Tuberculosis has had many names, including consumption, scrofula and the great white plague, but whatever you call it, this disease still claims one life every 10 seconds and global mortality rates are increasing despite the use of chemotherapy (Drugs versus bugs: in pursuit of the persistent predator Mycobacterium tuberculosis. 2008 Nature Reviews Microbiology 6: 41-52). Why have we not progressed further towards the eradication of this disease? There are many answers, including politics and poverty, and some less shameful excuses such as HIV and drug resistance. Whatever the reason, without new weapons in the armory against TB, the disease will continue to make ground.
Two factors, persistence and resistance, make the treatment of Mycobacterium tuberculosis infections particularly difficult. The term persistence describes the survival of the causative organism despite the use of antibiotics. The local concentration of antibiotics in lesions such as granulomas might not be adequate to kill the cells, or some bacteria might adopt a physiological state that renders them less susceptible to antibiotics. For these reasons, drug treatments must be extended. Currently, even the most effective regimes require a combination of at least 3 drugs and last for six months. Because patients feel better within 1 2 weeks, they have little motivation to continue with therapy, so the current World Health Organization guidelines call for treatment to be directly observed (DOTS). This can be difficult to provide in much of the world, including the areas where tuberculosis rates are highest.
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There is an excellent chance that patients who have tuberculosis can be cured using currently available drugs if they complete the required course of therapy. But what characteristics should new drugs have to improve on current treatments?
Oral bioavailability: to avoid the need for injections.
Good tolerance: to avoid unwanted side-effects might cause treatment to be abandoned.
Widespread usability: including AIDS patients, young children and pregnant women.
Compatibility with anti-retroviral drugs: because co-infection with HIV and TB is common.
Infrequent dosing: once a day drugs improve treatment compliance.
Activity against drug-resistant TB strains: possibly the most important factor with the rise of MDR and XDR-TB.
Rapid clearance of chronic infection: so that treatment times can be shortened.
Affordability: so they can be used in the areas of the world where TB is most prevalent.
Mycobacterium tuberculosis has no significant animal or environmental reservoirs and shows limited genetic diversity. In spite of this, TB continues to be a widespread and devastating disease. The need for new faster-acting drugs is clear. Recent work by my colleague Dr Mark Carr from the School of Biological Sciences at the University of Leicester might help in future drug development. The M. tuberculosis ESAT-6/CFP-10 complex consists of two proteins which, together, allow the bacteria to survive inside white blood cells. Removal of the genes for these proteins from the TB genome renders the bacteria unable to cause disease. Similarly, studies of the structure of the protein complex have shown that removal of a “long arm” from the molecule prevents the complex s ability to bind to the outer surface of human white blood cells. In the structure of the ESAT-6/CFP-10 complex above, the “long arm” is in red on the right side of CFP-10. When this is intact, it allows the complex to attach to the outside of host white blood cells. When the long arm is cleaved off, the complex shows greatly reduced attachment. This data provides an insight into the important components of this complex. Mark Carr says: “Current work is attempting to identify the exact components of the human white blood cells that this complex is targeting. Once found, this should give us a greater knowledge of the action of these molecular weapons of TB and give us the edge in the war against an ancient, reawakened foe.”
Mycobacterium tuberculosis can persist in the host for decades after infection before reactivating to cause disease. The bacterial and host factors that contribute towards latent TB infection and reactivation disease have long remained enigmatic. However, there is considerable circumstantial evidence to suggest that the persisting organisms may include bacteria in physiological states that are characterized by impaired culturability (i.e. colony-forming ability). These observations suggest a plausible link between an intrinsic microbiological property of M. tuberculosis the ability to enter into a state of dormancy from which culturability can be restored and the clinically defined phenomenon of latent infection. M. tuberculosis contains five resuscitation-promoting factor (Rpf)-like proteins, RpfA-E, that are implicated in resuscitation of this organism from dormancy via a mechanism involving hydrolysis of the peptidoglycan by Rpfs and partnering proteins. In this study, the rpfA-E genes were shown to be collectively dispensable for growth of the organism in broth culture. The defect in resuscitation of multiple mutants from a non-culturable state induced by starvation under anoxia was reversed by genetic complementation or addition of culture filtrate from wild-type organisms confirming that the phenotype was associated with rpf-like gene loss and that the non-culturable cells of the mutant strains were viable. Other phenotypes uncovered by mutagenesis revealed a functional differentiation within this protein family.
Researchers have yet to unravel the complexities which underpin the in vivo phenotypes and relate them to the various in vitro phenotypes associated with rpf-like gene loss. The fact that some Rpfs interact with other proteins in the cell to form protein complexes that may cleave distinct forms of peptidoglycan further adds to the complexity of Rpf function and regulation. However, the collection of mutant strains reported in this and earlier studies are an important resource for future biochemical, microbiological and physiological studies on this fascinating family of proteins.
In August, most people took a holiday and this was the quietest month of the year in terms of visitors, but we still managed to fit in Hendra, chikungunya and Marburg viruses.
In this article in Microbiology Today, Hazel Dockrell describes the role of gamma interferon in the fight against TB and predicts a complex future.
Mycobacterium tuberculosis is an intracellular pathogen, choosing to live within macrophages, where it inhibits antibacterial processes such as phagosome-lysosome fusion. It also expresses haemolysin-like molecules that might, like Listeria, enable its escape into the cytoplasm, although confirmed evidence of this is still lacking. It induces granuloma formation within the lungs, which can progress to causing necrosis, enabling its spread by coughing, and resulting in the destruction of lung tissue. The classic test for infection, the Mantoux skin test, measures recruitment and activation of antigen-specific T cells in a delayed-type hypersensitivity test. This focus on cell-mediated immunity has led to a major interest in the role of gamma interferon.
The infection-induced suicide of host cells (apoptosis) following invasion by intracellular pathogens is an ancient defense mechanism observed in multicellular organisms of both the animal and plant kingdoms. It is therefore not surprising that persistent pathogens of viral, bacterial, and protozoal origin have evolved to inhibit the induction of host cell death. Mycobacterium tuberculosis, the etiological agent of tuberculosis, has latently infected about one third of the world’s population and can persist for decades in the lungs of infected, asymptomatic individuals.
The survival and persistence of M. tuberculosis depends on its capacity to manipulate multiple host defense pathways, including the ability to actively inhibit the death by apoptosis of infected host cells. The genetic basis for this anti-apoptotic activity and its implication for mycobacterial virulence have not previously been determined. Using a novel gain-of-function genetic screen, a recent paper demonstrated that inhibition of infection-induced apoptosis of macrophages is controlled by multiple genetic loci in M. tuberculosis. Characterization of one of these loci in detail revealed that the anti-apoptosis activity was attributable to the type I NADH-dehydrogenase of M. tuberculosis, and was mainly due to the subunit of this multicomponent complex encoded by the nuoG gene. A mutant of M. tuberculosis in which nuoG was deleted triggered a marked increase in apoptosis by infected macrophages, and subsequent analysis of this mutant in the mouse tuberculosis model provided direct evidence for a causal link between the capacity to inhibit apoptosis and bacterial virulence. The discovery of anti-apoptosis genes in M. tuberculosis could provide a powerful approach to the generation of better attenuated vaccine strains, and may also identify a new group of drug targets for improved chemotherapy.
Vitamin D was used to treat tuberculosis in the pre-antibiotic era. Prospective studies to evaluate the effect of vitamin D supplementation on antimycobacterial immunity have not previously been performed. A double-blind randomized controlled trial was conducted in 192 healthy adult tuberculosis contacts in London, UK. Participants were randomized to receive a single oral dose of 2.5 mg vitamin D or placebo and followed up at 6 weeks. A single oral dose of 2.5 mg vitamin D significantly enhanced the ability of participants’ blood to restrict the growth of recombinant reporter mycobacteria in vitro without affecting antigen-stimulated interferon-gamma responses. Clinical trials should be performed to determine whether vitamin D supplementation prevents reactivation of latent tuberculosis infection.
Hepatitis C virus receptor Hepatitis C virus (HCV) is a leading cause of cirrhosis and liver cancer worldwide. A better understanding of the viral life cycle, including the mechanisms of entry into host cells, is needed to identify novel therapeutic targets. Although HCV entry requires the CD81 co-receptor, and other host molecules have been implicated, at least one factor critical to this process remains unknown. Claudin-1 (CLDN1), a tight junction component that is highly expressed in the liver, is essential for HCV entry. CLDN1 is required for HCV infection of human hepatoma cell lines and is the first factor to confer susceptibility to HCV when ectopically expressed in non-hepatic cells. Discrete residues within the first extracellular loop (EL1) of CLDN1, but not protein interaction motifs in intracellular domains, are critical for HCV entry. Antibodies directed against an epitope inserted in the CLDN1 EL1 block HCV infection. The kinetics of inhibition indicate that CLDN1 acts late in the entry process, after virus binding and interaction with the HCV co-receptor CD81. With CLDN1 we have identified a novel key factor for HCV entry and a new target for antiviral drug development.
Mixing it: why two antibiotics may be better than one The rapid evolution of bacterial drug resistance and the slowdown in development of new antibiotics is possibly the most worrying aspect of present-day microbiology. One possible answer is to develop effective multidrug combinations that it is much harder for pathogens to become resistant to than single drug treatments, but a paper just published in Nature shows how difficult this can be.
Drug combinations can be classed as synergistic, additive or antagonistic, depending whether the combined effect of the drugs is larger than, equal to or smaller than the effect of their individual activities. In some cases the effect of certain drug combination is less than that of one of the drugs by itself. But the new paper shows that developing resistance to one drug can sometimes do invading bacteria more harm than good. Although the molecular mechanisms underlying drug interactions are complex, suppression between antibiotics is not uncommon. This work shows that picking the right drug combinations needs careful research.
World Tuberculosis Day is observed on March 24 each year and commemorates the date in 1882 when Robert Koch announced the discovery of Mycobacterium tuberculosis, the bacterium which causes TB. Worldwide, tuberculosis remains one of the leading causes of death from infectious disease. An estimated 2 billion people (one third of the world’s population) are infected with M. tuberculosis. Each year, approximately 9 million people become ill with TB and nearly 2 million die from the disease, with new infections occurring at a rate of one every second. Tuberculosis has plagued humans for as long as we’ve been on this planet, probably evolving alongside us. In the UK, the Health Protection Agency has just announced that tuberculosis has increased another 2% to over 8000 cases in 2006. Since the late 1980s the number of people in the UK diagnosed with TB has risen every year, although in the USA CDC has just reported a 3.2% decline in cases in 2006.
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Tuberculosis is spread by aerosol droplets expelled by people with infected lungs when they cough, sneeze, speak, or spit. The disease most commonly affects the lungs (pulmonary TB), but can also affect the central nervous system, most other organs, bones, and even the skin. Worldwide, TB is the leading cause of death in terms of curable infectious diseases.
Although there are several drug-resistant forms of the organism, such as multi-drug resistant or MDR-TB (strains which are resistant to at least two of the main first-line TB drugs such as isoniazid and rifampin) and the virtually untreatable extreme drug resistant XDR-TB (strains which are resistant to all the current the front-line drugs, and three or more of the six classes of second-line drugs), the majority of TB strains are not resistant to drugs in the way that other bacteria such as MRSA are resistant to most antibiotics.
Patients with TB typically have to take four different antibiotics for two months and then continue with two of these antibiotics for an additional four months. DOTS (Directly Observed Treatment Short-course), the WHO-recommended TB control strategy includes directly observed treatment for at least the first two months in order to ensure compliance. Poor adherence to therapy results in the emergence of MDR and XDR strains, hence the need for new drugs to shorten treatment of drug-sensitive TB, and for effective treatment of MDR- and XDR-TB.
Why is such long treatment needed? Traditionally the answer was thought to lie in the fact that Mycobacterium tuberculosis enters a dormant or non-replicating state in an infected person. Because most of antibiotics act only on replicating bacteria, the dormant state of TB was thought to render it resistant to treatment. But that notion has now been challenged by a paper in PLoS Medicine (Why is long-term therapy required to cure tuberculosis? 2007 PLoS Med 4: e120).
Soon after the discovery of streptomycin, the first effective drug against TB, it became clear that while many patients treated with this antibiotic initially improved dramatically, most subsequently developed streptomycin-resistant strains so that there was little improvement in mortality over untreated patients. The development of new antibiotics led to the realization that there were two requisites for an effective cure: treatment with multiple antibiotics and a long course of therapy.
TB displays multiple mechanisms of drug resistance when growing in vivo. Some of these are classic genetic mechanisms, but others are phenotypic and reversible, such as growth within enclosed granulomas and in biofilms. In tuberculosis, more than with other bacterial infections, these phenotypic mechanisms seems to be of great importance.
New drugs which target non-replicating bacteria are likely to revolutionize TB therapy. Such agents have the potential not only to treat MDR and XDR strains but also to dramatically shorten the duration of therapy, and shorter treatment times should allow higher patient adherence, reduced transmission of infection, and decreased drug resistance, leading in turn to fewer deaths and the ultimate prospect of controlling one of the human race’s oldest enemies, tuberculosis.
Tuberculosis has had many names, including consumption, scrofula and the great white plague, but whatever you call it, this disease still claims one life every 10 seconds and global mortality rates are increasing despite the use of chemotherapy (Drugs versus bugs: in pursuit of the persistent predator Mycobacterium tuberculosis. 2008 Nature Reviews Microbiology 6: 41-52). Why have we not progressed further towards the eradication of this disease? There are many answers, including politics and poverty, and some less shameful excuses such as HIV and drug resistance. Whatever the reason, without new weapons in the armory against TB, the disease will continue to make ground.
We started January off with 
