Tuberculosis is a popularly known disease due to its high mortality rate. Even though tuberculosis has a live attenuated vaccine of its own, it’s still known to affect many people worldwide. About two million people die each year from it. It is actually known to be one of the oldest known human diseases. Tuberculosis affects many parts of the body such as the bones, the central nervous system, the brain, and many other organisms part of the body, however, it is mostly known as a pulmonary disease. The causative agent for this pulmonary disease is Mycobacterium tuberculosis. The bacteria is a non-motile, acid fast, obligate aerobe. Their size ranges from two to four micrometers in length and they have a slow generation time which ranges from fifteen to twenty hours. Their cell wall is composed of mainly acidic waxes, and of those acidic waxes, mycolic acids specifically compose their wall. Since the bacteria is known to be resistant to drying and chemicals, they are much easily transmitted from individuals. The way that the bacteria is spread from individual to individual is through aerosol droplets into the nuclei. There is a higher risk of obtaining the bacteria when individuals are in environments where they have to experience close contact with other individuals without any type of good air circulation. It has been reported that there are some M. tuberculosis strains which are more virulent than others which could lead in disease progression. This is supported by the fact that there has been a higher rate of morbidity and mortality in individuals who have been infected with M. tuberculosis. In order to develop new antitubercular agents, it is a necessity to study the physiology and the genetics of M. tuberculosis along with how it interacts with host cells.
There are different stages of human tuberculosis based off of how severe the infection is on the patient and the involved organ. When tuberculosis occurs in the spine, it is known as Pott’s disease, which back in the 18th-century, Hippocrates found that it had many similarities to pulmonary tuberculosis. When tuberculosis is seen to be developing in the central nervous system, meningitis takes the predominant form of it. Tuberculosis can also take place in the form of lupus vulgaris when it occurs cutaneously, in the urogenital tract, and the digestive system. Now-a-days however, tuberculosis infections are usually initiated by the route of exposure to milk products through the respiratory tracts. It was in 1978 when it was discovered that tuberculosis mostly affected the pulmonary system. The progression and the resolution of the disease was divided into four different stages by Wallgren. In the first stage, he observed M. tuberculosis the first three to eight weeks that it was found in inhaled aerosols which then became implanted in in alveoli. During this stage, M. tuberculosis the Ghon complex is created through the spreading of the bacteria to the lymph nodes in the lung by lymphatic circulation. Once the bacteria is spread, tuberculin reactivity occurs. The second stage lasts about three months and this is the time when the bacteria produces blood and it is circulated to many organs and other places which are included in the lungs. The second stage is when Wallgren observed that affected individuals experienced acute and sometimes fatal diseases which occurred as tuberculosis meningitis or disseminated tuberculosis. The third stage lasts three to seven months and it is the stage in which inflammation of the pleural surfaces occur. Since the pleural surfaces of the lungs are affected, it can also lead to chest pains. This stage can last up to two years. Pleural infection can occur due to the spread of blood particles from the bacteria to pleural space from subpleural concentrations of bacteria in the lung. The released particles interact with CD T4 lymphocytes and are proliferated to release inflammatory cytokines. In the last stage, which is also known as the resolution of the primary complex, more extrapulmonary lesions which are being slowly developed start to appear. This stage could last up to three years, but this stage is rare to appear to affected individuals.
There are two different events which occur during the infectious process of the bacteria, the early process and the late process. The early process usually affects children rather than it does adults. In the early processes of infection, also known as primary tuberculosis, it is observed that M. tuberculosis affects the individual through aerosol entrance. Although it is known that M. tuberculosis causes tuberculosis through the entrance of alveolar passages, it is also known that they can affect individuals by being ingested by alveolar epithelial type II pneumocytes. Since these pneumocytes are found in a greater amount than macrophages are in alveoli, the bacteria is able to infect and grow inside of them. Another factor that plays a part in the early infectious process are dendritic cells. The reason being that unlike macrophages, dendritic cells are migratory. Their quality of being migratory helps with dissemination of the bacteria. Knowing the purpose of dendritic cells, it is easier to observe the interaction of M. tuberculosis interacting with macrophages.
Through the observation, a process occurs in which bacterial contact with the macrophage mannose causes phagocytosis of the bacteria. A glycoprotein, surfactant protein A, then enhances the binding and uptake of the bacteria on alveolar surfaces by regulating mannose receptor activity. There are a few factors that can cause antimicrobial growth to occur. Once the bacteria enters a host macrophage, they take shelter in an endocytic vacuole called the phagosome. If phagosomal mutation occurs as it normally does, the bacteria can end up entering a very hostile environment. The hostile environment includes having a low pH value, reactive oxygen intermediates, and toxic peptides. However, since exposure to the hostile environment is very common, the bacteria has adapted qualities which help it to avoid the hostile environment.
When M. tuberculosis enters the human body, it has been observed that Ca2+ signaling is not able to happen. This is important to know because the job of Ca2+ is to stimulate host responses to infections. Since Ca2+ is not able to send out signals to the host cell, it allows the bacteria to take over the host and reproduce from it.
The late process of infection is known as secondary tuberculosis. Secondary tuberculosis usually occurs due to reactivation of the latent primary infection. There is a lot of information on how M. tuberculosis is able to enter and infect the host cell, but there is not that much information on how it stays intracellular and its survival and growth. It is observed that the macrophages that are infected in the lungs produce chemokines which will attract inactivated antigens such as monocytes, lymphocytes, and neutrophils, none of which are strong enough to kill the bacteria entirely. After they are all brought together, granulomatous focal lesions start to form. This procedure occurs in order to contain the spread of the bacteria and build up cellular immunity. As cellular immunity is getting stronger, macrophages that are filled with the bacilli are killed and the granuloma is formed. The tissue becomes strong because it is surrounded with fibroblasts, lymphocytes, and blood-driven monocytes. Although the tissue has become stronger, M. tuberculosis may still be able to remain dormant and alive for many decades. Whether or not the host cell progresses to the next stage of tuberculosis or remains is all dependent on the strength of the host cellular immune response. This type of infection is known as an enclosed infection. It is latent and asymptomatic. Individuals that have a strong immune system and experience enclosed infection may be able to permanently rid of the bacteria. However, if an individual with a weak immune system was not able to control the bacteria from the primary infection stage, their immune system continues to weaken and the granuloma center can become liquefied. Now that the granuloma is liquefied, it can serve as a rich medium for the bacteria to revert back to being virulent. Once the bacteria has become virulent, it can escape from the granuloma center and spread throughout the lungs, thus becoming active tuberculosis, and also spread through the lymphatic system, and blood. Once this happens, the individual is now infectious and requires antibiotics in order to survive.
Although it is now known in the present day that M. tuberculosis is contagious, it was not always believed so. In the 5th century BCE, the Greek physician, Hippocrates, had made the claim that tuberculosis was the most widespread disease of his time. He also made the claim that the disease was hereditary, and many people had agreed with him. One person who did not agree with him however was the philosopher, Aristotle. Aristotle was under the belief that the disease was contagious. However, neither of them had any type of proof to determine whether it was hereditary or contagious. A few centuries after, Europe had split into two different bodies of agreement on the etiology of the disease. Northern Europe was under the belief that it was hereditary and southern Europe was under the belief that it was contagious. This division of belief had occurred due to the large distribution of tuberculosis. One reason that people thought that tuberculosis had spread geographically was due to Indo-European cattle herders. They had traveled throughout Europe and Asia, bringing along with them the disease. The peak of the spread of the disease in Europe was during the 15th-17th century due to the increase in population and low sanity levels. The reason being that the more people are in a place, the easier it is for the bacteria to spread from one host to another. In 1720, it was made by Benjamin Marten that the bacteria was infectious, but it took many years after that to determine whether his observation was correct or not. Rene Theophile Hyacinthe Laennec was the inventor of the stethoscope and was the one that was responsible for one of the first evidence to prove that M. tuberculosis was pathogenic. In 1865, Jean-Antoine Villemin showed that a rabbit has the ability to be infected by M. tuberculosis by an infected cadaver. In 1882, Robert Koch had presented his discovery of the tubercle bacillus. Tuberculosis rates started to decline within the 19th and 20th century due to an improvement in sanitation and also due to the many discoveries of the bacteria and how it works.
However, determining whether or not the bacteria is virulent is still yet to be determined. There is not enough knowledge on how the bacteria is able to cause the disease, however, its virulence is able to be measured. Two words which represent its virulence are: mortality and morbidity, mortality being the percent of infected individuals that experience fatality. Another factor which helps determine its virulence is bacterial load which is observing the amount of bacteria that is found inside the infected host. Determining what kind of bacteria is inside the host helps to determine the types of bacterial strains that are able to survive host responses while they are infected. Hosts that are have a low amount of bacteria inside of them experience different types of growth curves. In one study, the bacteria was grouped with different bacteria and it was observed how they are controlled by different types of bacteria. In order to understand the mortality and morbidity that is caused by M. tuberculosis, the pathogenesis that is associated with tuberculosis should also be understood. When the bacteria is not able to be controlled, it leads to prolonged lung damage that will ultimately lead to death due to suffocation. There are other types of untreated forms of tuberculosis such as tubercular meningitis which can result in death due to inflammation in the brain tissue which could end up leading to seizures.
There are two different kinds of models for measuring the virulence of M. tuberculosis. One of them being animals. Animal models are better to use in relation to macrophage models because scientists are able to study all stages of the tuberculosis. The three different types of animals that are normally chosen for study are: mice guinea pigs and rabbits. Mice are chosen usually for vivo models because they have well-studied genetics. One quality that their genetics has are known as inbred strains. A benefit of this is that the strains show a wide range of different levels of resistant that M. tuberculosis has. With that quality, scientists were able to discover that M. tuberculosis acquires a complex trait that other types of loci would have to play a role in. The loci help to determine different kinds of resistance that hosts have on M. tuberculosis infections. A negative aspect to using mice models is that the progression of tuberculosis in mice is not the same as it would be in humans. In mice, the granulomas that are formed are not as differentiated, however, since mice are not as sensitive to certain diseases as other animals are, their stage of infection is similar to that of humans. Guinea pig models are used mostly because they experience sensitivity to the infection caused by M. tuberculosis. During the time that the granulomas are forming is the stage that guinea pigs experience similarity with infected humans. The disadvantage of using guinea pig models however is that unlike the mice, they do not have the inbred strains and reagents; they also cost way more to maintain. Lastly, the model of the rabbit attains one great advantage over the other two animal models, and that is that the granulomas that are formed in the rabbit’s lungs show the same type of progression throughout the stages that a human would experience. However, just like the mice and the guinea pig, they also have a disadvantage and that is that their maintenance is even more expensive than both the mice and the rabbit. The second type of model that is used as described earlier are macrophages. Since the bacteria is known to be an intracellular pathogen, it mostly infects macrophages. Scientists use the infected macrophages to analyze the virulence of the bacteria’s strains and also its mutants.
Moving on to the genetics of M. tuberculosis. The bacteria itself is known to be part of the Bacillus genus which means that is rod shaped. It ranges between two to four micrometers and has a slow generation time of fifteen to twenty hours, which explains why an individual may experience mild symptoms or they may be asymptomatic. The M. tuberculosis genome actually contains four thousand genes within it. Two hundred of those genes are known to be encoding enzymes for the metabolisms within fatty acids. Within those two hundred genes, one hundred of them are used to work in the fatty acid’s beta-oxidation. In comparison to other bacteria, the fatty acids of M. tuberculosis carry twice as many enzymes used for fatty acids. This predicts the strong ability for the pathogen to grow in tissues of infected hosts. The amount of fatty acid enzymes are not the only unusual thing of the bacteria’s genome. Another quality is the amount of unrelated Pro-Glu (PE) and Pro-Pro-Glu (PPE) sequences.
There are different methods of genetic analysis that is used when trying to inactivate the genes of M. tuberculosis. The gene disruption techniques are divided into: directed and global methods. They usually require a phenotype that has resistance to antibiotics. Directed gene inactivation requires an antibiotic resistant cassette to be inserted into the middle of the chosen gene. Once it is inserted, the DNA of the antibiotic is transformed into mycobacteria forming either a linear or a circular molecule through the help of electroporation. This procedure should result in replacement of the alleles in the original chromosomal gene with that of the mutated one. Using a single-stranded linear shaped DNA is more beneficial because it increases the chance of allelic replacement. The single-stranded linear shaped DNA also makes cloning easy since only a short amount of the DNA fragment is used. However, there is a disadvantage as to using the sing-stranded linear DNA ad that is that it requires a certain kind of restriction enzyme site. This site would not need to be used if only one is able to create more of a manipulation of the DNA sequence. Global gene inactivation is another use of inactivation. When this is used, foreign DNA is inserted into multiple sites in the genome of the bacteria.
When the infection of the animals and macrophages occurs, this is the time when the virulence of the bacteria can be properly measured in order to create mutations inside of the bacteria’s genes. Those methods lead to the identification of many genes that are of the importance for pathogenicity of the bacteria known as virulence factors. First is the cell secretion and the envelope function of the proteins. These proteins have a role in synthesizing different kind of cell surface molecules. The cell wall and the envelope of the bacteria are very complex in which M. tuberculosis is able to be separated from its external environment. The cell wall is composed of acidic waxes, specifically that of mycolic acids. One of the proteins that are found in the environment that M. tuberculosis grows in are known as culture filtrate proteins. The studies of these proteins show that live forms of attenuated M. tuberculosis vaccines are preferred over vaccine shat are made from heat-killed cells because when they are grown, the culture filtrate proteins are released and they stimulate host immune mechanisms. Another virulence factor would be the enzymes that are involved for general cellular metabolism. These enzymes are mutated by researchers in order to observe how they protect the bacteria against oxidative stress, given that the bacteria is a non-motile aerobe. One last virulence factor would be transcriptional regulators. The job of the transcriptional regulators is to control the transcription of the bacteria’s genes. It is known to inactivate regulatory genes because it is classified as a mutational strategy and through this procedure, the virulence of M. tuberculosis can be figured out. One of the strategies that is used by prokaryotes is known as sigma factors and M. tuberculosis uses this to transcribed genes to be used for virulence.
It is good to understand how the bacteria works and how it is able to spread because it explains who is affected and why they are affected. The people that are mostly at risk in being infected with M. tuberculosis are usually that of the younger age. However, people that have an even higher risk are those that have had close contact with an infected individual within a year. If they happen to be infected and experience symptoms, their symptoms would be in relation to that of a flu. The individual would then not suspect that they have tuberculosis, and they will leave the infection untreated. If the infection is left untreated, then this is the time that M. tuberculosis will infect and multiply itself with pulmonary alveolar macrophages in the lungs. Once the host has been invaded, they will migrate towards enlarged lymphocytes. This procedure will then cause an immune response to occur from T-helper cells and that will cause inflammation to occur at multiple sites. During this time, the infected individual will be able to take a tuberculin skin test and either test positive or negative. If the test is positive, a bump of the size of a pencil eraser will appear on the arm. Another way that one can determine if they have a positive test result is getting an x-ray of their lungs. If there are opacities seen in the resulting x-ray, it most likely means that the individual has tuberculosis.
So all in all, after researching about what the bacteria is and how it works and also what the disease is, it is also important to know ways to prevent it. There is no way to prevent infection of M. tuberculosis, however, there are ways to prevent it from spreading or from getting worse after the primary infection stage. One way would be through “identification and cure”. Once the individual has tested positive on the tuberculin skin test, then they should take action to making their immune system stronger and also taking any type of antibiotics that they can to help arrest the bacteria. By strengthening their immune system, they are preventing primary tuberculosis from becoming secondary tuberculosis. A second way that they can prevent the bacteria from spreading or the disease itself from spreading is by improving air filtration. By doing this, there is less opportunity for the bacteria to travel from individual to individual just as it did in 15th-17th century Europe. The last way that someone will be able to prevent M. tuberculosis from infecting their host or from spreading would be to just live a healthy life style. Doing so will not only help the invidvual physically, but will also help strengthen their immune system. By living a healthy life style, if the individual does happen to get infected, the strong immune system will prevent the primary tuberculosis from immediately moving onto the secondary tuberculosis and it could also rid of the bacteria for good during the primary tuberculosis stage.