Deletion of a mycobacterial gene encoding a reductase leads to an altered cell wall containing β-oxo-mycolic acid analogues, and the accumulation of longchain ketones related to mycolic acids.

Mycolic acids are essential components of the mycobacterial cell wall. In this study we show that a gene encoding a reductase involved in the final step of mycolic acid biosynthesis can be deleted in Mycobacterium smegmatis without affecting cell viability. Deletion of MSMEG4722 (ortholog of Mycobacterium tuberculosis Rv2509) altered culture characteristics and antibiotic sensitivity. The ΔMSMEG4722 strain synthesized α-alkyl, β-oxo intermediates of mycolic acids which were found esterified to cell wall-arabinogalactan. While the precursors could not be isolated directly due to their inherent instability during base-treatment, their presence was established by prior reduction of the β-oxo group by sodium borohydride. Interestingly, the mutant also accumulated unsaturated ketones, similar to tuberculenone from M. tuberculosis, which were shunt products derived from spontaneous decarboxylation of α-alkyl, β-oxo fatty acid precursors of mycolic acids

Tuberculosis in Dr Granville's mummy: a molecular re-examination of the earliest known Egyptian mummy to be scientifically examined and given a medical diagnosis

‘Dr Granville's mummy’ was described to the Royal Society of London in 1825 and was the first ancient Egyptian mummy to be subjected to a scientific autopsy. The remains are those of a woman, Irtyersenu, aged about 50, from the necropolis of Thebes and dated to about 600 BC. Augustus Bozzi Granville (1783–1872), an eminent physician and obstetrician, described many organs still in situ and attributed the cause of death to a tumour of the ovary. However, subsequent histological investigations indicate that the tumour is a benign cystadenoma. Histology of the lungs demonstrated a potentially fatal pulmonary exudate and earlier studies attempted to associate this with particular disease conditions. Palaeopathology and ancient DNA analyses show that tuberculosis was widespread in ancient Egypt, so a systematic search for tuberculosis was made, using specific DNA and lipid biomarker analyses. Clear evidence for Mycobacterium tuberculosis complex DNA was obtained in lung tissue and gall bladder samples, based on nested PCR of the IS6110 locus. Lung and femurs were positive for specific M. tuberculosis complex cell-wall mycolic acids, demonstrated by high-performance liquid chromatography of pyrenebutyric acid–pentafluorobenzyl mycolates. Therefore, tuberculosis is likely to have been the major cause of death of Irtyersenu

Pathophysiological Implications of Cell Envelope Structure in Mycobacterium tuberculosis and Related Taxa

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, where they are accompanied 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

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 Hódmezővásárhely-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

Positive Diagnosis of Ancient Leprosy and Tuberculosis Using Ancient DNA and Lipid Biomarkers

Diagnosis of leprosy and tuberculosis in archaeological material is most informative when based upon entire genomes. Ancient DNA (aDNA) is often degraded but amplification of specific fragments also provides reliable diagnoses. Cell wall lipid biomarkers can distinguish ancient leprosy from tuberculosis and DNA extraction residues can be utilized. The diagnostic power of combined aDNA and lipid biomarkers is illustrated by key cases of ancient leprosy and/or tuberculosis. Human tuberculosis was demonstrated in a woman and child from Atlit-Yam (~9 ka) in the Eastern Mediterranean and in the 600 BCE Egyptian “Granville” mummy. Both aDNA and lipids confirmed Pleistocene tuberculosis in a ~17 ka bison from Natural Trap Cave, Wyoming. Leprosy is exemplified by cases from Winchester (10th–12th centuries CE) and Great Chesterford (5th–6th centuries CE). A mixed infection from Kiskundorozsma, Hungary (7th century CE) allowed lipid biomarkers to assess the relative load of leprosy and tuberculosis. Essential protocols for aDNA amplification and analysis of mycolic, mycolipenic, mycocerosic acid, and phthiocerol lipid biomarkers are summarized. Diagnoses of ancient mycobacterial disease can be extended beyond the reach of whole genomics by combinations of aDNA amplification and lipid biomarkers, with sole use of the latter having the potential to recognize even older cases

The Distribution and Origins of Ancient Leprosy

Human leprosy is primarily caused by Mycobacterium leprae, but also by the related ‘M. lepromatosis’. Ancient leprosy can be recognised in archaeological materials by the paleopathology associated with multi-bacillary or lepromatous forms of the disease. Whole M. leprae genomes have been obtained from human skeletons, and diagnostic aDNA fragments have been recovered. The derived M. leprae phylogenies, based on single nucleotide polymorphisms, mirror past human migrations, as M. leprae is usually an obligate pathogen. The detection of M. leprae in historical leprosy cases is assisted by the hydrophobic M. leprae cell envelope, which is composed of unusual lipids that can be used as specific biomarkers. Lipid biomarkers are more stable than aDNA and can be detected directly without amplification. Indigenous human leprosy is extinct in Western Europe, but recently, both M. leprae and ‘M. lepromatosis’ were found in British red squirrels. Leprosy may also be found in nine-banded armadillos (Dasypus novemcinctus) where it can cause a zoonotic human infection. Certain leprosy-like diseases, caused by uncultivable species in cats, for example, may be related to M. leprae. The closest extant relatives of leprosy bacilli are probably members of the M. haemophilum taxon, emerging pathogens with genomic and lipid biomarker similarities

MKAN27435 is required for the biosynthesis of higher subclasses of Lipooligosaccharides in Mycobacterium kansasii

Lipooligosaccharides are glycolipids found in the cell wall of many mycobacterial species including the opportunistic pathogen Mycobacterium kansasii. The genome of M. kansasii ATCC12478 contains a cluster with genes orthologous to Mycobacterium marinum LOS biosynthesis genes. To initiate a genetic dissection of this cluster and demonstrate its role in LOS biosynthesis in M. kansasii, we chose MKAN27435, a gene encoding a putative glycosyltransferase. Using Specialized Transduction, a phage-based gene knockout tool previously used to generate null mutants in other mycobacteria, we generated a MKAN27435 null mutant. The mutant strain was found to be defective in the biosynthesis of higher LOS subspecies, viz LOS-IV, LOS-V, LOS-VI and LOS-VII. Additionally, a range of low abundance species were detected in the mutant strain and mass spectroscopic analysis indicated that these were shunt products generated from LOS-III by the addition of up to six molecules of a pentose