This infectious bacterial disease is mainly caused by Mycobacterium tuberculosis which was first described by Koch back in 1882.The mycobacteria species are characterized as acid fast as they are able to retain the carbol fuchsin due to the lipid rich cell wall and alcohol fast which can be demonstrated in the Ziehl -nielson staining procedure (Leitch, 2008; Yates and Rook, 2008). Mycobacterium tuberculosis and Mycobacterium leprae are one of the few mycobacteria species which are pathogenic and also obligate human parasites(Leitch, 2008).
Mycobacterium tuberculosis has been one of the successful pathogen in mankind(Stewart et al., 2003). It can be easily transmitted via the airbourne route by the inhalation of a single bacilli containing in a droplet nuclei (Leitch, 2008). This is due to the fact that Mycobacterium tuberculosis is carried in an airborne particles called the droplet of nuclei which are generated and released into the environment by a person infected with a pulmonary tuberculosis during aspiration(Gupta and Chatterji, 2005; Leitch, 2008) .The droplet of nuclei containing the bacteria will first enter the respiratory tract infecting the lung to establish an initial infection(Gupta and Chatterji, 2005). In the lung, the bacteria will first encounter the alveolar macrophages initiating the body innate defense mechanism which under normal circumstances will cause the engulfment and the killing of any potential pathogen. However, Mycobacteria tuberculosis have evolved survival strategies and developed resistance towards the killing mechanism of these macrophages enabling them to survive, persist and replicate under such unfavourable environment(Gengenbacher and Kaufmann, 2012). The Mycobacteria tuberculosis are initially phagocytosed by the alveolar macrophage which causes the invagination of the membrane forming a phagosome encapsulating the bacteria. Typically, the phagosome will initiate the destruction and digestion of the engulfed microbes by causing them to experience increased acidification, reactive oxygen,nitrogen species and hydrolytic enzymes (Gengenbacher and Kaufmann, 2012). This acidic pH will activate enzymes that are responsible in degrading the bacterial lipids and protein thus affecting the overall microbial metabolism(Gengenbacher and Kaufmann, 2012). The presence of reactive oxygen and nitrogen species generated by the phagosomal enzymes will modify the microbes .Further damage will be induced by the permeabilization of the cell membrane causing cellular rupture .The whole process of bacterial elimination and destruction will end with phagolysososome fusion. Subsequenly,the residual content of the phagosome can be further processed for antigen presentation initiating the adaptive immune system. The components of the bacteria will then be processed and presented by macrophages and dendritic cells to activate the T lymphocytes to mount the appropriate adaptive immune response(Gengenbacher and Kaufmann, 2012; Gupta and Chatterji, 2005; Stewart et al., 2003).
However Mycobacteria tuberculosis have developed ways to adapt to the intracellular environment of macrophage to sustain survival. These bacilli under stressful condition will alter their metabolism to assure prolonged survival. Due to the unfavourable condition of the phagosome, the microbes will utilize lipid as the source of nutrient which can originate from the host-derived cholesterol(Gengenbacher and Kaufmann, 2012). Additionally, the bacteria has acquired ability to block the maturation of this phagosome preventing the acidification of the internal environment to below pH 6.4 and are able to inhibit the fusion with the acidic (pH4.8) lysosomes which normally will kill any invaders with the proteolytic activities(Apt and Kondratieva, 2008). The ability to evade the host immune system by preventing phagosome maturation and lysosome fusion can be mediated by cell wall lipid as the mycobacterial species are able to secrete membrane protein of the phagosome itself(Apt and Kondratieva, 2008). In addition the unique composition of the cell wall exist in the mycobacterial speices could also mediate the resistance and offer protection against the microbicidal mechanisms of macrophage by producing enzymes to detoxify the oxidative radicals(Gengenbacher and Kaufmann, 2012). Consequently, the onset of the innate immune system after phagosome formation will initiate the production of several inflammatory cytokines and chemokine which will recruit and attract immune cells from the surrounding tissue and bloodstream causing infiltration of immune cells like neutophils and macrophages to the infection site(Apt and Kondratieva, 2008). For instance the tissue necrotic factor alpha is one of the first few substances that are produced to activate the macrophages during inflammation (Apt and Kondratieva, 2008).
Simultaneously, in response to the cytokine and chemokine, mononuclear phagocytes will be recruited to the infection site and will start to differentiate into tissue macrophage capturing the newly formed mycobacteria which are released after the degradation of the infected alveolar macrophages forming tuberculous granuloma (Apt and Kondratieva, 2008).After few days of infection, the adaptive immune system will be initiated with the presentation of the antigen derived from the bacteria by the macrophages activating the T lymphocytes .These activated T lymphocytes will arrest the mycobacteria tuberculosis inside a tubercle forming a granuloma lesion which comprises of a central area of infected cells surrounded by phagocytic cells and foamy giant cells with the lymphocytes found at the periphery(Apt and Kondratieva, 2008; Gengenbacher et al., 2010).
In most clinical cases, the interplay between the innate and adaptive immune system is a host response to contain the infection and resist infection dissemination causing the bacteria to remain dormant or clinically known as the latent tuberculosis infection (LTBI)(Stewart et al., 2003) .In such situation, the patients will show no signs and symptoms of the diseases as the individuals are able to mount a secondary immune response and the disease will manifest when the immune system falters for instance with the onset of AIDs, aging or malnutrition (Gupta and Chatterji, 2005).
On the other hand, in an active tuberculosis, the bacilli will be continuously replicating and are spreading locally and systemically as the immune system failed to mount a defence mechanism(Gupta and Chatterji, 2005). The activated macrophages will release lytic enzyme destroying healthy neighbouring tissue liquefying the infected portion of the lung tissue affecting the respiratory capacity hence developing symptoms like persistent coughing up of blood (Gupta and Chatterji, 2005).
Conventionally, in a clinical setting generally a chest radiography, tuberculin test or mantoux test , interferon-gamma release assay (IGRA) and several lab tests can be recommended to patients who are suspected with tuberculosis (Yates and Rook, 2008). The chest radiography will detect any cavities as a result of tissue necrosis that occurs in active tuberculosis. In addition chest radiograph can be employed to diagnose LTBI which can detect scars from previous tuberculosis infection.Whereas the tuberculin test depends on the delayed type hypersensitivity to the mycobacterial antigens which is mediated by the lymphocytes and can be used to diagnose both active tuberculosis and LTBI .It involves intradermal injection of purified protein derivative (PPD)(Yates and Rook, 2008).However such test is not sensitive as there is a likelihood of a false positive result as a positive test can result from BCG vaccination or from previous exposure with environmental mycobacteria .This is similar to mantoux test where of purified protein derivative (PPD) is also intradermally injected on the forearm and the result will be read after 24-72 hours in which the diameter of the area of induration will be measured. Similar to tuberculin test,the mantoux test is also not sensitive and a false positive result is inevitable(Yates and Rook, 2008).Alternatively,the IGRA could be a reliable test to identify latent tuberculosis as it quantifies the interferon gamma released by the T lymphocytes as a result of stimulation by antigens unique to Mycobacterium tuberculosis.
On the other hand, laboratory testing mainly includes acid-fast bacilli (AFB) smear microscopy method which can be employed as a preliminary test to diagnose a mycobacterial infection as Mycobacterium tuberculosis is a slow growing organism. However, traditionally, culturing of the bacteria is the gold standard along with the drug susceptibility testing to confirm the tuberculosis diagnosis. Despite of being more sensitive as compared to the smear microscopy, this method takes time resulting in further delay in initiating the appropriate antitubercular treatment(Rodrigues and Vadwai, 2012). Hence more advanced methods have now been developed involving molecular techniques like PCR for a rapid diagnosis.
In the treatment asprect,the antitubercular treatment are usually designed to cater to different individuals based on the age, gender and health status. Generally, tuberculosis can be treated with the continuous intake of combination of drugs for 6-9 months(St. Georgiev, 1994) .The first line of anti tuberculosis drugs forms the core of the treatment regimen and this includes isoniazid, rifampin, ethambutol and pyrazinamide(St. Georgiev, 1994). The tuberculosis treatment comprised of the initial phase which takes up to 2 month followed by the continuation phase of either 4 -7 month depending on the patient response towards the prescribed treatment(St. Georgiev, 1994). Generally for newly diagnosed patients including adult and children, the treatment regimen comprised of the 6 month daily intake of isoniazid and rifampicin, supplemented with pyrazinamide for the initial 2 month phase. However there are variations in the continuation phase depending on level of drug resistance in the initial phase(St. Georgiev, 1994). However for the case of LTBI, treatment is vital to eliminate and prevent the progression into the active infection .Treatment mainly composed of combination of drugs like the isoniazid,rifampin and rifepentine over a period of 4 month or more as elimination of these persisting bacilli pose great challenge(Lobue and Menzies, 2010) .
Preventive measures can be taken to reduce tuberculosis incidence for instance BCG vaccination which was proven to be effective in reducing tuberculosis incidence in neonates but however has reduced or no effect on adult pulmonary disease(Leitch, 2008; Stewart et al., 2003). The BCG vaccine is a live attenuated strains derived from Mycobacterium bovis and is able to induce an immune response mimicking a real infection. It is only effective in containing infection by suppressing the immediate threat of active infection but however it does not eliminating subsequent infection(Stewart et al., 2003).
Overall, the success of Mycobacterium tuberculosis are based on three key features; firstly the survival of the bacteria in the host as a result of the macrophage reprogramming upon primary infection; secondly the formation of granuloma creating a confined environment causing the bacteria to persist in the host until the host immune system fail to contain the bacteria resulting in the manifestation of the disease. Finally,Mycobacterium tuberculosis are able to persist by transiting to the dormant stage rendering the bacteria resistance to the host immune system and drug treatment(Gengenbacher and Kaufmann, 2012).This hence suggest that persistence is the hallmark of tuberculosis as the bacterial incubation period could vary from a few weeks to a lifetime before the manifestation of the disease(Stewart et al., 2003). There is a need to understand the mechanisms of persistence as majority of the reported infected case belong to the LTBI and these patients are asymptomatic and harbour dormant bacilli which will restore virulence under weakened immune system. At present,most of the antibacterial drugs are potent in targeting actively replicating culture for instance isoniazid; a cell wall synthesis inhibitor and rifampicin; a transcriptional inhibitory(Gomez and McKinney, 2004; Gupta and Chatterji, 2005).The effectiveness of a preventive therapy for future recurrence mostly depends on the time course of the treatment .Moreover these drugs are ineffective in targeting the dormant bacilli thus resulting in a longer course of treatment as subpopulation of these bacteria are phenotypically drug resistant(Gomez and McKinney, 2004). Hence it is essential to understand the molecular mechanism of dormancy in mycobacterium to further understand the persistence issue .This could eventually lead to discovery of new drugs which could target these persisting bacilli with effective short courses therapies(Gupta and Chatterji, 2005).
Several studies have established working model to mimic the in vivo environment of the infected granulamatous lesion which are mainly hypoxic and nutrionally deprived(Gupta and Chatterji, 2005; Zahrt, 2003). The Wayne dormancy model was developed to replicate the hypoxic in vivo environment. Principally, bacteria will be grown anerobically in a non-agitated culture. This will allow a uniform and stable bacterial population with the non-replicating settling at the bottom and the replicating population in the upper layer of the tube due to their different oxygen content(Hayes, 1996).
In our study, we would like to dissect the dormancy mechanism in a nutrionally deprived bacteria hence Loebel model culture will be employed which is analogous to the nutrionally deprived in vivo environment. Bacteria will be grown in DPBS supplemented with Tween 80 to prevent bacterial aggregation in a well aerated and agitated roller bottle. Martin et al have employed the loebel model to investigate the dormancy issue in nutritionally starved Mycobacterium tuberculosis(Gengenbacher et al., 2010). Similar to the Wayne model demonstrated by Koul et al, both models demonstrated a fivefold drop in the ATP level when the bacteria transit to the quiescent non-replicating state with a stable CFU of 107 range (Gengenbacher et al., 2010; Koul et al., 2008).However these dormant bacteria are still viable and are able to survive under such hostile environments. Relatively, Loebet et al has previously demonstrated a gradual shutdown in rate of respiration indicating low metabolic rate when the bacteria were transferred from nutrient rich medium to PBS but the bacteria remained viable and were able to resuscitate to its replicating state after enrichment(Loebel et al., 1933). This suggests that despite of the different condition set in the different in vitro models, all the models have shown the generation of quiescent non-replicating bacteria. Additionally, the models also demonstrated reduction of the intracellular ATP level and phenotypic drug resistance which can be identified as core features of dormancy(Gengenbacher and Kaufmann, 2012). Furthermore, transcriptomic studies have shown a global downregulation of carbohydrate metabolism and replication machinery in nutrient starved mycobacterium tuberculosis(Joanna C. Betts and Duncan, 2002). Accordingly, this could explain the phenotypic resistance of these dormant bacilli towards drugs rendering chemotherapy ineffective in treating LTBI. Such phenomenon can be explain by the adverse changes the bacteria undergo during this transition .These changes could include the downregulation of energy metabolism accompanied by the loss of cell wall permeability, the absence of protein biosynthesis, remodelling of the mycobacterial cell envelope and cell wall thickening(Sarathy et al., 2013). Mycobacterium tuberculosis has also been shown to undergo morphological change and staining properties in vitro (Smeulders et al., 1999). Some of these dormant bacilli tend to grow in aggregate to maximize the limited nutrients available (Smeulders et al., 1999). Moreover, with recent advancement in proteomic profiling, it has provided a greater opportunity in understanding persistence in TB by studying the protein expression during dormancy. A study which involved the analysis of the proteome of Mycobaterium tuberculosis bacteria has revealed a global downregulation of protein involving in metabolism and respiration after nutrient starvation(Albrethsen et al., 2013) .While relatively there were upregulation of various functional proteins including virulence, detoxification and adaptation, lipid metabolism, cell wall and cell processes which could be indispensable for the survival of the dormant bacilli hence providing possible candidates for drug targets to treat LTBI(Albrethsen et al., 2013).
Our research aims to investigate if such responses observed in nutritionally starved Mycobacterium tuberculosis are conserved in the nutritionally deprived Mycobacterium smegmatis. If such responses are conserved in the fast growing model, a working model can then be established and can be used for future studies to further understand the molecular mechanism of dormancy and could possibly find potential drug target to treat LTBI. Mycobacterium smegmatis is a perfect model organism for any mycobacterial related studies. This is mainly due to its fast growing rate as the generation time of Mycobacterium smegmatis is only 2-3 hrs with 2-4 days to yield visible colonies on the nutrient agar(Gupta and Chatterji, 2005). This is different as compared to Mycobacterium tuberculosis which has a generation time of 24 hours and 3-4 weeks to yield visible colonies. Furthermore, unlike Mycobacterium tuberculosis,Mycobacterium smegmatis is non pathogenic thus fewer constraints are imposed on its handling and manipulation as no Biosafety Level 3 (BSL3) containment is needed(Gupta and Chatterji, 2005).
For the purpose of this study Mc 1552 will be used and our approach is to employ the nutrient starvation model by growing the bacteria in DPBST in a roller bottles with agitation for 40 days .Throughout this experiment, analysis will be done on consecutive days to determine the bacterial viability by measuring the plated CFU for survival and the OD600 for growth. Consecutively, the intracellular of the ATP level will be measured on consecutive days throughout the 40 days experiment. The drug susceptibility test will then be determined by exposing a known drug concentration to the non-replicating culture on the day when the bacteria transit to their quiescent non-replicating state. This value can then be compared to the maximum bactericidal concentration (MBC) value of the replicating Mycobacterium smegmatis to demonstrate if those dormant bacilli are as phenotypically drug resistant as the dorman tMycobacterium tuberculosis. Finally, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) will be performed to observe the protein expression of the Mycobacterium smegmatis bacteria in a normal log-phase growth and after two weeks of nutrient starvation.
In summary, the main objective of our study is to replicate the simplest model of Mycobacteriun tuberculosis dormancy in the fast growing Mycobacterium smegmatis.We aim to investigate if the growth of the dormant M.smegmatis is similar to the Mycobacteriun tuberculosis in the in vitro model which closely reminisce the bacterial growth in vivo. Once the survival of the non replicating culture has been established in the model organism, we could then investigate and characterize those dormant cells by analyzing the kinetics of the intracellular ATP levels and the drug susceptibility to observe for any phenotypic drug resistant.
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