Leprosy: Ancient and Modern In medieval Europe, leprosy was greatly feared: Sufferers had to wear bells and were shunned and kept isolated from society. Although leprosy largely disappeared from Europe in the 16th century, elsewhere in the world almost a quarter of a million cases are still reported annually, despite the availability of effective drugs. Schuenemann et al. (p. 179 , published online 13 June; see the 14 June News story by Gibbons , p. 1278 ) probed the origins of leprosy bacilli by using a genomic capture-based approach on DNA obtained from skeletal remains from the 10th to 14th centuries. Because the unique mycolic acids of this mycobacterium protect its DNA, for one Danish sample over 100-fold, coverage of the genome was possible. Sequencing suggests a link between the middle-eastern and medieval European strains, which falls in line with social historical expectations that the returning expeditionary forces of antiquity originally spread the pathogen. Subsequently, Europeans took the bacterium westward to the Americas. Overall, ancient and modern strains remain remarkably similar, with no apparent loss of virulence genes, indicating it was most probably improvements in social conditions that led to leprosy's demise in Europe.
Background: An impediment to the rational development of novel drugs against tuberculosis (TB) is a general paucity of knowledge concerning the metabolism of Mycobacterium tuberculosis, particularly during infection. Constraint-based modeling provides a novel approach to investigating microbial metabolism but has not yet been applied to genome-scale modeling of M. tuberculosis.
Regulation of the expression of heat-shock proteins plays an important role in the pathogenesis of Mycobacterium tuberculosis. The heat-shock response of bacteria involves genome-wide changes in gene expression. A combination of targeted mutagenesis and whole-genome expression profiling was used to characterize transcription factors responsible for control of genes encoding the major heat-shock proteins of M. tuberculosis. Two heat-shock regulons were identified. HspR acts as a transcriptional repressor for the members of the Hsp70 (DnaK) regulon, and HrcA similarly regulates the Hsp60 (GroE) response. These two specific repressor circuits overlap with broader transcriptional changes mediated by alternative sigma factors during exposure to high temperatures. Several previously undescribed heat-shock genes were identified as members of the HspR and HrcA regulons. A novel HspR-controlled operon encodes a member of the low-molecular-mass α-crystallin family. This protein is one of the most prominent features of the M. tuberculosis heatshock response and is related to a major antigen induced in response to anaerobic stress.
Mycobacterium tuberculosis is one of most successful pathogens of mankind, infecting one-third of the global population and claiming two million lives every year. The ability of the bacteria to persist in the form of a long-term asymptomatic infection, referred to as latent tuberculosis, is central to the biology of the disease. The persistence of bacteria in superficially normal tissue was recognized soon after the discovery of the tubercle bacillus, and much of our knowledge about persistent populations of M. tuberculosis dates back to the first half of the last century. Recent advances in microbial genetics and host immunity provide an opportunity for renewed investigation of this persistent threat to human health.
The pathogenesis of mycobacterial infection is associated with an ability to interfere with maturation of the phagosomal compartment after ingestion by macrophages. Identification of the mycobacterial components that contribute to this phenomenon will allow rational design of novel approaches to the treatment and prevention of tuberculosis. Microarray-based screening of a transposon library was used to identify mutations that influence the fate of Mycobacterium bovis bacille Calmette-Guérin (BCG) following uptake by macrophages. A screen based on bacterial survival during a 3-d infection highlighted genes previously implicated in growth of Mycobacterium tuberculosis in macrophages and in mice, together with a number of other virulence genes including a locus encoding virulence-associated membrane proteins and a series of transporter molecules. A second screen based on separation of acidified and non-acidified phagosomes by flow cytometry identified genes involved in mycobacterial control of early acidification. This included the KefB potassium/proton antiport. Mutants unable to control early acidification were significantly attenuated for growth during 6-d infections of macrophages. Early acidification of the phagosome is associated with reduced survival of BCG in macrophages. A strong correlation exists between genes required for intracellular survival of BCG and those required for growth of M. tuberculosis in mice. In contrast, very little correlation exists between genes required for intracellular survival of BCG and those that are up-regulated during intracellular adaptation of M. tuberculosis. This study has identified targets for interventions to promote immune clearance of tuberculosis infection. The screening technologies demonstrated in this study will be useful to the study of pathogenesis in many other intracellular microorganisms.
A system for the tetracycline-inducible regulation of gene expression in mycobacteria has been developed. We have sub-cloned the tetRO region from the Corynebacterium glutamicum TetZ locus into a mycobacterial shuttle plasmid, making expression of genes cloned downstream of tetRO responsive to tetracycline. Using the luxAB-encoded luciferase from Vibrio harveyi as a reporter (pMind-Lx), we observed a 40-fold increase in light output from Mycobacterium smegmatis cultures 2 h after adding 20 ng ml−1 of tetracycline. Similarly, exposure to the drug resulted in up to 20-fold increase in relative light units from M.bovis BCG carrying the reporter construct, and a 10-fold increase for M.tuberculosis. Tetracycline induction was demonstrated in log and stationary phase cultures. To evaluate whether this system is amenable to use in vivo, J774 macrophages were infected with M.bovis BCG[pMind-Lx], treated with amikacin to kill extracellular bacteria, and then incubated with tetracycline. A 10-fold increase in light output was measured after 24 h, indicating that intracellular bacteria are accessible and responsive to exogenously added tetracycline. To test the use of the tetracycline-inducible system for conditional gene silencing, mycobacteria were transformed with a pMind construct with tetRO driving expression of antisense RNA for the ftsZ gene. Bacterial cells containing the antisense construct formed filaments after 24 h exposure to tetracycline. These results demonstrate the potential of this tetracycline-regulated system for the manipulation of mycobacterial gene expression inside and outside cells.
Studying ancient DNA allows us to retrace the evolutionary history of human pathogens, such as Mycobacterium leprae, the main causative agent of leprosy. Leprosy is one of the oldest recorded and most stigmatizing diseases in human history. The disease was prevalent in Europe until the 16th century and is still endemic in many countries with over 200,000 new cases reported annually. Previous worldwide studies on modern and European medieval M. leprae genomes revealed that they cluster into several distinct branches of which two were present in medieval Northwestern Europe. In this study, we analyzed 10 new medieval M. leprae genomes including the so far oldest M. leprae genome from one of the earliest known cases of leprosy in the United Kingdom—a skeleton from the Great Chesterford cemetery with a calibrated age of 415–545 C.E. This dataset provides a genetic time transect of M. leprae diversity in Europe over the past 1500 years. We find M. leprae strains from four distinct branches to be present in the Early Medieval Period, and strains from three different branches were detected within a single cemetery from the High Medieval Period. Altogether these findings suggest a higher genetic diversity of M. leprae strains in medieval Europe at various time points than previously assumed. The resulting more complex picture of the past phylogeography of leprosy in Europe impacts current phylogeographical models of M. leprae dissemination. It suggests alternative models for the past spread of leprosy such as a wide spread prevalence of strains from different branches in Eurasia already in Antiquity or maybe even an origin in Western Eurasia. Furthermore, these results highlight how studying ancient M. leprae strains improves understanding the history of leprosy worldwide.
The ability of the tubercle bacillus to arrest phagosome maturation is considered one major mechanism that allows its survival within host macrophages. To identify mycobacterial genes involved in this process, we developed a high throughput phenotypic cell-based assay enabling individual sub-cellular analysis of over 11,000 Mycobacterium tuberculosis mutants. This very stringent assay makes use of fluorescent staining for intracellular acidic compartments, and automated confocal microscopy to quantitatively determine the intracellular localization of M. tuberculosis. We characterised the ten mutants that traffic most frequently into acidified compartments early after phagocytosis, suggesting that they had lost their ability to arrest phagosomal maturation. Molecular analysis of these mutants revealed mainly disruptions in genes involved in cell envelope biogenesis (fadD28), the ESX-1 secretion system (espL/Rv3880), molybdopterin biosynthesis (moaC1 and moaD1), as well as in genes from a novel locus, Rv1503c-Rv1506c. Most interestingly, the mutants in Rv1503c and Rv1506c were perturbed in the biosynthesis of acyltrehalose-containing glycolipids. Our results suggest that such glycolipids indeed play a critical role in the early intracellular fate of the tubercle bacillus. The unbiased approach developed here can be easily adapted for functional genomics study of intracellular pathogens, together with focused discovery of new anti-microbials.
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