|Year : 2002 | Volume
| Issue : 2 | Page : 61--68
Microbial pathogenesis: An insight into Mycobacterium tuberculosis
S Manjula, V Sritharan
Dept. of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderbad-500 046, India
Dept. of Animal Sciences, School of Life Sciences, University of Hyderabad, Hyderbad-500 046
Tuberculosis, as yet, is far from being controlled. Several reasons can be attributed to this, a major contributing factor being the development of resistance to the currently available drugs due to the successful adaptation of the pathogen. Most of the inferences about the pathogen are based on the observation of mycobacteria grown in synthetic media in vitro and of the mycobacteria maintained in macrophages simulating the in vivo conditions. Molecular studies in mycobacteria had been slow to come due to the difficulty in the generation of mutants. However, new technologies that have now been developed for studying in vivo expressed molecules in other bacterial systems are being successfully applied to mycobacteria, especially the pathogenic M. tuberculosis. Additionally, an equally important factor in the study of the disease is the genetic predisposition of population to the infection. New findings link the Nramp1 and Toll receptor polymorphisms to susceptibility to infectious diseases.
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Manjula S, Sritharan V. Microbial pathogenesis: An insight into Mycobacterium tuberculosis.Indian J Med Microbiol 2002;20:61-68
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Manjula S, Sritharan V. Microbial pathogenesis: An insight into Mycobacterium tuberculosis. Indian J Med Microbiol [serial online] 2002 [cited 2020 Jul 14 ];20:61-68
Available from: http://www.ijmm.org/text.asp?2002/20/2/61/8348
Tuberculosis (TB) must have been a scourge since mankind existed, as reports of this dreadful disease are recorded centuries back. Today, tuberculosis is a major global public-health problem. In 1997, it was estimated that worldwide there were 7.96 million new cases of tuberculosis (136/100,000 persons), in addition to the 8.22 million existing cases, and that 1.87 million people died of tuberculosis. In India, there is an estimated 3.5 million people who are sputum positive with about 2.2 million new cases every year and 0.5 million people dying from TB every year.
[Figure:1], [Figure:2], [Figure:3]
Mycobacterium tuberculosis, the causative organism has been one of the most successful pathogens in humans. Despite extensive research and the discovery of several drugs, this pathogen during the chronic state is found to exist in diverse metabolic states that are not targeted by the conventional antimycobacterial agents. The current antimycobacterial agents need to be used for a considerably long period to effectively ensure the killing of all the persistent bacteria. Further, the currently available drugs target only a small number of bacterial processes, such as cell wall formation and chromosomal replication with the additional and imminent threat of emergent drug resistant strains. In chronic TB infections, the bacilli adapted to the environment of the macrophages colonize in them and undergo rapid replication, in spite of a competent T cell response. Though a vast wealth of knowledge on the structure, biology and the complete genome sequence of this pathogen are available, man is still unable to control it. WHO had declared tuberculosis a global emergency in 1993. Efforts are being made worldwide to understand the mechanisms of survival of this pathogen. Overall, it is a two-pronged attempt: one aiming to study the adaptation of the pathogen under in vivo conditions and the second, an equally important aspect, is the susceptibility/resistance of the host to infection.
What is microbial pathogenicity?
A microbe becomes a pathogen when its biochemical pathways, either individually or acting in concert with one another, causes disease in a host. In microbial pathogenicity, two terms encountered are infection and disease. Infection refers to the multiplication (or colonization) or the persistence of the organism within the host environment, while disease refers to the significant damage caused by the organism in the host due to the infection.
Microbial pathogenicity is usually not attributed to a single contributing factor. It is multifactorial and is the culmination of several adaptations by the organism in order to survive within the hostile environment of the host. In order to devise methods for combating an invading pathogen, it is necessary to identify its determinant(s) of virulence. A study of these determinants with insight into their structure, biological action and their expression in vivo will aid in developing new targets for the attack of these pathogens. In order to identify virulence determinants, the following sequence of events could be followed
1. Compare the products of strains of high and low virulence.
2. Identify products related to a specific biological function.
3. Relate the biological function/effect to infection.
4. Determine the chemistry of the factor.
Today, it is becoming more and more clear that the products identified under the artificially created in vitro conditions do not simulate the natural in vivo conditions and thus may not give the true direction to the problem. It would therefore not suffice to extrapolate the in vitro observations to in vivo conditions.
New technologies for identifying virulence determinants
Microbial pathogenesis is being better understood with the deciphering of several genome sequences and the development of several new techniques [Table:1] for identifying the molecules in play under in vivo conditions. The principles of some of these techniques are explained in Box 1, 2 and 3. They have been used to identify a number of virulence determinants in several bacteria.
Mycobacterium tuberculosis: how much do we know about the pathogen?
An important aspect of TB infection is to understand the ability of the tubercle bacillus to invade and successfully multiply within the macrophages of the host. The macrophage, though it provides the nutrients for the intracellular mycobacteria to grow, also exerts considerable environmental stress. The intracellular M. tuberculosis thus has adapted to the hostile environment and devised ways of withstanding or evading the stress imposed by the host macrophages. What is interesting and troubling at the same time is the ability of the M. tuberculosis organisms to lie dormant within this environment for prolonged periods, only to be awakened subsequently to cause reactivation of the earlier infection in the host. Little is known about the mechanisms of reactivation and how these bacteria survive for such long periods.
Results had been slow to come in mycobacterial research due to the long generation time of the pathogens (approx. 24 hours for M. tuberculosis) and due to the difficulty in the production of mutants. Earlier, not much success was achieved in the generation of mutants in mycobacteria both by allelic exchange or transposon mutagenesis. However, with the recent development of techniques for the genetic manipulation of M. tuberculosis, progress is being made in adapting these techniques to identify virulence determinants in this pathogen.
Techniques discussed above have been adapted to the study of pathogenesis of TB infection. [Table:2] lists some of the amplification methods in the identification of genes expressed by M. tuberculosis under in vivo conditions. Of the several thousand M. tuberculosis mutants generated by STM, seven mutants were found to be attenuated due to disruption of a locus involved in the synthesis of phthiocerol dimycocerosate, a constituent lipid found in significant amount in the cell wall. This effect would result in the alteration of cell wall fluidity and thus the consequence of such a mutation would be indirect, probably due to alteration in the secretion of a protein, ion or other molecules. Barry, in analysing the role of the cell wall components as virulence factors in M. tuberculosis explains that though the bacterial cell surface is going to contribute to the outcome of infection by the interaction with several host molecules, the effects of mutation on the cell wall components should be viewed as indirect effects.
Iron acquisition in mycobacteria
Iron is an obligate cofactor for at least forty different enzymes encoded by the M. tuberculosis genome. Iron is required by all aerobic micro-organisms, except lactobacilli. Iron acquisition is mediated by iron-chelators called siderophores and their receptors on the cell surface, called iron regulated proteins/envelope proteins (IRPs/IREPs). The synthesis of these components of the iron acquisition machinery is regulated at the molecular level by iron concentration. In addition, iron controlled the expression of certain virulence genes as seen in the expression of aerobactin in E.coli, hemolysin in Vibrio cholerae, anguibactin in Vibrio anguillarum, diphtheria toxin in Corynebacterium diphtheriae, exotoxin A in Pseudomonas aeruginosa and others. Several reviews have been written on iron and micro-organisms, including mycobacteria.,,,
Iron regulation of gene expression has been extensively studied in E. coli due to the ease of generating mutants. In E. coli and other Gram negative bacteria the repressor protein Fur is the best studied iron-responsive transcriptional regulator. Encoded by the Fur gene, this protein binds its co-repressor, ferric iron to form a complex that binds to operator sequences, thereby repressing transcription. Upon iron starvation, the Fur protein is no longer able to bind to these sequences and the corresponding genes become de-repressed. The corresponding repressor in Gram positive bacteria is the DtxR protein, first identified in C. diphtheriae. M. tuberculosis contains as many as four such iron-dependant regulators, of which the IdeR (Rv2711) is the only protein that has been identified, expressed and shown to influence the expression of the synthesis of the siderophores. This repressor, which shows sequence similarity with the DtxR protein differs from the Fur protein and binds different operator sequences. In mycobacteria, the IdeR is a major regulator of the iron acquisition apparatus. But, the regulation is not complete as it does not account for all the adaptive responses to iron availability. Additional mechanisms are thus a possibility. It is interesting to note that the M. tuberculosis genome contains two genes furA and furB that encode proteins similar to the E. coli Fur and a third gene SirR, whose protein product is similar to the iron-dependant regulator SirR from Staphylococcus epidermidis.. Whether these proteins are expressed in vivo and whether they influence the iron assimilation is not known. Immediately downstream of the furA is the katG gene, encoding the catalase-peroxidase enzyme. It is not known if FurA plays a role in the regulation of katG expression; however, iron and oxidative stress are found to be linked in M. smegmatis.
The role of siderophores in the virulence in mycobacteria has been doubtful due to the failure to identify them from pathogenic mycobacteria recovered from in vivo conditions. Now, the generation of mutants has identified mycobactin as a virulence determinant in M. tuberculosis.,, These mutants, defective in the biosynthesis of mycobactin T, failed to exhibit normal growth both in vitro and inside the macrophages.
Oxidative stress response and iron
Iron levels and oxidative stress are closely linked in aerobic organisms. In actively respiring cells, the super oxides and hydrogen peroxide must be inactivated before they generate the more harmful ROS (reactive oxygen species). These ROS would cause extensive damage to DNA, proteins and lipids. In enteric bacteria, the transcriptional regulators OxyR, SoxR/SoxS and RpoS, which activate the expression of anti-oxidant enzymes are well studied. Mycobacteria produce catalase-peroxidase (katG), superoxide dismutase (sodA) and an alkyl hydroperoxide reductase (Ahr) but relatively little is known about the regulation of these genes. In M. tuberculosis, there is an oxyR homologue linked to the ahpCF operon (ahpCF is the subunit of ahr) but the OxyR product is non functional, as it has many deletions. In M. smegmatis an increase in sensitivity to hydrogen peroxide in IdeR mutants was observed due to the decreased activity of catalase-peroxidase (KatG) and superoxide dismutase (SodA) activity, implying that IdeR is most likely to play a central role in oxidative stress., Manabe et al  by the introduction of a dominant Dtx (E175K) iron-dependant repressor into M.tuberculosis proved that the IdeR repressor controlled the events which influenced the virulence in a murine model of infection.
Another component of the iron acquisition machinery is the IRPs/IREPs. In enteric bacteria, these surface receptors and their genes are well characterised.20 Mycobacteria also express proteins under iron starvation conditions. The 29 kDa protein functioning as the receptor for ferri-exochelin in M. smegmatis is found in several mycobacterial species. In M. neoaurum, the major 21 kDa protein, exochelin MN and mycobactin were co-ordinately regulated by iron levels. These IREPs are elaborated not only by the in vitro grown mycobacteria but are also expressed from in vivo derived bacteria, as seen in M. avium and M. leprae recovered from infected tissues. Whether these proteins are the same as that expressed under in vitro conditions and what role they play is unknown. In M. tuberculosis, several proteins were identified in different iron concentrations of which some like the 10 kDa protein IrpA (irpA) and the CtpC (ctpC) are reported as related to iron transport based on their sequence comparison with other known sequences. It would be worth examining if the repressor IdeR controlled the expression of any of the IREPs and if uptake of iron from the ferri-siderophore can be blocked by the generation of mutants.
Host susceptibility to infection
Earlier, most of the attempts to control a disease focussed on ways to combat the causative organism. But, it is now becoming clear that the development of a disease depends not merely on the efficiency of a pathogen to establish an infection but equally on the host's susceptibility to the specific pathogen. The role of certain unique host proteins and sequences at the molecular level in influencing the host's susceptibility to infection is unfolding from observations from several studies.
NRAMP1 and susceptibility to intracellular pathogens: what is its role in tuberculosis?
In humans, it is now evident that susceptibility to infectious diseases is under genetic control. Animal models provide an ideal tool to study the genetic component of susceptibility and to identify candidate genes that can then be tested for association or linkage studies in human populations from endemic areas of disease. In mice, innate resistance/susceptibility to infection with several unrelated pathogens like Leishmania, Mycobacteria and Salmonella was shown to be controlled by the locus Ity/Lsh/Bcg, which, later by positional cloning was designated as NRAMP1. The NRAMP1 gene codes for the 'natural resistance associated macrophage protein', belonging to a family of divalent metal transporters that play important roles in the homeostasis of iron and other metals. NRAMP1, though expressed in spleen, liver and lungs is highly expressed in circulating monocytes / macrophages and polymorphonuclear leucocytes, being distributed throughout the plasma membrane of these cells., This protein is found not only in the mammalian system, but homologues are found in insects, plants, bacteria.
The macrophage NRAMP1, of molecular mass 60 kDa, is a highly hydrophobic protein and shows structural similarities to other ion channels and transporters. Though the exact mechanism of action of this protein is not known, it is implicated in the control of growth of intracellular pathogens. Gruenheid et al established that the Nramp1 protein which is recruited to the membrane of the endocytic compartments in resting macrophages, is routed to the membranes of phagosomes upon phagocytosis, thereby controlling the replication of intracellular pathogens by altering the intra-vacuolar environment of the microbe-containing phagosome. Hackam et al, in their studies with Mycobacterium bovis demonstrated that the pH of phagosomes containing live bacilli was significantly more acidic in Nramp1- expressing macrophages than in mutant cells. These authors suggest that Nramp1 affects intracellular mycobacterial replication by modulating the phagosomal pH implicating a central role for Nramp1 in this process. Others, propose that Nramp1 affects the intra phagosomal microbial replication by modulating iron levels and other divalent cations in this organelle, in the milieu of which both mammalian and bacterial transporters may compete for the same substrate.
In the majority of humans, an effective immune response develops after infection with M. tuberculosis, thereby preventing the multiplication of the pathogen. In the remaining percentage of individuals, there appears to be a complex interaction of genetic and environmental factors that probably influences the outcome of an infection. The correlation between Nramp1 gene variations and disease susceptibility points to this gene as a strong candidate for human tuberculosis. Susceptibility to infection in inbred strains of mice correlated with NRAMP1 mutations with single glycine-to-aspartic acid substitution at position 169 of the predicted trans membrane domain 4 of the Nramp protein. A strong association between NRAMP1 polymorphisms and susceptibility to tuberculosis was shown by Bellamy et al in West Africans, while in South Vietnam correlation was seen between NRAMP1 mutations and susceptibility to leprosy. Further, this genetic predisposition was evident as a dominant trait among the members of a single large aboriginal family in Canada, an observation that came into light during an epidemic of tuberculosis that occurred in a community of Aboriginal Canadians during the period 1987-89.
As mentioned earlier, Nramp1 are found in several systems including bacteria. The Nramp1 protein homologue in mycobacteria (referred to as MRamp) is found in M.tuberculosis, M. leprae and BCG. The Mramp protein, functioning as a transporter of Zn2+ and Fe2+ probably helps in the detoxification of the superoxides and hydrogen peroxide within the macrophage. Further, it is thought to add to those mechanisms that inhibit the acidification of the phagosomes thereby permitting the multiplication of intracellular mycobacteria. A competition thus ensues between the host NRAmp1 and the mycobacterial Mramp in controlling the pH of the phagosome, the outcome of which probably depends on the efficiency of the two transporters.
Toll-like receptors and CpGs
Host defense consists of two types of immune responses, innate and adaptive immunity. B and T lymphocytes invoke the adaptive immune response, while the innate immunity is mainly mediated by the macrophages and dendritic cells. Mycobacterium bovis BCG, an attenuated form of M. bovis is a safe vaccine administered to children to protect them against tuberculosis. Today, BCG is a widely used effective treatment of bladder cancer due to its immunogenic activity. An interesting observation about the active component of BCG that was responsible for enhancing the innate immunity of the host was in fact, the DNA itself. It was a CpG motif, unique to bacteria (but not found in vertebrate system) that functioned as an immuno-stimulatory sequence. In the efforts to understand the host immune cells like the macrophages/monocytes and the polymorphonuclear cells, receptors called as pattern recognition receptors (PRR) were identified. Among these receptors, there were motifs called pathogen associated molecular patterns (PAMPs) on bacteria and viruses. Among this family of PRR of importance is the Toll-like receptors (TLRs) of which as many as ten have been identified. It is the TLR system that detects the CpG dinucleotide sequences. TLR signaling pathways, first identified in Drosophila system has been shown to play significant roles in host defense.
Means et al studied how human Toll-like receptors mediate cellular activation by M. tuberculosis. In Gram negative bacteria LPS stimulated TLR-4 activation. But, mycobacteria are devoid of LPS and shown to act via TLR-2 and TLR-4. Pathogenic mycoabcteria including M. tuberculosis produce mannose capped LAM in contrast to the arabino furanosyl-capped LAM of fast growing non-pathogenic mycobacteria. These authors suggest that part of the survival strategy of M. tuberculosis may depend on these bacilli entering the host macrophage without causing a strong antimicrobial response. It is thought that CD14 ligands present in the cell walls of Gram positive and Gram negative bacteria, which activates TLR signaling resulting in significant antimicrobial responses, may in fact be seen with M. tuberculosis.
It is obvious that man, despite developing many new technologies to understand the host-pathogen inter-relationship is still far from conquering the microbes. One of the most successful pathogens has been M. tuberculosis, that has overcome not only the natural barriers within the human host but is continually adapting to the challenges put forth by man in the battle against it by developing resistance to the multitude of drugs currently available. There is thus a great need to understand how exactly the pathogen survives under the in vivo conditions and identify pathways unique to the intracellular environment that could be utilized for the development of new and better drugs. Yet, it is clear that the disease progression is not just related to exposure to the pathogen. The great variability in the susceptibility among persons emphasizes the need for a better understanding of the genetic susceptibility of the host population to this dreaded disease. The combination of the pathogen's adaptation to new challenges and the susceptibility of specific population makes this a formidable pathogen to fight. However, the fight has to continue with man attempting to control this pathogen, if not to eradicate it altogether.
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