Experimental Models Used to Study Human Tuberculosis

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3 Experimental Models Used to Study Human Tuberculosis Ronan O’Toole

Contents

Abstract

I. Introduction II. Use of Surrogate Models to Study Tuberculosis III. In vitro Models of Mycobacteriology A. Fast-growing mycobacterial species B. The M. tuberculosis complex IV. In vivo Models of Tuberculosis A. Macrophages and cell cultures B. Rodent models C. Nonhuman primates V. The Study of Tuberculosis Pathogenesis in Human Patients VI. Conclusions and Future Prospects Acknowledgments References

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Mycobacterium tuberculosis causes more deaths in humans than any other bacterial pathogen. The most recent data from the World Health Organization reveal that over 9 million new cases of tuberculosis occur each year and that the incidence appears to be increasing with population growth. Despite the global burden of tuberculosis, we are still reliant on relatively dated measures to prevent, diagnose, and treat the disease. New, more effective tools are needed to diminish the incidence of tuberculosis. M. tuberculosis lacks a natural host beyond humans and, hence, surrogate models have been employed in the study of the

School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand Advances in Applied Microbiology, Volume 71 ISSN 0065-2164, DOI: 10.1016/S0065-2164(10)71003-0

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2010 Elsevier Inc. All rights reserved.

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pathogen. The discovery and development of new vaccines, diagnostics, or antitubercular drugs are dependent upon the validity of any experimental model used and its relevance to tuberculosis in humans. In this review, a range of experimental models, from in vitro studies with fast-growing low-pathogenic species of mycobacteria to the infection of nonhuman primates with virulent M. tuberculosis, will be discussed.

I. INTRODUCTION Tuberculosis (Tb) is the leading cause of death from a single infectious organism (National Institute of Allergy and Infectious Diseases, 2006). In 2007 alone, 9.27 million people developed tuberculosis and 1.78 million died of the disease (WHO, 2009). The causative agent, Mycobacterium tuberculosis, is transmitted via aerosols and enters the lung from where it can cause active clinical Tb or persist in latent form over the lifetime of the host (Parrish et al., 1998). Predisposing factors that influence the onset of clinical disease include HIV infection, diabetes, smoking, alcoholism, malnutrition, and overcrowded living conditions (Lonnroth et al., 2009). Clinical tuberculosis invariably commences with the pulmonary form of the disease; however, the pathogen can subsequently disseminate via the circulatory or lymphatic systems and multiply in extrapulmonary host sites such as the skin, lymph nodes, central nervous system, genitourinary tract, and skeleton (Kritski and de Melo, 2007). The earliest direct evidence of tuberculosis in humans originates from several thousand years ago. Traces of M. tuberculosis DNA, mycolic acid lipids, and paleopathological tubercular lesions have been identified in skeletal remains excavated from the submerged site of Atlit-Yam in the Eastern Mediterranean which dates from 9250 to 8160 BP (Hershkovitz et al., 2008). Indirect evidence is derived from the analysis of phylogenetic markers present in animal and human mycobacterial isolates. This has resulted in an estimation that the most common ancestor of the M. tuberculosis complex emerged approximately 40,000 years ago from its progenitor in East Africa, a time which is believed to coincide with the expansion of ‘‘modern’’ human populations from this area (Wirth et al., 2008). Despite our long association with M. tuberculosis, we have not yet been able to effectively control or eradicate this pathogen. Previous targets of halving the incidence of tuberculosis between 2006 and 2015 or of eliminating the disease by 2050 will need to be reexamined (Lonnroth and Raviglione, 2008). The World Health Organization has concluded that new preventative, diagnostic, and treatment measures are needed to bring tuberculosis under control (WHO, 2006). The capacity of

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researchers to deliver new tools to control tuberculosis is acutely dependent on the relevance of the experimental models they use to study the disease and its etiological agent.

II. USE OF SURROGATE MODELS TO STUDY TUBERCULOSIS M. tuberculosis has a very limited host range with no known natural hosts beyond humans (Brosch et al., 2002). Despite this, the pathogen does not require a zoonotic or environmental reservoir for persistence between episodes of clinical disease. The World Health Organization has estimated that one-third of the world’s population is latently infected with the pathogen (Dye et al., 1999). This high-carriage rate provides a reservoir for subsequent disease in susceptible hosts whereby the age-weighted lifetime risk of the development of clinical tuberculosis in a latently infected individual has been estimated to be 12% (Vynnycky and Fine, 2000). This risk is higher in individuals coinfected with HIV (Selwyn et al., 1992), and notably, 1.3 million cases of tuberculosis in 2007 occurred in individuals coinfected with HIV (WHO, 2009). Given the host specificity of M. tuberculosis and the ethical and regulatory restrictions limiting tuberculosis studies in humans, alternative models of the disease have to be devised. With any model, it is imperative that it has a sufficient degree of applicability to human tuberculosis. A variety of experimental models have been developed for the study of tuberculosis. Many of these models are in vitro based and involve the study of M. tuberculosis in isolation. Such models, although useful, are limited in their ability to account for host–pathogen interactions. Other models include the use of species of mycobacteria that are viewed as nonpathogenic, and hence, their application to pathogenic M. tuberculosis is sometimes questioned. Whichever model is chosen, it is unrealistic to expect that any one model can provide a basis for the study of all, or even most, aspects of human tuberculosis. In this review, a range of experimental models used to investigate tuberculosis will be examined.

III. IN VITRO MODELS OF MYCOBACTERIOLOGY A. Fast-growing mycobacterial species The value of using fast-growing species of mycobacteria in tuberculosis research was recognized by Selman Waksman in the 1940s. Waksman found that screening for antimycobacterial compounds using the pathogenic M. tuberculosis was a slow process (Sneader, 2005). He decided to screen against the fast-growing nonpathogenic species, M. phlei, and this

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ultimately led to the discovery of streptomycin, the first antibiotic effective in the treatment of tuberculosis. Since then, other fast-growing species of mycobacteria have been used in the study of tuberculosis including members of the taxonomic groups M. chelonae/abscessus, M. fortuitum, and M. smegmatis (Brown-Elliott and Wallace, 2002). The most widely used of these has been M. smegmatis, which has served as a model for the study of processes such as mycobacterial biofilm formation and sliding motility (Recht and Kolter, 2001; Recht et al., 2000), cell division and morphology (Anuchin et al., 2009; Casart et al., 2008; Jakimowicz et al., 2007), starvation and hypoxia response (Dick et al., 1998; Mayuri et al., 2002; O’Toole et al., 2003; Smeulders et al., 1999), and gene regulation. Many of the regulators of gene expression in M. tuberculosis have counterparts in M. smegmatis. A review of the partially completed M. smegmatis genome in 2002 identified orthologs for 6 of the 11 M. tuberculosis two-component systems (Tyagi and Sharma, 2002). Completion of the M. smegmatis genome sequence in 2004 identified a total of 26 sigma factors in M. smegmatis including orthologs of 10 of the 13 M. tuberculosis sigma factors (Waagmeester et al., 2005). A multigenome homology comparison with a protein similarity cutoff of 40% reveals that a high number, i.e. 3002, of M. tuberculosis H37Rv proteins, have orthologs encoded by the M. smegmatis mc2155 genome (comparison performed using the http://cmr.jcvi.org/cgi-bin/CMR/shared/MakeFrontPages. cgi?page¼circular_display online resource provided by the J. Craig Venter Institute). As can be seen, most of the studies involving M. smegmatis have focused on the genetics or physiology of mycobacteria. The use of M. smegmatis in virulence studies is less well established. M. smegmatis survives poorly in macrophages (Anes et al., 2006; Jordao et al., 2008) and exhibits no detectable pathogenicity in mice (Bange et al., 1999). Questions have therefore arisen as to the relevance of M. smegmatis to M. tuberculosis as fundamental differences exist in crucial areas such as growth rate and pathogenicity. A letter published in Trends in Microbiology in 2001 entitled ‘‘Mycobacterium smegmatis: an absurd model for tuberculosis?’’ (Reyrat and Kahn, 2001) provoked a discussion on the issue (Barry, 2001; Tyagi and Sharma, 2002) and consensus has not been reached to date as to the limitations or usefulness of M. smegmatis in the study of tuberculosis. M. smegmatis has long been regarded as an environmental saprophyte of no clinical significance. However, in 1986, pleura-pulmonary disease caused by M. smegmatis was reported (Vonmoos et al., 1986). Subsequently, other researchers reported the characterization of 22 clinical isolates of M. smegmatis from a range of disease cases in humans (Wallace et al., 1988). As reviewed in 2002 (Brown-Elliott and Wallace, 2002), community-acquired diseases caused by M. smegmatis included cellulitis, soft tissue necrosis, localized abscesses, osteomyelitis of a

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wound site, and lipoid pneumonia. Healthcare-associated diseases included catheter sepsis, infected pacemaker site, sternal wound infection following cardiac surgery, and infection following plastic surgery (Brown-Elliott and Wallace, 2002). It is becoming apparent that it may not be possible to strictly define M. smegmatis as a nonpathogenic species of mycobacteria. The association of M. smegmatis with human disease may in part support the use of this species as an in vitro model of tuberculosis. The identification of one of the antitubercular drugs currently in clinical trials, the diarylquinoline TMC207 (R207910), from high-throughput screening using M. smegmatis (Andries et al., 2005) has increased its level of acceptance as an antitubercular drug-screening model (Barry, 2009).

B. The M. tuberculosis complex The ease with which fast-growing species of mycobacteria can be cultivated and manipulated has led to their popularity for a variety of uses. However, any findings obtained from the use of M. smegmatis and other fast growers are generally expected to be validated in M. tuberculosis or another member of the M. tuberculosis complex (MTBC). Where biosafety level 3 containment facilities are not available, nonpathogenic alternatives include the tuberculosis vaccine strain M. bovis BCG and more recently, M. tuberculosis H37Ra, which is an avirulent derivative of strain H37Rv. Early in vitro models include a starvation model described by Nyka. He discovered that M. tuberculosis cells deprived of nutrients in vitro exhibited some of the same characteristics, such as altered morphology and loss of acid fastness have been observed for mycobacteria isolated from human tuberculosis lung tissue (Nyka, 1967, 1974). Some researchers proposed that M. tuberculosis is deprived of certain nutrients in the host lung, and this view has been supported by the use of auxotrophic mutants in mouse infections (Gordhan et al., 2002; Hampshire et al., 2004; Hondalus et al., 2000; Smeulders et al., 1999). Recently, global analyses of transcription and protein expression were performed on 7-dayold cultures of M. tuberculosis that were resuspended and maintained in phosphate-buffered saline for 6 weeks to identify genes that may play a role in mycobacterial survival in the host (Betts et al., 2002). Subsequent analysis of the genes expressed in the nutrient-starvation model revealed that many of these genes detected were also expressed in M. tuberculosis isolated from mouse macrophages, lung tissue, or hollow fibers applied subcutaneously (Murphy and Brown, 2007). A second in vitro model is the Wayne dormancy model. This model entails the gradual self-induced depletion of oxygen in a deep liquid medium combined with gentle stirring to maintain an even dispersion of the bacterial cells (Wayne and Hayes, 1996). In this model, M. tuberculosis shuts down its growth but maintains cell viability. It was proposed

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that the ability of M. tuberculosis to enter one of two nonreplicating persistent states, microaerophilic or anaerobic persistence, enables the pathogen to stay dormant in the host for long periods while retaining the capacity to reactivate and cause disease at a later stage in the host’s lifetime (Wayne and Hayes, 1996). This hypothesis has been supported by work that has found that tuberculosis lesions in humans exhibit hypoxia (Tsai et al., 2006; Wayne and Sohaskey, 2001). Wayne proposed that the in vitro hypoxia model could also be useful in screening drugs for the ability to kill M. tuberculosis in different stages of nonreplicating persistence (Wayne and Hayes, 1996). Indeed, several research groups have employed the Wayne dormancy model to screen in high-throughput chemical libraries for compounds that may be active against M. tuberculosis in vivo (Cho et al., 2007; Khan and Sarkar, 2008; Kharatmal et al., 2009) and to further characterize promising new antitubercular drugs such as PA-824 and TMC207 (Koul et al., 2008; Lenaerts et al., 2005) Most in vitro models have tended to focus on a single physicochemical condition that M. tuberculosis is believed to encounter during human infection. However, recently an in vitro dormancy model has been developed for M. tuberculosis that uses multiple stress conditions simultaneously, that is, low oxygen (5%), high CO2 (10%), low nutrient (10% Dubos medium), and acidic pH 5.0 (Deb et al., 2009). While an in vitro model may never be able to adequately substitute for an in vivo model, the incorporation of multiple stresses encountered by M. tuberculosis during infection should enhance the quality of data obtained.

IV. IN VIVO MODELS OF TUBERCULOSIS A. Macrophages and cell cultures As with in vitro models of tuberculosis, a variety of cell culture models have been used to study the disease. Based on the phagocytosis of M. tuberculosis at an early stage of infection of the host lung, many of the cell culture models have focused on the use of murine or human monocyte-derived macrophages or dendritic cells (de Chastellier, 2009). The use of cell cultures enables researchers to isolate the bacterial processes, which are central to uptake and survival and, in addition, the nonspecific immune responses that are effected by macrophages. For example, the use of macrophage cell cultures has established that live M. tuberculosis cells survive phagocytosis by specifically preventing phagosome maturation and, hence, fusion with lysosomes ( Jordao et al., 2008; Russell, 2001). It has also been found that the pathogen can persist and replicate within the phagosome (de Chastellier, 2009).

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The macrophage cell culture model has been used to perform singlegene and genomewide assays for M. tuberculosis genes which are expressed post phagocytosis (Dellagostin et al., 1995; Haydel and ClarkCurtiss, 2004; Rohde et al., 2007; Waddell and Butcher, 2007). In addition, transposon mutant libraries have been successfully screened for mycobacterial genes which are required to prevent phagolysosomal fusion (Pethe et al., 2004) and early acidification of the phagosome (Stewart et al., 2005). Host cell signaling events, such as NF-kB activation, have also been established as being important to phagosome maturation and mycobacterial killing using macrophage cell cultures (Gutierrez et al., 2008). Macrophages have served as important surrogates to measure the antimicrobial activity of new antitubercular compounds (Khan et al., 2008; Lougheed et al., 2009). Such use has been expanded to highthroughput/content screening for new compounds which inhibit phagocytosed M. tuberculosis (Christophe et al., 2009). This assay offers added advantages over conventional in vitro high-throughput screening, as it can potentially detect compounds that require modification within a host cell for antimycobacterial activity as well as compounds that modulate the macrophage to improve its killing effect on infecting mycobacteria.

B. Rodent models The use of animal models to study tuberculosis dates back to Robert Koch’s early work on the causative agents of a number of important human diseases in the late 1800s. Koch used guinea pigs in applying his set of postulates to establish M. tuberculosis as the cause of tuberculosis in humans (Brock, 1999), work for which he was awarded the Nobel Prize in Physiology or Medicine in 1905. Although guinea pigs do not acquire tuberculosis naturally, animals inoculated with pure cultures of the pathogen quickly develop active tuberculosis exhibiting a number of the clinical signs of tuberculosis that are observed in humans (Brock, 1999). Guinea pigs became the most widely used animal model to study tuberculosis in the 1800s and early 1900s (McMurray, 1994) in part due to their high susceptibility to experimental infection with M. tuberculosis (Brock, 1999) and their physiological and immunological similarities to humans. In particular, the immune response in guinea pigs of the lung and skin to inflammatory stimuli is similar to that of humans (McMurray, 1994). Furthermore, guinea pigs develop primary pulmonary lesions following inoculation with a small number of tubercle bacilli, which resemble lesions found in humans (Gupta and Katoch, 2005). Today, guinea pigs are used in a range of assays including testing the potency of tuberculosis vaccines (Gupta and Katoch, 2009; Skeiky et al., 2009; Williams et al., 2009), measuring the in vivo efficacy of antitubercular drugs (Ahmad et al., 2009;

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Lenaerts et al., 2007), and determining the virulence of mutant derivatives of M. tuberculosis (Converse et al., 2009; Drumm et al., 2009). In addition to guinea pigs, Koch also experimented with a number of other animal models of tuberculosis. The discovery of streptomycin highlighted the need for a more cost-effective model of tuberculosis because of the large number of animals needed in trials with the drug (Orme and Collins, 1994). Mice offered the advantage of lower costs and eventually replaced guinea pigs as the most widely used animal model of tuberculosis. A major drawback with the use of the mouse model, however, includes the relatively small window of protection seen for vaccination with BCG. In the mouse model, immunization with BCG vaccine achieves only an approximate 1 log reduction in peak lung bacillary load compared to a 2–3 log reduction seen for guinea pigs (Gupta and Katoch, 2009). In addition, tuberculosis in mice is associated with diffuse cellular infiltration rather than the caseous necrotic lesions found in human tuberculosis (Young, 2009), and lesions in mice have not been found to be hypoxic (Aly et al., 2006). In an effort to mimic hypoxic granulomas in mice, a model based on encapsulating M. tuberculosis bacilli in hollow fibers and implanting them subcutaneously in mice was established (Klinkenberg et al., 2008). While the hypoxia-activated drug metronidazole did not show antitubercular activity in this model, the hollow-fiber model did identify a higher percentage of hypoxia-attenuated mutants than both the guinea pig and standard mouse infection models (Klinkenberg et al., 2008).

C. Nonhuman primates A nonhuman primate model was developed by Joanne Flynn and colleagues at the University of Pittsburgh using cynomolgus macaques to better resemble tuberculosis in humans (Flynn et al., 2003). A low dose of 15–25 colony-forming units of M. tuberculosis was introduced via bronchoscope into the right lower lobe or middle lobe of the lung of the monkeys. In terms of pathogenesis, one monkey rapidly developed tuberculosis, nine exhibited active or chronic tuberculosis, and the remaining monkeys were judged to have a latent tuberculosis infection. Both caseous necrotic and solid nonnecrotic granulomas were observed in a manner that was considered to correlate with the extent of disease (Flynn et al., 2003). Furthermore, the presence of hypoxic lesions has been confirmed in the primate model (Via et al., 2008). The nonhuman primate model therefore appears to reproduce a number of different outcomes that are observed with respect to M. tuberculosis infection in humans. The capacity of the primate model to generate latent infections provides a valuable experimental model for the study of latent tuberculosis in humans. A more extensive study, conducted with a larger number of monkeys, determined

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that similarities also exist between the primate model and humans in terms of gross pathology, microscopic histopathology, and immune response (Lin et al., 2009). In addition, most reagents that are used to study human immunology can be used in primates (Young, 2009). It is apparent that nonhuman primates provide models of both active and latent tuberculosis that closely resemble human tuberculosis, and therefore can provide data that may not be attainable using other models of the disease.

V. THE STUDY OF TUBERCULOSIS PATHOGENESIS IN HUMAN PATIENTS There is little doubt that the more an experimental model recreates the events that occur during the natural infection of humans by M. tuberculosis, the more relevant the data generated will be to the human disease. Improvements can be made to animal models of tuberculosis is to based on our knowledge of the natural history of tuberculosis in humans (Smith et al., 2000). For example, humans acquire tuberculosis through aerosol infection and only a small number of viable bacilli are required to establish an infection. Therefore, models that incorporate the introduction of a small inoculum of M. tuberculosis into the respiratory tract would be preferable. Additional improvements require new knowledge derived from direct examination of tuberculosis infections in humans (Young, 2009). Studies have been made on tissue samples taken from tuberculosis patients. It was demonstrated some time ago that M. tuberculosis cells present in caseous lesions of lungs were surgically removed from patients who lost their acid-fastness in the standard Ziehl–Neelsen staining procedure (Nyka, 1967). Lung tissues from a pathology archive have also been investigated in histopathological and immunohistochemical studies. Unlike in mice, caseous necrosis and multinucleated giant cells were found to be prominent in human tuberculosis lung tissue with some necrotic areas exhibiting hypoxia (Tsai et al., 2006). The mature human tubercular lesions were determined to consist of a central zone of necrotic tissue bordered by T lymphocytes and macrophages and an area that included aggregates of B cells (Tsai et al., 2006). The extraction of lung tissue from patients provides a retrospective look at events that occur in a host during infection. Recently, advances have been made in the study of M. tuberculosis infections in patients in real time. Barry and colleagues at the National Institutes of Health, USA and South Korea’s National Masan Tuberculosis Hospital have developed a technique to visualize granulomas in tuberculosis patients. Positron emission tomography (PET) scans identify areas of inflammation using a radiolabeled tracer, [18F]-fluorodeoxyglucose (FDG) (Barry et al., 2009).

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FDG is a glucose analog and its uptake acts as a marker of metabolic activity and, indirectly, immune cell activity and inflammation. Computed tomography (CT) takes high-resolution X-ray images of the body which are then compiled into a three-dimensional reconstruction of the scanned region. Granuloma number and size of tuberculosis patients can be quantified using this technique and the effect of current or new chemotherapy regimens on the extent of patient lesions can be determined over time. There are clearly other potential applications of PET/CT and these are likely to produce valuable knowledge on the progression of tuberculosis in humans.

VI. CONCLUSIONS AND FUTURE PROSPECTS It is clear that a large number of in vitro and in vivo models have been developed for the study of human tuberculosis. As well as the biological questions to be addressed, the choice of model will often depend on the financial resources and facilities available to a researcher. It is important that new tuberculosis research is not impeded by local limitations in technology or expertise. The sharing of experimental models to study human tuberculosis will likely hasten progress towards more effective measures to eradicate the disease.

ACKNOWLEDGMENTS The support of the Health Research Council of New Zealand (grant number 07/379), the Wellington Medical Research Foundation (grant no. 2006/121), and the University Research Fund, Victoria University of Wellington (grant no. 26211/1496), is gratefully acknowledged.

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