturases have been proposed to be involved in this process. Here we show that one of these genes, desA1, is essential for growth of the saprophytic Mycobacterium smegmatis. Depletion of desA1 in a M. smegmatis conditional mutant led to reduction of mycolic acid biosynthesis and loss of viability. The DesA1-depleted cells exhibited two other phenotypes: using 14[C]-labelling, we detected the accumulation of minor mycolic acid-related species that migrated faster in a silver TLC plate. Spiral Time of Flight Mass Spectroscopic analysis suggested the presence of species with sizes corresponding to what were likely monoenoic derivatives of α-mycolic acids. Additionally, conditional depletion led to the presence of free fatty acyl species of lengths ~C26-C48 in the lysing cells. Cell viability could be rescued in the conditional mutant by Mycobacterium tuberculosis desA1, highlighting the potential of desA1 as a new drug target in pathogenic mycobacteria.

Osteological, biomolecular and geochemical examination of an early anglo-saxon case of lepromatous leprosy.

We have examined a 5th to 6th century inhumation from Great Chesterford, Essex, UK. The incomplete remains are those of a young male, aged around 21-35 years at death. The remains show osteological evidence of lepromatous leprosy (LL) and this was confirmed by lipid biomarker analysis and ancient DNA (aDNA) analysis, which provided evidence for both multi-copy and single copy loci from the Mycobacterium leprae genome. Genotyping showed the strain belonged to the 3I lineage, but the Great Chesterford isolate appeared to be ancestral to 3I strains found in later medieval cases in southern Britain and also continental Europe. While a number of contemporaneous cases exist, at present, this case of leprosy is the earliest radiocarbon dated case in Britain confirmed by both aDNA and lipid biomarkers. Importantly, Strontium and Oxygen isotope analysis suggest that the individual is likely to have originated from outside Britain. This potentially sheds light on the origins of the strain in Britain and its subsequent spread to other parts of the world, including the Americas where the 3I lineage of M. leprae is still found in some southern states of America.

Human tuberculosis predates domestication in ancient Syria.

The question of pre-neolithic tuberculosis is still open in paleopathological perspective. One of the major interests is to explore what type of infection could have existed around the early stage of animal domestication. Paleopathological lesions evoking skeletal TB were observed on five human skeletons coming from two PPNB sites in Syria, which belongs to the geographical cradle of agriculture. These sites represent respectively pre-domestication phase (Dja'de el Mughara, Northern Syria, 8800-8300 BCE cal.) and early domestication phase (Tell Aswad, Southern Syria, 8200-7600 BCE cal.). MicroCT scan analyses were performed on two specimens (one per site) and revealed microscopic changes in favor of TB infection. Detection of lipid biomarkers is positive for two specimens (one per site). Initial molecular analysis further indicates the presence of TB in one individual from Dja'de. Interestingly, no morphological evidence of TB was observed on animal remains of wild and newly domesticated species, discovered in these sites. These observations strongly suggest the presence of human tuberculosis before domestication and at its early stages.

Ancient DNA analysis - An established technique in charting the evolution of tuberculosis and leprosy.

Many tuberculosis and leprosy infections are latent or paucibacillary, suggesting a long time-scale for host and pathogen co-existence. Palaeopathology enables recognition of archaeological cases and PCR detects pathogen ancient DNA (aDNA). Mycobacterium tuberculosis and Mycobacterium leprae cell wall lipids are more stable than aDNA and restrict permeability, thereby possibly aiding long-term persistence of pathogen aDNA. Amplification of aDNA, using specific PCR primers designed for short fragments and linked to fluorescent probes, gives good results, especially when designed to target multi-copy loci. Such studies have confirmed tuberculosis and leprosy, including co-infections. Many tuberculosis cases have non-specific or no visible skeletal pathology, consistent with the natural history of this disease. M. tuberculosis and M. leprae are obligate parasites, closely associated with their human host following recent clonal distribution. Therefore genotyping based on single nucleotide polymorphisms (SNPs) can indicate their origins, spread and phylogeny. Knowledge of extant genetic lineages at particular times in past human populations can be obtained from well-preserved specimens where molecular typing is possible, using deletion analysis, microsatellite analysis and whole genome sequencing. Such studies have identified non-bovine tuberculosis from a Pleistocene bison from 17,500 years BP, human tuberculosis from 9000 years ago and leprosy from over 2000 years ago.

Morphological and biomolecular evidence for tuberculosis in 8th century AD skeletons from Bélmegyer-Csömöki domb, Hungary.

Macromorphological analysis of skeletons, from 20 selected graves of the 8th century AD Bélmegyer-Csömöki domb, revealed 19 cases of possible skeletal tuberculosis. Biomolecular analyses provided general support for such diagnoses, including the individual without pathology, but the data did not show coherent consistency over the range of biomarkers examined. Amplification of ancient DNA fragments found evidence for the Mycobacterium tuberculosis complex DNA only in five graves. In contrast, varying degrees of lipid biomarker presence were recorded in all except two of the skeletons, though most lipid components appeared to be somewhat degraded. Mycobacterial mycolic acid biomarkers were absent in five cases, but the weak, possibly degraded profiles for the remainder were smaller and inconclusive for either tuberculosis or leprosy. The most positive lipid biomarker evidence for tuberculosis was provided by mycolipenic acid, with 13 clear cases, supported by five distinct possible cases. Combinations of mycocerosic acids were present in all but three graves, but in one case a tuberculosis-leprosy co-infection was indicated. In two specimens with pathology, no lipid biomarker evidence was recorded, but one of these specimens provided M. tuberculosis complex DNA fragments.

Lipid biomarkers provide evolutionary signposts for the oldest known cases of tuberculosis.

Studies on the evolution of tuberculosis, and the influence of this disease on human and animal development and interaction, require the accumulation of indisputable biomarker evidence. Ideally, the determination of full genomes would provide all the necessary information, but for very old specimens DNA preservation may be compromised and only limited DNA amplification may be a possibility. Mycobacterium tuberculosis is characterised by the presence of unusual cell envelope lipids, with specific biomarker potential. Lipid biomarker recognition has been decisive in pinpointing the oldest known cases of human and animal tuberculosis; the former are a woman and child from a pre-pottery settlement at Atlit-Yam, Israel (∼9,000 ka) and the latter is an extinct Bison antiquus from Natural Trap Cave, Wyoming (∼17,000 ka). Including some new data, it is demonstrated how analysis of a combination of mycolic, mycocerosic and mycolipenic acid and phthiocerol biomarkers provide incontrovertible evidence for tuberculosis in these landmark specimens.

Development of sample clean up methods for the analysis of Mycobacterium tuberculosis methyl mycocerosate biomarkers in sputum extracts by gas chromatography-mass spectrometry.

A proof of principle gas chromatography-mass spectrometry method is presented, in combination with clean up assays, aiming to improve the analysis of methyl mycocerosate tuberculosis biomarkers from sputum. Methyl mycocerosates are generated from the transesterification of phthiocerol dimycocerosates (PDIMs), extracted in petroleum ether from sputum of tuberculosis suspect patients. When a high matrix background is present in the sputum extracts, the identification of the chromatographic peaks corresponding to the methyl derivatives of PDIMs analytes may be hindered by the closely eluting methyl ether of cholesterol, usually an abundant matrix constituent frequently present in sputum samples. The purification procedures involving solid phase extraction (SPE) based methods with both commercial Isolute-Florisil cartridges, and purpose designed molecularly imprinted polymeric materials (MIPs), resulted in cleaner chromatograms, while the mycocerosates are still present. The clean-up performed on solutions of PDIMs and cholesterol standards in petroleum ether show that, depending on the solvent mix and on the type of SPE used, the recovery of PDIMs is between 64 and 70%, whilst most of the cholesterol is removed from the system. When applied to petroleum ether extracts from representative sputum samples, the clean-up procedures resulted in recoveries of 36-68% for PDIMs, allowing some superior detection of the target analytes.

Ancient mycobacterial lipids: Key reference biomarkers in charting the evolution of tuberculosis.

Mycobacterium tuberculosis has a cell envelope incorporating a peptidoglycan-linked arabinogalactan esterified by long-chain mycolic acids. A range of "free" lipids are associated with the "bound" mycolic acids, producing an effective envelope outer membrane. The distribution of these lipids is discontinuous among mycobacteria and such lipids have proven potential for biomarker use in tracing the evolution of tuberculosis. A plausible evolutionary scenario involves progression from an environmental organism, such as Mycobacterium kansasii, through intermediate "smooth" tubercle bacilli, labelled "Mycobacterium canettii"; cell envelope lipid composition possibly correlates with such a progression. M. kansasii and "M. canettii" have characteristic lipooligosaccharides, associated with motility and biofilms, and glycosyl phenolphthiocerol dimycocerosates ("phenolic glycolipids"). Both these lipid classes are absent in modern M. tuberculosis sensu stricto, though simplified phenolic glycolipids remain in certain current biotypes. Dimycocerosates of the phthiocerol family are restricted to smaller phthiodiolone diesters in M. kansasii. Diacyl and pentaacyl trehaloses are present in "M. canettii" and M. tuberculosis, accompanied in the latter by related sulfated acyl trehaloses. In comparison with environmental mycobacteria, subtle modifications in mycolic acid structures in "M. canettii" and M. tuberculosis are notable. The probability of essential tuberculosis evolution taking place in Pleistocene megafauna, rather than Homo sapiens, is reemphasised.

7000 year-old tuberculosis cases from Hungary - Osteological and biomolecular evidence.

This study derives from the macroscopic analysis of a Late Neolithic population from Hungary. Remains were recovered from a tell settlement at Hódmezovásárhely-Gorzsa from graves within the settlement as well as pits, ditches, houses and as stray finds. One of the most important discoveries from these remains was evidence of tuberculosis. Pathological analysis of the seventy-one individuals revealed numerous cases of infections and non-specific stress indicators on juveniles and adults, metabolic diseases on juveniles, and evidence of trauma and mechanical changes on adults. Several cases showed potential signs of tuberculosis and further analyses were undertaken, including biomolecular studies. The five individuals were all very young adults and included a striking case of Hypertrophic Pulmonary Osteopathy (HPO) with rib changes, one case with resorptive lesions on the vertebrae, two cases with hypervascularisation on the vertebrae and periosteal remodelling on the ribs, and one case with abnormal blood vessel impressions and a possible lesion on the endocranial surface of the skull. The initial macroscopic diagnosis of these five cases was confirmed by lipid biomarker analyses, and three of them were corroborated by DNA analysis. At present, these 7000-year-old individuals are among the oldest palaeopathological and palaeomicrobiological cases of tuberculosis worldwide.

Tuberculosis origin: The Neolithic scenario.

This paper follows the dramatic changes in scientific research during the last 20 years regarding the relationship between the Mycobacterium tuberculosis complex and its hosts - bovids and/or humans. Once the M. tuberculosis and Mycobacterium bovis genomes were sequenced, it became obvious that the old story of M. bovis evolving into the human pathogen should be reversed, as M. tuberculosis is more ancestral than M. bovis. Nevertheless, the timescale and geographical origin remained an enigma. In the current study human and cattle bone samples were examined for evidence of tuberculosis from the site of Atlit-Yam in the Eastern Mediterranean, dating from 9250 to 8160 (calibrated) years ago. Strict precautions were used to prevent contamination in the DNA analysis, and independent centers used to confirm authenticity of findings. DNA from five M. tuberculosis genetic loci was detected and had characteristics consistent with extant genetic lineages. High performance liquid chromatography was used as an independent method of verification and it directly detected mycolic acid lipid biomarkers, specific for the M. tuberculosis complex. These, together with pathological changes detected in some of the bones, confirm the presence of the disease in the Levantine populations during the Pre-pottery Neolithic C period, more than 8000 years ago.

A migration-driven model for the historical spread of leprosy in medieval Eastern and Central Europe.

Leprosy was rare in Europe during the Roman period, yet its prevalence increased dramatically in medieval times. We examined human remains, with paleopathological lesions indicative of leprosy, dated to the 6th-11th century AD, from Central and Eastern Europe and Byzantine Anatolia. Analysis of ancient DNA and bacterial cell wall lipid biomarkers revealed Mycobacterium leprae in skeletal remains from 6th-8th century Northern Italy, 7th-11th century Hungary, 8th-9th century Austria, the Slavic Greater Moravian Empire of the 9th-10th century and 8th-10th century Byzantine samples from Northern Anatolia. These data were analyzed alongside findings published by others. M. leprae is an obligate human pathogen that has undergone an evolutionary bottleneck followed by clonal expansion. Therefore M. leprae genotypes and sub-genotypes give information about the human populations they have infected and their migration. Although data are limited, genotyping demonstrates that historical M. leprae from Byzantine Anatolia, Eastern and Central Europe resembles modern strains in Asia Minor rather than the recently characterized historical strains from North West Europe. The westward migration of peoples from Central Asia in the first millennium may have introduced different M. leprae strains into medieval Europe and certainly would have facilitated the spread of any existing leprosy. The subsequent decline of M. leprae in Europe may be due to increased host resistance. However, molecular evidence of historical leprosy and tuberculosis co-infections suggests that death from tuberculosis in leprosy patients was also a factor.

Osteological and biomolecular evidence of a 7000-year-old case of hypertrophic pulmonary osteopathy secondary to tuberculosis from neolithic hungary.

Seventy-one individuals from the late Neolithic population of the 7000-year-old site of Hódmezovásárhely-Gorzsa were examined for their skeletal palaeopathology. This revealed numerous cases of infections and non-specific stress indicators in juveniles and adults, metabolic diseases in juveniles, and evidence of trauma and mechanical changes in adults. Several cases showed potential signs of tuberculosis, particularly the remains of the individual HGO-53. This is an important finding that has significant implications for our understanding of this community. The aim of the present study was to seek biomolecular evidence to confirm this diagnosis. HGO-53 was a young male with a striking case of hypertrophic pulmonary osteopathy (HPO), revealing rib changes and cavitations in the vertebral bodies. The initial macroscopic diagnosis of HPO secondary to tuberculosis was confirmed by analysis of Mycobacterium tuberculosis complex specific cell wall lipid biomarkers and corroborated by ancient DNA (aDNA) analysis. This case is the earliest known classical case of HPO on an adult human skeleton and is one of the oldest palaeopathological and palaeomicrobiological tuberculosis cases to date.

Genome-wide comparison of medieval and modern Mycobacterium leprae.

Leprosy was endemic in Europe until the Middle Ages. Using DNA array capture, we have obtained genome sequences of Mycobacterium leprae from skeletons of five medieval leprosy cases from the United Kingdom, Sweden, and Denmark. In one case, the DNA was so well preserved that full de novo assembly of the ancient bacterial genome could be achieved through shotgun sequencing alone. The ancient M. leprae sequences were compared with those of 11 modern strains, representing diverse genotypes and geographic origins. The comparisons revealed remarkable genomic conservation during the past 1000 years, a European origin for leprosy in the Americas, and the presence of an M. leprae genotype in medieval Europe now commonly associated with the Middle East. The exceptional preservation of M. leprae biomarkers, both DNA and mycolic acids, in ancient skeletons has major implications for palaeomicrobiology and human pathogen evolution.

Detection and strain typing of ancient Mycobacterium leprae from a medieval leprosy hospital.

Nine burials excavated from the Magdalen Hill Archaeological Research Project (MHARP) in Winchester, UK, showing skeletal signs of lepromatous leprosy (LL) have been studied using a multidisciplinary approach including osteological, geochemical and biomolecular techniques. DNA from Mycobacterium leprae was amplified from all nine skeletons but not from control skeletons devoid of indicative pathology. In several specimens we corroborated the identification of M. leprae with detection of mycolic acids specific to the cell wall of M. leprae and persistent in the skeletal samples. In five cases, the preservation of the material allowed detailed genotyping using single-nucleotide polymorphism (SNP) and multiple locus variable number tandem repeat analysis (MLVA). Three of the five cases proved to be infected with SNP type 3I-1, ancestral to contemporary M. leprae isolates found in southern states of America and likely carried by European migrants. From the remaining two burials we identified, for the first time in the British Isles, the occurrence of SNP type 2F. Stable isotope analysis conducted on tooth enamel taken from two of the type 3I-1 and one of the type 2F remains revealed that all three individuals had probably spent their formative years in the Winchester area. Previously, type 2F has been implicated as the precursor strain that migrated from the Middle East to India and South-East Asia, subsequently evolving to type 1 strains. Thus we show that type 2F had also spread westwards to Britain by the early medieval period.