Mycobacterium tuberculosis is a remarkably successful human pathogen. The interaction with the human host is complex and much remains unknown. Recent advances in systems biology have allowed the integration of data from humans and animal models into computational approaches. For example, mathematical models provide a platform for in silico manipulation of host-pathogen interactions to gain insight into this infection across temporal and biologic scales. This article reviews recent studies on global approaches toward identifying comprehensive responses of both host and bacillus during infection, and the potential for incorporation of these data into many types of useful computational systems. Systems biology approaches provide a unique opportunity to study interventions that may improve therapy and vaccines against this major killer.
Mycobacterium tuberculosis was first isolated more than 125 years ago. Although a huge amount of research has been devoted to it over this time, tuberculosis still represents a major public health threat in many countries. The main hindrances in fighting this disease include a lack of understanding of the human infection, its establishment and progression, as well as the host-pathogen interactions that determine the different outcomes. Furthermore, the treatment regimen of six months administration of up to four drugs has not evolved in more than four decades, and recent years have seen an alarming increase in multi-drug resistant (MDR) and extensively drug-resistant (XDR) strains. It is clear then that novel and imaginative approaches are needed to speed up both basic and translational research in tuberculosis, especially in the areas of vaccine and drug development.
Optimisation of Bioluminescent Reporters for Use with Mycobacteria. 2010 PLoS ONE 5(5): e10777. doi:10.1371/journal.pone.0010777 Mycobacterium tuberculosis, the causative agent of tuberculosis, still represents a major public health threat in many countries. Bioluminescence, the production of light by luciferase-catalyzed reactions, is a versatile reporter technology with multiple applications both in vitro and in vivo. In vivo bioluminescence imaging (BLI) represents one of its most outstanding uses by allowing the non-invasive localization of luciferase-expressing cells within a live animal. Despite the extensive use of luminescent reporters in mycobacteria, the resultant luminescent strains have not been fully applied to BLI.
One of the main obstacles to the use of bioluminescence for in vivo imaging is the achievement of reporter protein expression levels high enough to obtain a signal that can be detected externally. Therefore, as a first step in the application of this technology to the study of mycobacterial infection in vivo, we have optimised the use of firefly, Gaussia and bacterial luciferases in mycobacteria using a combination of vectors, promoters, and codon-optimised genes. We report for the first time the functional expression of the whole bacterial lux operon in Mycobacterium tuberculosis and M. smegmatis thus allowing the development of auto-luminescent mycobacteria. We demonstrate that the Gaussia luciferase is secreted from bacterial cells and that this secretion does not require a signal sequence. Finally we prove that the signal produced by recombinant mycobacteria expressing either the firefly or bacterial luciferases can be non-invasively detected in the lungs of infected mice by bioluminescence imaging.
While much work remains to be done, the finding that both firefly and bacterial luciferases can be detected non-invasively in live mice is an important first step to using these reporters to study the pathogenesis of M. tuberculosis and other mycobacterial species in vivo. Furthermore, the development of auto-luminescent mycobacteria has enormous ramifications for high throughput mycobacterial drug screening assays which are currently carried out either in a destructive manner using LuxAB or the firefly luciferase.
With an estimated annual incidence of over nine million cases, tuberculosis (TB) is believed to be responsible for more adult deaths each year than any other single infectious agent. The highest burden of disease is currently borne by the less developed countries of Africa and Asia where efforts to control TB are hampered by weak health systems and in some settings, by the high prevalence of co-infection with HIV. The recent emergence of multidrugresistant stains that cannot be cured with standard treatments has served to emphasize the urgency of the situation. Control of TB in high burden countries relies on the detection and treatment of infectious cases, most usually by testing patients attending a health clinic that report a cough of at least three weeks duration. The diagnostic tests available in these settings are sputum smear microscopy, an insensitive technique requiring a skilled practitioner and chest radiography, a technique lacking in specificity as well as sensitivity. World Health Organization estimates suggest that in 2006 there were 4 million individuals with undiagnosed tuberculosis. More effective interventions are required to detect and treat infectious cases earlier in the transmission chain, particularly in vulnerable communities with a high prevalence of HIV.
Mycobacterium tuberculosis, the causative agent is spread from person to person via infected aerosols created by patients with respiratory forms of the disease. Bacilli released into the airways following necrosis and destruction of lung tissue may be expelled from the lungs and if released in the form of aerosols may remain airborne and available for inhalation and infection of a new host. Despite being the major mode of transmission there is little data available regarding the exhalation of M. tuberculosis. This paper describes testing of a novel device that utilizes immunosensor and bio-optical technology to detect M. tuberculosis antigen in the breath of humans.
Field test of a novel detection device for Mycobacterium tuberculosis antigen in cough. BMC Infectious Diseases 2010, 10: 161 doi:10.1186/1471-2334-10-161
Tuberculosis is a highly infectious disease that is spread from person to person by infected aerosols emitted by patients with respiratory forms of the disease. We describe a novel device that utilizes immunosensor and bio-optical technology to detect M. tuberculosis antigen (Ag85B) in cough and demonstrate its use under field conditions during a pilot study in an area of high TB incidence.
The TB Breathalyzer device (Rapid Biosensor Systems Ltd) was field tested in the outpatient clinic of Adama Hospital, Ethiopia. Adults seeking diagnosis for respiratory complaints were tested. Following nebulization with 0.9% saline patients were asked to cough into a disposable collection device where cough aerosols were deposited. Devices were then inserted into a portable instrument to assess whether antigen was present in the sample. Demographic and clinical data were recorded and all patients were subjected to chest radiogram and examination of sputum by Ziehl-Nielsen microscopy. In the absence of culture treatment decisions were based on smear microscopy, chest x-ray and clinical assessment. Breathalyzer testing was undertaken by a separate physician to triage and diagnostic assessment.
Sixty individuals were each subjected to a breathalyzer test. The procedure was well tolerated and for each patient the testing was completed in less than 10 min. Positive breath test results were recorded for 29 (48%) patients. Of 31 patients with a diagnosis of tuberculosis 23 (74%) were found positive for antigen in their breath and 20 (64%) were smear positive for acid fast bacilli in their sputum. Six patients provided apparent false positive breathalyzer results that did not correlate with a diagnosis of tuberculosis.
We propose that the breathalyzer device described warrants further investigation as a tool for studying exhalation of M. tuberculosis. The portability, simplicity of use and speed of the test device suggest it may also find use as a tool to aid early identification of infectious cases. We recommend studies be undertaken to determine the diagnostic sensitivity and specificity of the device when compared to microbiological and clinical indicators of tuberculosis disease.
Endospores are unique among bacterial spores in that they are produced inside of another cell (the mother cell) and, upon maturation, are released as free spores by lysis of the mother cell. They are readily recognized under phase-contrast microscopy by their phase bright (refractile) appearance. They also exhibit diagnostic features under electron microscopy, such as a protein shell consisting of an inner coat and an electron dense, outer coat. Endospores are composed of numerous molecules found, thus far, only in bacterial endospores. These molecules include most of the proteins that encase the spore in a protective shell (called the coat), a family of DNA-protective proteins known as SASP that are bound to the chromosome, and a unique small molecule, dipicolinic acid. All previously known examples of endospore-forming bacteria are members of the low G+C group of Gram-positive bacteria (Firmicutes) belonging either to Bacilli or to Clostridia, and in all cases in which a genome sequence is available, orthologs of genes involved in endospore formation are readily seen. The Mycobacterium genus is a member of the high G+C group of Gram-positive bacteria (Actinobacteria) for which there are no prior claims of endospore formation. Certain members of the group, such as Streptomyces, do produce spores, but spores of a fundamentally different kind that are not produced inside a mother cell.
A recent publication in PNAS reported that M. marinum and M. bovis bacillus Calmette–Guérin produce a type of spore known as an endospore, which had been observed only in the low G+C group of Gram-positive bacteria. Evidence was presented that the spores were similar to endospores in ultrastructure, in heat resistance and in the presence of dipicolinic acid. This paper reports that the genomes of Mycobacterium species and those of other high G+C Gram-positive bacteria lack orthologs of many, if not all, highly conserved genes diagnostic of endospore formation in the genomes of low G+C Gram-positive bacteria. It also failed to detect the presence of endospores by light microscopy or by testing for heat-resistant colony-forming units in aged cultures of M. marinum. Finally, we failed to recover heat-resistant colony-forming units from frogs chronically infected with M. marinum. It concludes that it is unlikely that Mycobacterium is capable of endospore formation.
Historically, tuberculosis caused by Mycobacterium bovis (bovine tuberculosis, bTB) was a major public health issue in the UK. bTB in humans was widespread in the UK before the introduction of pasteurisation of milk in the 1960s: in the 1930s, 40% of dairy cows were infected and 0.5% had tuberculous mastitis. During this period, approximately 2500 people died annually from bTB. Therefore, measures were introduced to eliminate bTB from the UK. As a result, by the 1970s, bTB was eliminated from most of Britain, with persistent infection limited to the southwest. Subsequently, bTB has re-emerged: in 2007, there were 4172 new herd breakdowns in England and Wales. The resurgence of bTB has resulted in public expenditure now approaching £100 million annually. More and more extreme measures are being proposed to stop the spread of the disease such as widespread badger culling programmes, despite scientific studies casting doubt on the efficacy of such practices.
This article argues that, apart from milk pasteurisation, these measures no longer make economic sense and hence are now resulting in gross misallocation of public resources. We are therefore of the opinion that there is no public health rationale for the multimillion bTB control programme in the UK provided that milk continues to be pasteurised. The logical conclusion arising from this is that without a public health perspective, bTB is essentially an endemic animal disease and hence any control programme should be economically effective in terms of improvements in animal health and welfare and industry profitability or viability.
Public health and bovine tuberculosis: what’s all the fuss about? Trends in Microbiology, Nov 25 2009
Bovine tuberculosis (bTB) in UK cattle is increasing rapidly. Consequently, the UK Government is spending escalating sums of money in attempts at disease control. We propose that bTB control in cattle is irrelevant as a public health policy. In the UK, cattle-to-human transmission is negligible. Aerosol transmission, the only probable route of human acquisition, occurs at inconsequential levels when milk is pasteurised, even when bTB is highly endemic in cattle. Furthermore, there is little evidence for a positive cost benefit in terms of animal health of bTB control. Such evidence is required; otherwise, there is little justification for the large sums of public money spent on bTB control in the UK.
Gender: biological and behavioral differences determining “the state of being male or female”)
Sex: biological differences in males and females
In most countries, tuberculosis (TB) notification is twice as high in men as in women. Although there is clear evidence that socioeconomic and cultural factors leading to barriers in accessing health care may cause undernotification in women, particularly in developing countries, biological mechanisms may actually account for a significant part of this difference between male and female susceptibility to TB. The role of biological gender has been determined in a number of infectious and noninfectious diseases. However, there is an absence of information on the role of biological gender in TB. Thus, investigations should be conducted to clearly understand the role of sexual hormones, sex-related genetic background and genetic regulations, and metabolism, among other factors, in susceptibility differences between men and women. This research may help not only to fully understand the obviously biased gender distribution among TB cases, but also to better adapt future intervention strategies at the community level. In this review, we expand on the various issues relating to TB notification and gender bias.
Large prevalence surveys have suggested that the sex bias observed in pulmonary TB cases may result partly from genuine biological differences in male and female susceptibility to M. tuberculosis infection or the development of TB disease. This finding would not be particularly surprising, as many studies in humans and experimentally infected animals have established clear links between sex-specific factors, including steroid hormones and genetic variants, and the differential susceptibility of males and females to a number of other infectious and noninfectious diseases. In particular, gender bias among pulmonary microbial diseases is not restricted to TB, and important sex differences in the incidence and severity of a number of respiratory tract bacterial infections have been reported in the literature. As a selected example, it has been shown that men have a four-times higher risk of developing nosocomial Legionella pneumophila infection than women. Only 5% to 10% of individuals exposed to M. tuberculosis develop TB, and up to 70% of those who do develop the disease are male. In other words, the human population as a whole is remarkably resistant to M. tuberculosis, but women seem to be even more resistant to the bacillus than men. So, why do only a minority of individuals, other than patients with HIV/AIDS, fail to control infection? Why are women less likely to develop TB than men? Why are some women more resistant to TB than other women exposed to a similar extent? Field research consortia including not only microbiologists, immunologists, and human geneticists, but also epidemiologists and sociologists, should be established to unravel the many faces of sexual inequality in TB, and to decipher the delicate mechanisms involved in natural and sex-associated resistance to TB. Such work would facilitate the design of future intervention strategies for combating the disease and the development of useful tools for evaluating prognosis and protection in future clinical trials.
Dr Helen McShane at the University of Oxford and the Oxford-Emergent Tuberculosis Consortium Ltd have received a Wellcome trust Strategic Award to part-fund the first trial to test efficacy of the new TB vaccine candidate, MVA85A, for potential efficacy in South African infants. In this film Dr McShane discusses the new vaccine candidate and these trials. www.wellcome.ac.uk
The causative agents of tuberculosis, grouped in the Mycobacterium tuberculosis complex (MTBC), have infected one-third of the present human population and a wide range of other mammals. However, questions, such as why, where and when the disease began and expanded, have largely remained unanswered. A new study provides genetic evidence indicating that the common ancestor of the tuberculosis complex emerged some 40,000 years ago in East Africa, the region from where modern human populations disseminated around the same period. This initial step was followed 10,000 to 20,000 years later by the radiation of two major lineages, one of which spread from human to animals. In more recent years (approximately 180 years ago), coinciding with the human population explosion and the industrial revolution, the human-associated pathogen lineages have strongly expanded. These results thus reveal the strikingly parallel demographic evolution between humans and one of their primary pathogens.
Using mycobacterial tandem repeat sequences as genetic markers, the authors show that the MTBC consists of two independent clades, one composed exclusively of M. tuberculosis lineages from humans and the other composed of both animal and human isolates. The latter also likely derived from a human pathogenic lineage, supporting the hypothesis of an original human host. These findings unveil the dynamic dimension of the association between human host and pathogen populations.
Mycobacterium tuberculosis is a remarkably successful human pathogen. The interaction with the human host is complex and much remains unknown. Recent advances in systems biology have allowed the integration of data from humans and animal models into computational approaches. For example, mathematical models provide a platform for in silico manipulation of host-pathogen interactions to gain insight into this infection across temporal and biologic scales. This article reviews recent studies on global approaches toward identifying comprehensive responses of both host and bacillus during infection, and the potential for incorporation of these data into many types of useful computational systems. Systems biology approaches provide a unique opportunity to study interventions that may improve therapy and vaccines against this major killer.
