Examining mycobacterium tuberculosis, its spread and effect

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Mycobacterium Tuberculosis is a pathogenic bacterium in the genes Mycobacterium. It was first found in 1882 by Robert Koch, Mycobacterium tuberculosis has a very specific waxy coating on the cell surface primarily made of mycolic acids which are resisting to Gram staining and therefore acid-fast detection techniques are used. Mycobacterium tuberculosis are highly aerobic bacterial cells, so requires certain amount of oxygen levels to survive. Therefore the respiratory system in mammal is the optimum place to begin infection. M. tuberculosis often found infecting the human lungs and is the causative agent of tuberculosis (TB), which is a well known disease in human beings. (RHEE, J.T, 1999) Tuberculosis is a fatal disease which causes death in about 3,000,000 people around the world every year, the deaths toll is more than malaria, other tropical disease, and AIDS all combined. About one third of the world's population in total are known to be infected with tuberculosis. This is the reason why tuberculosis easily becomes the most lethal infectious disease in the world. (RHEE, J.T, 1999) The transmission of tuberculosis is mainly by the aerosol route but it may also be able to transmit through the gastrointestinal tract. Coughing by people who are infected with active tuberculosis will produces droplet nuclei, which containing infectious organisms and will be able to remain suspended or survive in the air for several hours. Infection occurs if a person inhales these droplets, it will result in the infectious organisms reaching the alveoli in the lungs of the person. In this stage, the infectious organisms are then taken up by the alveolar macrophages which are then carried to lymph nodes system. This is where it can spread to multiple organs by lymph system around the body. (RHEE, J.T, 1999) After infection of the tuberculosis in two to eight weeks, the cell mediated immunity (CMI) and hypersensitivity (DTH) will develop, this will be able to detect by the tuberculin test due to the characteristic reaction. In immunocompetent individuals, this can be check or test by containment of infection. Inflammatory immune responses often result in lung damage of the infected person. There are only about 10% of immunodeficient people in the world infected with M. tuberculosis will develop active disease in their life. The other 90% of people do not develop disease and will not transmit the organisms but in some groups such as infants or the immunodeficient the proportion which will develop different clinical tuberculosis is much higher than others. (CAWS, M, 2008) There are a lot of studies regarding the genome of the M. tuberculosis, the most common used strain is called CDC1551. It often used in laboratory to compare different laboratory strains such as strain H37Rv. The reason of studying the genome of the M. tuberculosis is try to understand the function of each gene within the genome, such as the virulence or the host immune response. (COLE, S.T, 1998) Understanding the genome of M. tuberculosis can help to study the genome of other members in the mycobacterium tuberculosis complex, such as Mycobacterium bovis, leprae and microti.

Mycobacterium Microti, is a member of the Mycobacterium tuberculosis complex. M. microti is the main causative agent of the tuberculosis that found in voles throughout Great Britain. Although M. microti often infect voles and other mammals such as house cats, (C.C.Frota 2004) but there are also few rarely cases that involve human infection of M. microti. It known to have causing infection in both immunocompetent and immunocompromised humans, these rare cases have been reported from these countries: Netherlands, Germany, Switzerland, England and Scotland. (F. X. Emmanuel 2007) Due to the increasing number of human infection cases, this indicated that M. microti has become more diverse than before. The genome of M. microti is closely related to Mycobacterium tuberculosis. M.Microti has very similar genome sequence as the normal type of Mycobacterium Tuberculosis, such as Mycobacterium Bovis. Recent findings shown that several specific genes were deleted in the genome of different strains of M.Microti which similar to M. bovis. (N.H.Smith, 2009) The genes that found to be deleted in M. microti are RD7, RD9, RD1mic and MiD4, but RD1mic and MiD4 genes are found to be intact in M. bovis genome which allow clear distinguish between these mycobacterium complexes.

Mycobacterium Bovis, is a pathogenic species in Mycobacterium which is similar to M.tuberculosis. M.bovis has significant importance in livestock and a wide range of wild animal species worldwide. It is also known to cause tuberculosis disease in humans with similarity to M. tuberculosis. Tuberculosis in animals is mostly known from cases in cattle and for which the disease is generally referred to be bovine tuberculosis. The strain causes infection in animals of bovine tuberculosis is by Mycobacterium bovis, which is a member of the Mycobacterium tuberculosis complex (Smith et al., 2006). M. Bovis infections are also found in natural environment, it can cause some influences for the ecosystem. On the other hand, animal tuberculosis may have a zoonotic potential therefore public health concern is also needed. (Renwick et al., 2007) Although animal test schemes have reduce the rate of infection of bovine tuberculosis significantly in many industrialized countries, but these expensive control programmes are often questioned with regards to their economic burden and increasing opposition by farmers. In the UK, several different mammals also have been found to be infected with M. bovis, but the frequency of infection cases is significantly less than the cases reported for the cattle and badgers. This disease is widely spread in cattle throughout the world. However in some countries, they have managed to reduce or limit the incidence of the infections of the disease through a process which select and pick or kill infected cattle from the cattle stock. Most of the Europe and different Caribbean countries including Cuba are almost free of M. bovis infections. However some infections cases might still exist in isolated parts of the Northern Territory. In the United States (US), there is an outbreak of M. bovis in white-tailed deer in the northeastern part of the US near Michigan and in northern Minnesota. There are also some cases shown that they were imported from Mexico. Recent finding shown that the genome of M. bovis have several genes deleted compare to M. tuberculosis that found in human, the genes that are deleted are RD4, RD7, RD9, RD12 and RD13.

Mycobacterium Leprae is an aerobic and is a bacterium that causes leprosy also known as Hansen's disease. It has a rod-shaped bacillus cell body. M.leprae was first discovered in 1873 by a Norwegian physician called Gerhard Armauer Hansen. It was discovered when he was examining the bacteria on the skin of patients with leprosy. It was also the first bacterium species identified that causes human’s disease. The leprosy patients that have diffuse lesions or lepromas will contain many M leprae bacilli cells or multibacillary lesions. These lesions are mostly found on the cooler part or surface of the body, such as the nasal mucosa and the peripheral nerve trunks at the elbow, wrist, knee, and ankle. The cell itself is surrounded by a form of thick waxy coating which is unique to mycobacteria species. The size and shape of M.leprae is very similar to Mycobacterium tuberculosis. Due to its thick waxy coating, M.leprae only can stains with a carbol fuscin rather than the original and traditional Gram stain. This complex and unique cell wall has caused the Mycobacterium genus difficult to destroy and express which caused slow replication rate. (HALL, B.G. 2010) Under observation of microscope, the microscopy shows M. leprae formed in clumps, gathered in masses or in bacilius form which lie side by side to each cell. It has never been successfully cultured in vitro, but some research shown that it has been grown in mouse foot pads and nine-banded armadillos. The difficulty in culturing it is because it appears to be an obligate intracellular pathogen or parasites that requires mainly on the environment of the host macrophage for survival, it is also being considered of lacking many necessary genes for independent survival. (AKAMA, T, 2010) In the genome of M. leprae with comparision to M. tuberculosis has found that several genes which involve metabolic activites have been deleted. (MARTINEZ, A.N, 2009)

Table 1: Shown the appearance of the genes in the genome of different species of Mycobacterium. (N.H.Smith, 2009)

There are several genetic techniques that may be used in the project such as 454 Sequencing, PCR and Sanger sequencing. The sample input and fragmentation of 454 sepuencing is the complete sequencing of the two systems GS FLX and GS Junior Systems which has four main steps, leading from purified DNA to analyzed results. These main steps involved the generation of a single-stranded template DNA library, clonal amplification the library with emulsion-based, sequencing-by-synthesis for data generation and the data obtained will perform a data analysis with different bioinformatics tools used. The 454 sequencing is based on minimize the process of pyrosequencing technique. It aims to give a reliable result at a low cost with less time, and the sequences produced are shorter than the sequences produced by classical methods. (Blazewicz. J, 2009)

C:\Users\Vincent Li\Downloads\nbt1485-F2.gifFigure 1: The process of 454 sequencing.

(a) Genomic DNA being isolated and separated into single stranded DNA. (b) The Fragment are then bound to beads by one fragment per bead strategy, the beads are isolated in the PCR reaction mixture in oil emulsion droplet and amplification occurs in each droplet, which causes each bead carry 10 million copies of DNA template. (c) The emulsion is now broken and the DNA strands are denatured. The beads that are carrying single stranded DNA templates will deposited into the wells of fibre-optic slide. (d)Some smaller beads which carry immobilized enzymes will need a solid phase pyrophosphate sequencing reaction, so they are deposited in each well. (e) Electron micrograph scanning performed for a portion of a fiber optic slide, which shows the cladding and wells before the beads deposition. (f) The 454 sequencing instrument is works by following system, a fluidic assembly (i), a flow cell that has the fiber-optic slide (ii) and a CCD camera based assembly which has its own fiber-optic bundle used to take image of the fiber-optic slide (iii) with a computer monitor and controls that provide user interface and instrument control (iii). (Jonathan.M.R, 2008)

Sanger sequencing which also known as “chain terminator sequencingâ€Â, was developed by Fred Sanger which allows several or large number of bases to be read and it works by inserting specific chain terminating nucleotide into the DNA sequence during DNA synthesis. (DAVIES, E., 2005) The extension is initiated at a specific part on the template DNA sequence by oligonucleotide 'primer', which is complementary to the template DNA at that specific part. This primer is extended by a DNA polymerase. There are four deoxynucleotide bases included with the polymerase and primer, which have a low concentration of a chain terminating nucleotide. Due to very little interaction between the DNA polymerase and the chain terminating nucleotide, it causes several of related DNA fragments that only terminated at specific positions where its specified nucleotide is used. The fragments are then able to perform a electrophoresis in a gel for size separation and data analysis. (PUSHKAREV, D, 2009)

Aims of the project

The aim of the project is to close the gaps between different short sequences that generated by PCR from the genome of Mycobacterium Microti. The scientific goal of it is to compare the genome of Mycobacterium Microti to Mycobacterium Tuberculosis and its complexes such as Mycobacterium Bovis and Mycobacterium Leprae. This will allow more information on studying the differences between different M. tuberculosis complexes such as the virulence factor on human and animals. The project will be outlined to four stages. The first stage is to design specific primers for each short sequence generated, so that it can close the gaps between the sequences. The second stage is to perform Polymerase Chain Reaction PCR with the genome of

M. microti to generate the specific short sequences. The third stage will be sequencing the PCR products generated by using sanger sequencing. The final stage is to combine the PCR products sequence with 454 sequencing. The predicted outcome of the project will be the identification of genes that present in M. microti which may not be found on the other M tuberculosis complexes, and found out the function of specific genes within the genome.

Proposed timetable

The whole project takes about 14 weeks, the planned time table is shown below:

1-3 weeks: Design primers for the genome

3 weeks: Perform PCR

2 weeks: Sequence PCR product using sanger sequencing

2 weeks: Combine PCR sequences by 454 sequencing

2 weeks: writing the project report and presentation preparation

2 weeks: for extra work or experiment that may need