DOSR a response regulator essential for hypoxic dormancy in mycobacterium bovis BCG 2

Chapter Introduction Tuberculosis (TB), a disease caused by the bacterium Mycobacterium tuberculosis (MTB) had been an agent of death and suffering for mankind since the days of the pharaohs, with a mortality rate of 40 to 60% if left untreated (Iseman and Madsen 1989) A new hope to eradicate the disease emerged when a live attenuated strain of Mycobacterium bovis was first used to vaccinate a child whose mother had died of TB (Calmette 1927) This vaccine called BCG or bacille Calmette-Guérin, named after its creators, is now the world’s most widely administered vaccine (WHO 1991) A further reprieve from the killer disease was granted by the introduction of antibiotics in the 1950’s, which significantly reduced the mortality rate Armed with a new vaccine and an arsenal of seemly invincible drugs, an atmosphere of complacency prevailed as shown by the U.S surgeon general statement “to close the book on infectious disease” cited in (Bloom and Murray 1992) This was a grave mistake Now TB is the leading cause of death among infectious diseases with global fatality rate of 23% and an estimated billion people (one-third of the world’s population) infected with MTB (Dye, Scheele et al 1999) This problem is further compounded by the inconsistent protective efficacy of the BCG vaccine in adults (McKinney, Jacobs et al 1998) Obviously, a more effective vaccine is required Furthermore, the pressing problems of multiple drug resistant TB strains (MDR TB) that have identical mortality rates to untreated TB has to be addressed (Iseman and Madsen 1989) Currently, a patient has to be treated for six months with a cocktail of drugs (isoniazid, rifampicin, ethambutol and pyrazinamide) before greater than 90% cure rates can be achieved (Bloom and Murray 1992) This long treatment time inevitably resulted in poor patient compliance One consequence is the emergence of MDR TB strains As such, shortening treatment time is of prime importance in the global control of TB [Global TB Alliance, 2001 #200] The key question is: ‘Why does it take so long?’ 1.1 Bacilli Persist in vivo MTB is predominantly transmitted through the inhalation of bacilli-containing droplets Once inhaled, the bacilli are internalized by macrophages and an infection of the lungs is established In an immunocompetent host, the infection is usually contained by the immune system and the bacilli are walled-off in granulomas The patient does not show any clinical symptoms and the disease enters a latent phase (Parrish, Dick et al 1998) Due to the denseness of the granulomas, the bacilli may be exposed to a hypoxic environment MTB is an obligate aerobe and as such requires oxygen for growth This suggests that the bacilli may enter a quiescent state within the granulomas Work by Wayne et al demonstrated that decade old granulomas contained acid-fast bacilli and up to 40% of them contains viable bacilli as shown by plating and viable count determination (Hurford and Valentine 1957),(Wayne and Salkin 1956) Since mRNA is rapidly degraded in dead cells due to its short half-life, it is a good indicator for viability and the presence of metabolic activity Indeed, expression of several MTB genes was detected in lung granulomas via RNA-RNA in situ hybridization (Fenhalls, Stevens et al 2002) Taken together, these findings suggest that bacilli are able to persist in the host for long periods of time in a viable but non-growing state There is a to 23% chance of reactivation of the disease in a lifetime but the risk increases to to 10% per year if the host becomes immunocompromised, for example upon co-infection with HIV (Parrish, Dick et al 1998) If bacilli residing in the granulomas are indeed non-growing, it is conceivable that conventional TB drugs, which target growth related functions such as cell wall synthesis (Schroeder, de Souza et al 2002), are not effective against them In other words, they might show phenotypic drug resistance as opposed to a genetic mutation or acquisition of a drug resistant gene (Wayne 1994) Thus, the development of phenotypic drug resistance could be a major contributing factor to prolonged chemotherapy Therefore it is conceivable that by targeting these bacilli, or preventing the occurrence of persistence, the lengthy chemotherapy regimes can be shorten (Dick 2001; Wayne and Sohaskey 2001) This requires detailed understanding of the molecular mechanisms of persistence for the identification of drug targets Hence, the next question is: “What is the trigger, and does hypoxia play a role in phenotypic drug resistance?’ 1.2 Hypoxia could be a Factor in Persistence As stated above, one working model suggests that non-growing bacilli in granulomas reside in hypoxic environments Here, they wait for the opportunity to reactivate when oxygen becomes available, perhaps during the liquification of the granulomas Interestingly, oxygen levels can be low in the lungs, even outside of granulomas, as superinfection with obligate anaerobes can occur in the tubercular cavities (Garay 1996) This indicates that anoxic environments exists in the latter In a review (Segal 1984), Segal had shown that MTB directly harvested from host tissues, shifted from oxygen dependent pathways to anaerobic metabolic pathways Indeed, the complete genome sequence of MTB H37Rv strain revealed several genes encoding components of anaerobic respiration They include narGHIJ and nirBD, for nitrate reduction and nitrite reduction respectively, frdABCD for furamate reduction and narX, encoding a ‘fused’ nitrate reductase (Cole, Brosch et al 1998) NarX was shown recently to be expressed by bacilli residing in human granulomas via RNA-RNA in situ hybridization (Fenhalls, Stevens et al 2002) and under hypoxic conditions in vitro (Hutter and Dick 1999; Sherman, Voskuil et al 2001) A study of the narGHJI cluster demonstrated that nitrate respiration is an important factor contributing to the virulence of BCG in immunodeficient SCID mice (Weber, Fritz et al 2000) Here, a mutant lacking the nitrate reductase subunit encoded by narG was generated Histological analysis showed that mice infected with the narG mutant had small granulomas with reduced numbers of bacilli when compared to those infected with the wild type strain In addition, infection of SCID mice with the latter resulted in death within 85 days while those infected with the narG mutant still survived after more that 200 days As such, nitrate respiration may be of importance to the virulence of MTB in vivo Further evidence that the bacilli in vivo encounter hypoxic condition came from work on the drug metronidazole, which is known to be selectively active against anaerobes (Edwards 1979) and MTB grown under hypoxic conditions (Wayne and Sramek 1994; Wayne and Hayes 1996; Lim, Eleuterio et al 1999) Brooks et al infected mice with MTB via aerosol administration and fed them metronidazole at 15mg/kg/day for 30 days (Brooks, Furney et al 1999) When treatment was started during the established disease state (100 days post infection), a moderate but significant decrease in bacterial counts was observed This is expected as metronidazole is only active against the bacilli under strictly anoxic conditions (Wayne 1994; Wayne and Hayes 1996) It is unlikely that anoxic environments are predominant in vivo, but rather the bacilli encounter varying degree of hypoxia In addition, mice are not good models of human tuberculosis due to the difference in the pathology of the disease, for instance in the nature of the granulomatous response (McMurray, Collins et al 1996; Barry 2001) Therefore, this small bactericidal effect of metronidazole observed by Brooks and co-workers is intriguing and argues that MTB encounters severe hypoxia in the mouse model of the disease Thus, these data indicate that a small proportion of the bacilli resides in anoxic pockets in vivo and further supports the working model that hypoxia is encountered in vivo In conclusion, these in vivo data point to a probable relationship between hypoxic environments and persistence However, it is difficult to identify and thus experiment with these persistent bacilli in vivo due to a lack of molecular markers Even if this could be achieved, the small amount of materials available makes this exercise rather difficult Hence, a culture system that can generate a large amount of these persistent bacilli in vitro has to be developed before the molecular dissection of hypoxia-induced persistence can begin 1.3 Discovery of the Dormancy Response: Wayne ’76 Culture Model Early observations by Fisher and Kirchheimer (Fisher and Kirchheimer 1952) found that standing cultures of tubercle bacilli grown in Dubos Tween Albumin and only agitated on occasion, displayed a net rate of arithmetic multiplication Volk and Myrvik (Volk and Myrvik 1953) later showed that this was a direct consequence of oxygen limitation as cultures grown with constant aeration exhibit logarithmic growth It is apparent that bacilli in standing cultures would slowly settle to the bottom of the culture vessel, forming a sediment (see figure 1.3.1) As the latter thickens, it is unavoidable that the bacilli in the lower levels of the sediment encounter selfgenerated hypoxic conditions Through labelling of cells with 14 C- uridine, it was revealed that bacilli in the upper liquid phase displayed a logarithmic decrease in radioactivity counts, signifying that the bacilli there are growing logarithmically (Wayne 1976) However, replication proceeded at a much slower generation time of 33 hours This slowing down in replication is attributed to the bacilli settling through a spatial oxygen gradient where oxygen levels decrease to very low levels as they approach the sediment In contrast, the sediment showed a short rise in radioactive counts (probably due to settling of labelled cells) followed by a stable plateau of counts As such these cells are not replicating or so at a very slow rate (Wayne 1976) Thus, the net arithmetic growth observed is a product of the combinatorial effect of slow logarithmic growth of settling bacilli in the upper phase and nonreplication in the sediment (Wayne 1976) In addition, when the sediment bacilli are reconstituted in fresh oxygen rich media, re-growth was observed, thereby demonstrating that the bacilli in sediment are viable (Wayne 1977) Since sudden exposure to hypoxia is lethal (Wayne 1976), it appears that the bacilli adapt to hypoxia during their journey across the spatial oxygen gradient In addition, when the sediment bacilli are exposed to isonazid and rifampin, a very limited effect was observed as compared to 90% death in logarithmic cultures (Wayne and Sramek 1994) However, they developed sensitivity to metronidazole (Wayne 1976) Thus, using the standing culture system one could generate hypoxia induced non-replicating, drug resistant bacilli that can grow when oxygen is provided The physiological state of these in vitro generated bacilli could be similar to those found in hypoxic environments in vivo Further examination of the bacilli in the sediment yielded several interesting findings A unique antigen (URB) was discovered when protein extracts from hypoxic nonreplicating bacilli was subtracted with anti-sera from aerobic logarithmically cells (Wayne and Sramek 1979) It was later identified as the 16kDa α-crystallin homologue (Yuan, Crane et al 1998; Desjardin, Hayes et al 2001) In addition, Wayne not only detected the resumption of growth when the sediment bacilli were diluted in fresh media, he observed that the growth was synchronous (Wayne 1977) Detailed analysis of the phenomenon by monitoring incorporation of tritium labeled uridine revealed that the first eight hours post reconstitution showed a marked increase in RNA synthesis before the first cell division However, DNA synthesis was seen only after the latter was completed This suggests that a set of molecules is required to be synthesized before cell division can proceed and that the bacilli probably arrested growth as diploids (Wayne 1977) It is likely that specific genes are expressed during the adaptation phase such that an orderly shut down of the cell machinery could be completed before growth arrest and that the bacilli have everything in place, for instance, stress protection and enzymes to satisfy metabolic needs during prolong survival under hypoxic conditions Indeed, a five-fold increase in the specific activity of isocitrate lysase, the first enzyme in the glyoxylate by-pass that converts isocitrate to glyoxylate and succinate, was detected in non-replicating synchronous bacilli (Wayne and Lin 1982) This enzyme was later shown to be important for persistence and virulence in vivo as mice infected by bacilli lacking this gene displayed reduced lung pathology and survived the infection (McKinney, Honer zu Bentrup et al 2000) The detection of a novel enzyme, glycine dehyhdrogenase in MTB, which catalyses the reductive amination of glyoxylate to glycine using NADH, prompted Wayne and colleagues to investigate its occurrence in sediment cells This is because the pathway may be employed by the bacilli to maintain the NAD pool such that sufficient ATP is generated for the completion of a final round of DNA replication before total shutdown of the cellular machinery (Wayne and Lin 1982) The fact that reconstituted bacilli only synthesizes DNA after the first cell division supports this concept Indeed, a 10-fold induction in specific activity of glycine dehydrogenase was observed (Wayne and Lin 1982) Further analysis revealed that a 60:1 ratio of NAD to NADH is sufficient to decrease the rate of NADH reduction by 50% as compared to 400:1 ratio of glycine to glyoxylate This suggests that the aim of the reaction is to produce NAD rather that glycine Taken together, the induction of these two enzymes gave a first glimpse of alternative metabolic pathways that may be utilized by MTB to maintain a sufficient level of metabolic activity required for long term survival In conclusion, the transition from an actively replicating cell to non-replication can be viewed as an entry to a new developmental state It involves the induction of alternative metabolic pathways, antigenic differences and the population of nongrowing bacilli is arrested at an identical stage of the cell cycle (i.e the culture is synchronous) In this defined physiological state, the bacilli maintain viability for extended periods of time and can be reactivated As such, these bacilli can be described as dormant where dormancy is defined as: ‘a reversible state of low metabolic activity in which cells can persists for extended periods without division’ (Kaprelyants, Gottschal et al 1993) Figure 1.3.1 The Wayne’76 standing culture model (Wayne 1976) The bacilli settle through a spatial oxygen gradient allowing adaptation to hypoxic conditions The bacilli in the sediment are non-replicating, drug resistant and synchronized 1.4 Temporal Analysis of Dormancy: Wayne’96 Dormancy Culture Model The work of Wayne and colleagues on standing cultures described above provided valuable insight to dormancy However, the Achilles' heel of the system is its heterogeneous nature due to the presence of two (or more) distinct populations of cells that exists in different physiological states within one culture tube Only the sediment consists of a homogenous population of cells (Wayne and Hayes 1996) Therefore, the standing culture system does not allow for the temporal dissection of the dormancy response Hence, in 1996 Wayne et al developed the Wayne in vitro dormancy culture system (Wayne and Hayes 1996), which brought the study of dormancy to a new dimension – Time The strength of the Wayne’96 dormancy culture system is that the culture at any time point is homogenous (Wayne and Hayes 1996) The bacilli are grown in sealed tubes with stirring which cause the creation of a temporal self-generated oxygen gradient that allows for adaptation to hypoxia This results in the shift down to a nonreplicating dormant state (see figure 1.4.1) As such, parameters that will affect the oxygen diffusion kinetics and its availability have to be defined and kept constant for the culture system to be reproducible The bacilli are grown with Dubos Tweenalbumin broth in sealed tubes measuring 20 by 125mm that have a total capacity of 25.5ml The logarithmically growing bacilli are diluted to A600 0.005 and dispensed into the tubes as 17 ml aliquots Thus, 8.5 ml of headspace remains, which corresponds to a 0.5 ratio of air to liquid volume In addition, the bacilli are uniformly distributed by constant stirring with 7mm magnetic stirring bars at a speed of 120rpm At this rate, the oxygen diffusion rate is slow and comparable to those of standing cultures, as the liquid surface is undisturbed With these conditions, three distinct growth phases were observed (Wayne and Hayes 1996) Initially, aerobic logarithmic growth (generation time of 18hrs) was sustained for 58 hours, after which the culture enters a transition phase when a microaerobic threshold is reached (10% oxygen saturation) This growth phase is characterized by a slow increase in turbidity which occurs without replication This phenomenon can be attributed to a 33 % increased in 10 To provide a direct link between hypoxic dormancy and persistence, then transcript level of dosR could be monitored in the low dose mouse model If an induction of dosR mRNA is observed upon entering the persistence stage of infection, but not in bacilli exposed to NO donors, then this would indicate that hypoxic dormant bacilli exist in vivo, since DosR function and expression is dormancy specific In addition, a reduction of bacillary load of the dosR mutant in the stationary phase of infection would provide conclusive evidence that adaptation and survival to hypoxic dormancy is important for MTB persistence in vivo However, the findings of Parish et al showed that the dosR mutant was hyper virulent in mice Here, mice infected with the dosR mutant succumbed to the infection after 30 days as compared to 43 days for those infected with the wild type strain (Parish, Smith et al 2003) However, it must be noted that in this study immunodeficient SCID were used, where the growth of the bacilli continues uncheck until the animal dies This is unlike the TB mouse model used by Shi et al., where the bacillary counts stabilized after infection and a clinically quiescent phase (stationary phase) is maintained (Orme 1988; Shi, Jung et al 2003) As such, the role of dosR in persistence remains to be determined Excitingly, an analysis of the dosR mutant in guinea pigs was conducted recently (Tyagi 2002) The latter was deemed to be the better animal model in terms of its granulomous response, which resembles the pathophysiology of human pulmonary TB (Turner, Basaraba et al 2003) It was shown that the dosR mutant is indeed severely attenuated in guinea pigs, displaying a one log decreased in viability (Tyagi 2002) HspX, Rv2626c and Rv2623 expression is DosR dependent and had been shown to respond to NO stress It is conceivable that the mutant is unable to protect itself from 131 the onslaught of the host immune system This suggests a direct relevance of the hypoxic dormant bacilli generated by the Wayne’96 culture system to pathogenesis in vivo, which in turn indicates a crucial role for dosR and its regulon members in the progression of the disease It is apparent that identifying the members of the dosR regulon is of prime importance Significantly, the dosR regulon would be an obvious choice for target based drug development that could hold great potential in relieving the current obstacle for effective treatment of the disease, which is persistence This can be achieved through utilizing the dosR mutant strain A rapid and complete description of its regulon can be acquired via comparative microarray analysis By harvesting mRNA from wild type bacilli in growing, transition and hypoxic stationary phase, one could identify genes induced and down regulated during these growth phases These data can then be compared to those obtained from the dosR mutant strain using mRNA from identical growth phases Hence, genes that demonstrate altered expression patterns i.e loss of induction or repression would indicate that they are regulated by DosR In the same context, one could predict that the current chemotherapy drugs would be more effective against the dosR mutant This is because the latter is unable to enter into the non-replicative dormant form, thus would be at the mercy of conventional drugs that targets actively replicating bacilli (or would die due to hypoxia) One possible model for this study would be using a modified Cornell mouse model as described in Scanga et al (Scanga, Mohan et al 1999) This model allows the study of the ability of the bacilli to persist Here, the mice are inoculated intravenously with MTB and the resulting infection is treated with standard TB drugs for 12 weeks, by 132 then the mice were deemed to be sterilized After which, the infected mice are immunosuppressed with cortisone thereby triggering the reactivation of persistent bacilli Hence, it follows that if the dosR mutant strain displays a reduced or lack of reactivation, one can deduce that hypoxic dormant bacilli have a major impact on persistence in vivo In conclusion, the dosR mutant represents the first mutant that shows a strong survival phenotype both in vivo (Tyagi 2002) and in hypoxic dormant bacilli generated by the Wayne culture system (shown in this work) Three of its regulon members were identified and it was shown by work from other groups that they may play a role in the progression and maintenance of the infection These results indicate the relevance and importance of the use of the Wayne dormancy culture system as a tool for the mechanistic dissection of persistence As such, DosR will have an instrumental role in 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Proteins that are specifically induced in the transition phase and/or during the hypoxic stationary phase (but not in the aerobic exponential phase) may have a role in adaptation and survival to hypoxic. .. added before use LB broth 1.0% bacto tryptone, 2% yeast extract, 1.0% NaCl Autoclaved 22 LB Agar 1.5% bacto agar dissolved in LB Autoclaved 2. 2 Mycobacterial Culture 2. 2.1 The Wayne Dormancy Culture... Products) for minute 25 µl of proteinase K was added and the lysate was incubated for 30 minutes at 55 oC Subsequently, 20 0 µl of Buffer AL was added and the mixture was incubated at 70oC for 10 minutes