Demonstration of the Putative Roles of PE3 and PE4 Proteins of Mycobacterium tuberculosis in Intracellular Survival and in Immune Modulation

Abstract

Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis (TB) and a major cause of death worldwide (Murray et al., 1990; Raviglione et al., 1995). Mtb was discovered by Robert Koch in 1882, earlier it was called as „Koch bacilli‟. Mtb is an obligate aerobic, non-motile and rod-shaped distantly related to the Actinomycetes with 2-4μm in length and 0.2-0.5μm in width. It is a facultative intracellular pathogen that can survive and multiply inside macrophages and inside other mammalian cells. Mtb is not classified as either gram-positive or gram-negative because it does not have the chemical characteristics of either, although the bacteria do contain peptidoglycan (murein) in their cell wall (Prescott et al., 1996). Mycobacterium species, along with members of a related genus Nocardia, are classified as acid-fast bacteria due to their impermeability by certain dyes and stains. Despite this, once stained, acid-fast bacteria will retain dyes when heated and treated with acidified organic compounds. The mycobacterial cell wall has unique composition and composed by a complex outer cell wall consisting of large amount of lipid. It consists of several unique components such as lipoarabinomannan, lipomannan, pthiocerol dimycocerostate, mycocerostate, mycolic acid, trehalose dimycolate and sulpholipids (Brennan et al., 1990; Bersa and Chatterjee, 1994). These components are suggested to be responsible for mycobacterial hydrophobicity, ability to form clumps or cords, ability to survive intracellularly and it is the cell wall that gives acid-fastness, enabling it to retain basic dyes in the presence of acid alcohol. TB is a very common and deadly infectious disease caused by Mtb or M. bovis and almost one-third of the world's population now has the TB bacterium in their body (WHO, 2006). In 2004, 14.6 million people had active TB and there were 8.9 million new cases and 1.7 million deaths, mostly in developing countries (WHO, 2006). A rising number of people in the developed countries contain tuberculosis because of their immune systems are compromised by immunosuppressive drugs or HIV/AIDS (MMWR Morb Mortal Wkly Rep 2006). In the past, TB was called “consumption”, because it seemed to consume people with symptoms such as bloody cough, fever, pallor and long relentless wasting. Other names are “phthisis” (Greek name for consumption), scrofula (in adults affecting the lymphatic system and resulting in swollen neck glands), tabes mesenterica (TB of the abdomen and lupus vulgaris), King‟s evil (because it was believed that a king's touch would heal scrofula). Military TB is an archaic term that is still occasionally used when the infection invades the circulatory system resulting in X-ray lesions with the appearance of millet seeds (Smith, 2003). More than 75% TB cases are of pulmonary TB because of having affected lung. With the disease progression, infection spreads from the lungs to extra pulmonary sites including the pleura, central nervous system in meningitis, lymphatic system in scrofula of the neck, genitourinary system in urinogenital TB and bones as well as joints in Pott's disease of the spine. Extra pulmonary forms are more common in immune-suppressed persons and in young children (CDC, 2000). 1.2. Mycobacterium tuberculosis complex Phylogenetic studies among mycobacteria by 16S rRNA sequencing showed that Mtb belongs to a group of „slow growers‟, also known as „Mtb complex‟. The bacteria require 3-4 weeks to form colonies, with generation time of almost 24 h in solid media. The Mtb complex comprises six members: M. tuberculosis, the causative agent of the vast majority of human TB cases; M. africanum, an agent of human TB in sub-Saharan Africa; M. microti, the agent of TB in voles; M. bovis, which infects a very wide variety of mammalian species including humans; BCG, an attenuated variant of M. bovis; and M. canettii, a smooth variant that is very rarely encountered but causes human disease. Prior to the introduction of pasteurization of milk, M. bovis was responsible for approximately 6% of total TB deaths in humans in Europe. These strains are derived from a common ancestor and among these some are exclusively human (Mtb, M. africanum, M. canettii) or rodent pathogens (M. microti), whereas others have a wide host spectrum (M. bovis) (Brosch et al., 2002). Because of the unusually high degree of conservation in their housekeeping genes, it has been suggested that the members of the Mtb complex underwent an evolutionary bottleneck at the time of speciation, estimated to have occurred roughly 15,000-20,000 years ago (Sreevatsan et al., 1997). Also, it has been speculated that Mtb, has evolved from M. bovis, the agent of bovine tuberculosis, by specific adaptation of an animal pathogen to the human host. M. bovis is the etiologic agent of TB in cows and rarely in humans. Both cows and humans can serve as reservoirs. Humans can also be infected by the consumption of unpasteurized milk. This route of transmission can lead to the development of extrapulmonary TB, exemplified in history by bone infections that led to hunched backs (Stead et al., 1995). The whole genome sequence of Mtb uncovered several variable genomic regions in the members of the Mtb complex (Cole et al., 1998). Among the fast growing mycobacterium species, M. indicus pranii is of great interest and is a non-pathogenic, soil derived, rapidly growing, atypical mycobacterial organism. Interestingly, M. indicus pranii shares a number of common B and T cell determinants with M. leprae and Mtb (Zaheer et al., 1993). Other human pathogens belonging to the Mycobacterium genus include M. avium which causes a TB-like disease especially prevalent in AIDS patients, and M. leprae, the causative agent of leprosy. 1.3. Clinical manifestations Most TB infections are initiated through the respiratory route at least in the developed world. Generally TB pathogenesis follows a general pattern as reported by Wallgren, who divided the progression of the disease into four stages (Wallgren, 1948; Smith, 2003):- First stage, lasting about 3-8 weeks after Mtb inhalation in alveoli. Then the bacteria are disseminated by the lymphatic circulation to regional lymph nodes in the lung, forming the so-called primary or Ghon complex. At this time, conversion to tuberculin reactivity occurs. Second stage, lasting about 3 months, bacteria disseminate through blood circulation to many organs including other parts of the lung. During this period patients may have tuberculosis meningitis or miliary tuberculosis. Third stage, lasting 3-7 months, inflammation of the pleural surfaces with severe chest pain, but this stage may last up to 2 years. This condition is caused by either haematogenous dissemination or the release of bacteria into the pleural space from sub pleural concentrations of bacteria in the lung. The free bacteria or their components are thought to interact with sensitized CD4 T-lymphocytes that are attracted and then proliferate and release inflammatory cytokines. Fourth stage, up to 3 years, in this stage slowly developing extra pulmonary lesions, such as in bones and joints, frequent chronic back pain. The pathogenesis of tuberculosis is due to a complex interaction between the causative organism, Mtb and the human host immune response. Several investigators established the nature and importance of host immune responses and specifically cell-mediated host immune responses, in the pathogenesis of tuberculosis and the immune responses could be elicited by serum-free cells (Landsteiner and Chase, 1942; Chase, 1945). The lymphocytes are the primary component of the cellular immune response and macrophages as effecter cells in host defence against Mtb (Schluger, 2005). Overall, our understanding of the pathogenesis of tuberculosis supports a framework of immune responses which include the initial response to inhaled organisms, granuloma formation and establishment of a state of latent infection and reactivation of latent infection. While a large group of epidemiological studies revealed that the 90% of persons with latent tuberculosis infection will never develop clinical illness.