The pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis, has been present in humans since antiquity. Bone finds provide the earliest evidence for tuberculosis in man and animals, with examples of spinal TB (Pott's disease) dating back to about 8000 BC (Herzog, 1998). The frequency of Egyptian skeletons revealing tubercular deformities are high, which suggests that the disease was common amongst the populations of the time. Similarly, deformed bones have been found in Neolithic sites in Italy and Denmark and countries in the Middle East. This archaeological evidence suggests that TB was prevalent throughout the world approximately 4000 years ago (Smith, 2003).
Thanks to new developments in the molecular analysis of ancient mycobacterial DNA, further advancements have been made in the detection and characterization of Mycobacterium tuberculosis. Recently, DNA from five M. tuberculosis genetic loci was detected in bone samples from a woman and infant who were buried together in a Neolithic settlement in the Eastern Mediterranean, dating from 9250 - 8160 years ago (Hershkovitz et al., 2008). Furthermore, molecular analysis based on the amplification and verification of the M. tuberculosis complex insertion sequence IS6110 was conducted on bone samples from an ancient Egyptian population. Analysis of samples from Upper Egypt (2120 - 500 BC) and the necropolis of Abydos (3000 BC) suggests that the occurrence of M. tuberculosis was relatively frequent in ancient Egypt (Zink et al., 2001).
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It is speculated that the first literature mentioning TB was that formulated by the Babylonian monarch Hammurabi between 1948 and 1905 BC and is engraved in cuneiform script on a stone pillar. This text mentions a chronic lung disease, which is thought to be TB. More conclusively, the Greek literature of the time of Hippocrates (460 - 370 BC) introduces the concept of phthisis, or consumption (other names for tuberculosis), as the most common disease of the period, combined with a high mortality rate. The opinion of the time was that phthisis was a hereditary, rather than infectious disease, even though Aristotle (384 - 322 BC) believed that it was contagious. Only later Galen (131 - 201 AD) suspected the contagious nature of phthisis, which then formed part of medical thinking for the next few hundred years (Herzog, 1998).
The 17th century saw the first detailed pathoanatomical description of consumption, with Sylvius de la Boë of Amsterdam (1617 - 1655), being the first to describe the characteristic tubercles as a constant in the lungs and other organs of consumptives (Herzog, 1998).
During the 19th century evidence of infectiousness began to grow. In 1882 Robert Koch (1843 - 1910) produced irrefutable evidence that a specific microbe is the fundamental cause of tuberculosis. Shortly after that, in 1895, another important contribution to the diagnosis of TB was presented in the form of X-rays by Wilhelm Conrad von Röntgen (1845 - 1923). This meant that the presence, development and severity of TB could be accurately monitored and studied for the first time (Herzog, 1998).
The Symptoms and Treatment of Tuberculosis
Tuberculosis can manifest in many forms, with pulmonary TB accounting for the vast majority of cases. Pulmonary TB has previously been described as consumption and phthisis, both these terms are indicative of severe wasting and the coughing up of blood in later stages of the disease. Spinal tuberculosis, also termed Pott's disease, is marked by spinal deformity, as well as other bone defects. A common manifestation of TB in the Middle Ages was scrofula, or cervical lymphadenitis, which is characterised by the swelling of lymph nodes in the neck. Other forms of extra pulmonary TB include disease of the central nervous system, the urogenital tract, the digestive system and continuously in the form of lupus vulgaris (Smith, 2003).
Treatment of tuberculosis is a relatively new development, considering the presence of the disease throughout the ages. The first treatment for tuberculosis was suggested by Hermann Brehmer in the middle of the 19th century. He suggested that TB, until then believed to be incurable, could be healed by bringing patients to an "immune place", a region where there were no known consumptives. With this belief he founded his sanatorium in Göbersdorf in 1856. Many other sanatoria opened later in the 19th century, all of which adopted the strict rest cure regime suggested by Peter Dettweiler in 1893. Even though the benefits of sanatorium treatment can be debated, it did remove infectious patients from their public surroundings, as well as enforce rest and a sensible diet. This regulated lifestyle improved the well-being of patients greatly, however, the long term results were not very encouraging. Due to these poor long-term results, sanatorium treatment was gradually being supplemented. In the 1930s these supplementary treatments included pneumothorax and thoracoplasty. These techniques produced consistently good results, including closure of cavities, conversion to negative sputum and well-preserved lung function (Herzog, 1998).
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In the early 1900s the Frenchmen Calmette and Guérin were able, by means of serial passaging, to create an attenuated strain of Mycobacterium bovis, now known as BCG (bacille Calmette-Guérin). BCG is still in use in children today, although the benefits of vaccination in adults do not produce concise data (Herzog, 1998).
The most recent development in the treatment of tuberculosis is drug treatment in the form of antibiotics and antituberculous drugs. Currently treatment for tuberculosis consists of an initial two month phase (Isoniazid, Rifampin, Pyrazinamide, Ethambutol), followed by a four month consolidation phase (Isoniazid and Rifampin) (Herzog, 1998). However, drug resistance has become a major factor in the treatment of tuberculosis, with the occurrence of multi-drug resistant tuberculosis (MDR) and extensively drug resistant tuberculosis (XDR) on the rise. As a result, tuberculosis is a major global health burden, causing 1.8 million deaths in 2008 and 9.4 million TB cases in the same year (WHO, 2009). The further risk of HIV/AIDS co-infection is potentially devastating, resulting in a high cost, both financially and in terms of lives.
1.3 The Evolution of the Mycobacterium tuberculosis complex
The Mycobacterium tuberculosis complex consists of a group of species sharing 99.9% similarity at nucleotide level and identical 16S rRNA sequences (Brosch et al., 2002). Despite these similarities, which provide evidence that they all evolved from a common ancestor, they differ greatly in their host organism, phenotype and pathogenicity. M. tuberculosis, M. africanum and M. canetti make up the human pathogens of the complex, while M. microti is a rodent pathogen. M. bovis can infect a wide variety of mammals, including humans, whereas in the attenuated form (BCG) it is used as a vaccine, which rarely causes disease (Cole, 2002).
It was originally thought that M. tuberculosis evolved from M. bovis, the agent of bovine tuberculosis, roughly 15Â 000 years ago, following the domestication of livestock. This new organisation of living in villages, as opposed to a nomadic lifestyle, defined the Neolithic period and it seemed as though the animal pathogen had merely adapted itself to the human host (Cole, 2002). However, whole genome sequencing of M. tuberculosis and comparative genomic studies have resulted in a different explanation. M. bovis has undergone numerous deletions relative to M. tuberculosis, resulting in a smaller genome. This indicates that M. bovis is the final member of a lineage separate from M. tuberculosis, but with a common progenitor (Brosch et al., 2002).
1.4 Phenotypic and Genomic characteristics of Mycobacterium tuberculosis
All mycobacteria can be divided into two groups based on their growth rates. So called fast growers will produce colonies in less than seven days when plated onto solid media, whereas slow growers will only form colonies in more than seven days. In addition, fast growers are usually non-pathogenic and slow growers, including Mycobacterium tuberculosis, are usually pathogenic (Prescott et al., 2002).
Mycobacterium tuberculosis is a rod-shaped, Gram-positive organism with a complex cell wall with very high lipid content (Prescott et al., 2002). The surface of the pathogen is also coated with a detergent labile capsule layer, which consists of non-covalently linked glycans, lipids and proteins (Sani et al., 2010).
The complete sequencing of the genome of Mycobacterium tuberculosis reference strain H37Rv in 1998 heralded a new era for tuberculosis research. The genome of M. tuberculosis H37Rv is approximately 4.4 x 106 bp in size and contains approximately 4000 genes (Cole et al., 1998). These 4000 genes account for 91% of the potential coding capacity of the genome and have a characteristically high G+C content of 65.5%. Only around 40% of the genes have known functions and only 16% bear a resemblance to known proteins (Prescott et al., 2002).
1.5 The ESX gene cluster regions
The genome of M. tuberculosis contains five copies of the ESX gene cluster region, designated ESX-1 to ESX-5 (Fig 1.1). These gene cluster regions encode for a novel secretion system, namely type VII secretion, which is directly involved in pathogenicity and phagosomal escape (Abdallah et al., 2007). These gene cluster regions contain genes encoding exported T-cell antigens, namely ESAT-6 (early secreted antigenic target of 6kDa) and CFP-10 (culture filtrate protein of 10kDa). Due to this, these regions are often referred to as the ESAT-6 or ESX gene cluster regions (Brodin et al., 2005). It has also been demonstrated that ESAT-6 and CFP-10 form a tight 1:1 complex, which contributes to pathogenicity, since this complex binds to the surface of host cells, thus implying a signalling role (Renshaw et al., 2002).
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The ESX-1 secretion system is directly implicated in pathogenicity. ESX-1, containing ESAT-6 and CFP-10, is located in the region of difference 1 (RD1) deletion region, which is absent in Mycobacterium bovis BCG vaccine strain and results in the attenuation of the organism (Pym et al., 2002).
In addition to ESX-1, the genome of M. tuberculosis contains four additional ESX gene clusters, ESX-2 to ESX-5, which are homologous to ESX-1 and also contain genes encoding for ESAT-6 and CFP-10 proteins (Fig 1.1). It has been shown that ESX-4 is the most ancestral of all the regions, present in all other species of Mycobacteria and even in other high G+C Gram-positive bacteria, such as Corynebacterium diphtheriae and Streptomyces coelicolor (Fig 1.1) (Gey van Pittius et al., 2001).
ESAT-6 region 4 contains seven genes (Rv3444c to Rv3450c). Sequence homology suggest these genes code for proteins with specific functions involved in transport (Table 1.1), with Rv3444c (esxT) being an ESAT-6 like protein and Rv3445c (esxU) being a CFP-10 like protein. Furthermore, it has been shown that the genes present in ESX-4 are not essential for the in vivo survival of M. tuberculosis (Singh et al., 2006).
Figure 1.1 Schematic representation of the five ESAT-6 gene cluster regions of Mycobacterium tuberculosis in order of duplication. Blocked arrows indicate ORFs, as well as the direction of transcription. The different colours reflect the specific gene family, while the lengths of the arrows reflect the relative gene length. Black arrows indicate unconserved genes present in these regions. Annotations of M. tuberculosis H37Rv genes according to Cole et al (1998). The Mycobacterium bovis BCG RD1 deletion region is indicated in ESAT-6 region 1. Homology between ESAT-6 region 4 and the regions in Corynebacterium diphtheriae and Streptomyces coelicolor is also shown. (Gey van Pittius et al., 2001)
Region 4 details.jpg
Figure 1.2 A schematic representation of ESX-4 of Mycobacterium tuberculosis. The traditional annotation of M. tuberculosis H37Rv (Cole et al., 1998) is given in bold and the new nomenclature, as proposed by Bitter et al (2009) is indicated in brackets. Gene length is indicated on the gene and suggested functions are indicated above the genes.
Studying protein-protein interactions in Mycobacteria
In order to study protein interactions in M. tuberculosis Singh et al. (2006) utilised a mycobacterial two-hybrid system, termed Mycobacterial Protein Fragment Complementation (M-PFC). According to this method proteins of interest are independently fused to domains of murine dihydrofolate reductase (mDHFR). Upon interaction of the proteins, functional reconstitution of the two mDHFR domains occurs, resulting in mycobacterial resistance against the antibiotic trimethoprim, thus allowing for selection (Fig 1.3).
To achieve this, genes of interest are cloned into two M-PFC vectors, namely pUAB300 and pUAB400. pUAB300 contains a resistance marker for the antibiotic hygromycin B, whereas pUAB400 contains a resistance marker for the antibiotic kanamycin. These vectors are then co-transformed into Mycobacterium smegmatis.
M. smegmatis is a fast-growing, non-pathogenic mycobacterium and also contains a functional ESX protein secretion pathway (Converse and Cox, 2005) (Fig 1.4). This provides an attractive model organism with the advantages of safety to researchers and the ability to study the protein interactions of M. tuberculosis in a closely related organism, thus overcoming some of the limitations of traditional two-hybrid systems where yeast is used (Singh et al., 2006).
Using this system, Singh et al. were able to identify interaction between ESAT-6 and CFP-10, as well as interaction between KdpD and KdpE of M. tuberculosis. The system was validated through a M. tuberculosis library screen to identify which proteins associated with CFP-10, by which ESAT-6 was again identified (Singh et al., 2006).
Figure 1.3 Mycobacterial protein fragment complementation (M-PFC) is based on fusing small murine dihydrofolate reductase domains (mDHFR) independently on two possible interacting proteins. If there is no interaction between the proteins (X and Y), growth on plates containing trimethoprim (TRIM) does not occur (a). Upon interaction of the two fused proteins, functional reconstitution of the two mDHFR domains occur and mycobacterial resistance against TRIM will allow growth to be observed (b).
Figure 1.4 The three ESX regions of M. smegmatis. Blocked arrows indicate ORFs, as well as the direction of transcription. The different colours reflect the specific gene family, while the lengths of the arrows reflect the relative gene length. Black arrows indicate unconserved genes present in these regions (Gey van Pittius et al., 2001).
1.7 Functional studies in Mycobacteria
In recent years many molecular genetic techniques have been developed to better study the functions of proteins in Mycobacteria. Central to these techniques is the ability to inactivate genes at will, whether it is generating specific, targeted gene knock-outs or random knockout mutants. These techniques must fulfil the requirements for molecular Koch's postulates, which state that, when testing whether a gene encodes a proposed function, expression of its functional protein must be abolished. This specific function must be restored upon re-introduction of the functional gene. This provides evidence to link a pathogenic phenotype to a causal genotype (Machowski et al., 2005).
The development of techniques to generate targeted gene knock-outs have come a long way since 1990, when Snapper et al., (1990) described a mutant of M. smegmatis mc26 (ATCC607), which presented increased electro transformability. This strain, mc2155, made the detection of rare events more likely, which enabled the identification of knock-out mutants in M. smegmatis.
The pyrF gene was the first gene targeted for knock-out, since, depending on the supplement included in the media - uracil or 5-fluoroorotic acid (5-FOA) - it could be selected either for, or against. pyrF mutants could be obtained by transforming a simple suicide plasmid, carrying pyrF, and disrupted by a kanamycin resistance gene (aph) into M. smegmatis (Husson et al., 1990). These initial attempts posed several technical difficulties, which included high frequencies of illegitimate recombination and unstable double crossover (DCO) events (Kalpana et al., 1991, Aldovini et al., 1993).
In 1995 the ureC gene of M. bovis BCG was successfully inactivated by using a suicide plasmid delivery system. This urease-negative mutant meant that the loss-of-function phenotype could be assayed in vitro (Reyrat et al., 1995). In an attempt to reduce the amount of illegitimate recombinations, a system was developed to deliver the mutant allele on a piece of linear DNA 40-50 kb in length. This system was used to generate leuD mutant auxotrophs of M. tuberculosis (Balasubramanian et al., 1996).
In 1996, Azad et al., managed to replace the mas gene of M. bovis with the hygromycin cassette. The result was mutants incapable of synthesizing mycocerosic acids. The same method was employed on the pps gene cluster of M. bovis, resulting in a mutant incapable of synthesizing phthiocerol dimycocerosates (Azad et al., 1997).
In contrast to the techniques developed to create targeted gene knock-outs, random knock-out mutants can be created by random transposon mutagenesis. This requires no prior knowledge of the function of the gene (McAdam et al., 2002).
Construction of targeted genetic knock-outs
Targeted genetic knock-outs can be constructed by means of allelic exchange based methods. The gene or genetic region to be disrupted is cloned into a suicide plasmid and delivered into cells by means of electroporation. Two recombination events are required to create an allelic exchange mutant. An initial homologous recombination event will result in a single cross over (SCO) recombinant. This recombination between the chromosome and the suicide substrate can occur either upstream or downstream of the mutation and will contain both the wild type and mutant versions of the gene. In addition, all other sequences carried on the vector will be retained.
This is followed by a second recombination event, which will result in a double cross over (DCO) recombinant, which will either produce allelic exchange mutants (knock-outs) or be a reversion to wild type (Machowski et al., 2005).
To better identify allelic exchange mutants, two step selection / counter-selection procedures have been developed. In 1999, Pavelka and Jacobs refined allelic exchange methodology to produce mutants of two substrains of M. bovis BCG, M. tuberculosis H37Rv and M. smegmatis in which the lysA gene had been deleted.
Primary recombinants were selected for on medium containing hygromycin. In addition to the hyg gene, the suicide plasmid, pYUB657, also contained a counter selectable marker in the form of the sacB gene, which confers sensitivity to sucrose. sacB is one of the most commonly used counter selectable markers, specifically in slow growing mycobacteria (Pelicic et al., 1997). Thus, SCOs were not only hygromycin resistant, but also sensitive to sucrose.
In the event of a second homologous recombination event between duplicated regions, the plasmid, (containing the hyg and sacB genes) is lost. This results in a DCO recombinant that can be either a knock-out or a reversion to wild type. Knock-outs are able to grow on media containing sucrose, but die on media containing hygromycin (Pavelka and Jacobs, 1999).
However, the use of sucrose resistance as a counter selectable marker is hampered by the occurrence of spontaneous sacB mutants, which results in false positives being present during counter selection on sucrose plates. To distinguish between spontaneous sacB mutants and true knock-outs, the lacZ gene can be cloned into the vector. This will allow for the selection of SCOs (which will be blue on a media containing X-gal) and DCOs (which will be white on media containing X-gal) (Parish et al., 1999).
Relatively little is known about the ESX-4 secretion system of M. tuberculosis and other (myco)bacteria. This region contains genes encoding for members of the family of exported T-cell antigens ESAT-6 and CFP-10, which in ESX-1 of M. tuberculosis is directly involved in the pathogenicity of the organism. ESX-4 contains the minimal number of genes required for ESX secretion, thus, studying the protein-protein interaction of ESX-4 could help to set up a model of the machinery of this secretion system. Since ESX-4 has also been shown to be the ancestral region, any information generated could provide further insights into the evolution of the ESX secretion systems. In addition, deciphering the original function of the ESX secretion systems will lead to a better understanding of the evolution of the mechanism of pathogenicity.