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Mycobacterium tuberculosis evolutionary pathogenesis and its putative impact on drug development Fabien Le Chevalier1,2, Alessandro Cascioferro1, Laleh Majlessi1, Jean Louis Herrmann3,4 & Roland Brosch*,1

ABSTRACT Mycobacterium tuberculosis, the etiological agent of human TB, is the most important mycobacterial pathogen in terms of global patient numbers and gravity of disease. The molecular mechanisms by which M. tuberculosis causes disease are complex and the result of host–pathogen coevolution that might have started already in the time of its Mycobacterium canettii-like progenitors. Despite research progress, M. tuberculosis still holds many secrets of its successful strategy for circumventing host defences, persisting in the host and developing resistance, which makes anti-TB treatment regimens extremely long and often inefficient. Here, we discuss what we have learned from recent studies on the evolution of the pathogen and its putative new drug targets that are essential for mycobacterial growth under in vitro or in vivo conditions. Pathogenic mycobacteria are important causes of human and animal disease. Despite the availability of antibiotics and chemotherapeutic agents that show activity against certain mycobacteria, different drug-resistant forms are on the rise, which requires new efforts in the search for novel active compounds and treatment strategies. Phylogenetically, the genus Mycobacterium is contained within the phylum Actinobacteria, with the genera Corynebacterium and Streptomyces as close relatives. Most of the approximately 130 defined mycobacterial species contain harmless saprophytes [1] , which is most evident for the large group of fast-growing mycobacteria that comprise only very few pathogenic species, such as the Mycobacterium abscessus–Mycobacterium massiliense–Mycobacterium bolletti complex. These latter mycobacteria represent emerging opportunistic pathogens that are more and more often being recognized as causative agents of acute and persistent lung infections in cystic fibrosis patients and are also virulent in animal infection models [2–5] . They pose severe difficulties for treatment in humans due to their extensive level of drug resistance [6–8] . In contrast to the phylogenetically more diversified fast-growing mycobacteria, the slow-growing mycobacteria form a subcluster in the 16S rRNA-based phylogenetic tree [9] and are well known to harbor major human pathogens, such as Mycobacterium leprae [10] and Mycobacterium tuberculosis [11,12] , and also a series of opportunistic human pathogens, such as Mycobacterium marinum [13] or Mycobacterium kansasii [14] , which are considered to be close relatives of M. tuberculosis. Overall, mycobacteria are considered to be high-GC content, Gram-positive bacteria, but in contrast to other Gram-positive bacteria, such as Staphylococcus or Bacillus species, which have no outer membrane, mycobacteria possess a lipid-rich cell envelope that contains a standard inner membrane and a particular outer membrane, named the mycomembrane, which is specific to mycobacteria and might fulfill a similar barrier function as the outer membrane of Gram-negative bacteria

KEYWORDS 

• attenuation • drug development • evolution • Mycobacterium tuberculosis • secretion systems • virulence factors

Institut Pasteur, Unit for Integrated Mycobacterial Pathogenomics, Paris, France University Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, Paris, France 3 EA 3647, University Versailles St Quentin in Yvelines, Garches, France 4 Microbiology Laboratory, Assistance Publique – Hôpitaux de Paris, Raymond Poincaré Hospital, Garches, France *Author for correspondence: [email protected] 1 2

10.2217/FMB.14.70 © 2014 Future Medicine Ltd

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Review  Le Chevalier, Cascioferro, Majlessi, Herrmann & Brosch [15–17] .

In addition, the mycobacterial cell is also covered by a polysaccharide-based capsule [18,19] . This overall envelope architecture is conserved between nonpathogenic and pathogenic mycobacteria and contributes to the enhanced ability of mycobacteria to resist and persist in different environments. The complex cell envelope necessitates efficient secretion systems that can ensure the transport of a range of biomolecules across this multilayer barrier. Despite the conservation of many core elements in the mycobacterial cell envelope (reviewed in [20]) (Figure 1) , over the course of evolution, individual changes in the composition of cell wall components, such as particular lipids or specific secretion and transport systems, have emerged that contribute to the specific lifestyles of pathogenic mycobacterial species. Evolution towards pathogenicity has often been accompanied by a reduction of genome size that is compensated in part by gene acquisition, gene duplications and diversification (Figure 2A) [11,13–14,21–23] . When M. tuberculosis is compared with M. marinum, which is characterized by an approximately 6.7-Mb genome and the more distantly related, fastgrowing Mycobacterium smegmatis that harbors an approximately 7-Mb genome, a core of 2450 genes that are conserved among the three species can be defined (Figure 2B) . This comparison also showed that approximately 600 genes are specific for M. tuberculosis and might contribute to the pathogenicity of M. tuberculosis

A

B CAPSULE Extractable lipids Mycolic-acid layer (outer membrane) Arabinogalactan

Peptidoglycan

Plasma membrane

Figure 1. Organization of the mycobacterial cell envelope. (A) Model of the cell envelope of Mycobacterium tuberculosis and other tubercle bacilli. (B) Cell envelope of Mycobacterium bovis BCG by cryoelectron microscopy of vitreous sections. (A) Adapted with permission from [15]; (B) Reproduced with permission from [16].

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relative to the other mycobacterial species [13] . When M. tuberculosis is compared within the group of tubercle bacilli (i.e., mycobacteria that cause TB in mammalian species), recent genome comparisons have shown that Mycobacterium canettii strains, which represent rare human isolates with smooth colony morphology from the region of the Horn of Africa, harbor somewhat larger genomes that differ from M. tuberculosis by 16,000–60,000 single-nucleotide poly­ morphisms and individual accessory genomes of up to 366 genes [23] , which argues for their ancestral status relative to M. tuberculosis. As these M. canettii strains were found to be less virulent and less persistent than M. tuberculosis sensu stricto in laboratory mouse models, their genomes provide very interesting gene repertoires for learning more about the ancestral gene content of the putative progenitor of M. tuberculosis and about possible gene transfers that might have contributed to the virulence gain of M. tuberculosis during evolution. Comparison of several M. canettii strains with M. tuberculosis revealed some 51 genes that were specifically present in M. tuberculosis and absent from M. canettii [23] , such as the gene rv1818 encoding a specific member (PE_PGRS33) of a large mycobacterial protein family named after the Pro–Glu motif in the N-terminus and the presence of polymorphic GC-rich repetitive sequences in the central part or the C-terminus of the proteins [11] . On the other hand, nine genes were found that were specifically absent from M. tuberculosis relative to M. canettii, such as the cobF gene encoding an enzyme in the vitamin B12 metabolic pathway [23,24] . The loss of cobF from M. tuberculosis might be compensated by substrates provided by the host, which is presently under investigation. As such, the different genes and their gene products that differ between M. canettii and M. tuberculosis can now be further investigated for their impact on the different phenotypes of the concerned phylogenetically diverging tubercle bacilli. As mentioned above, genome comparisons between fast-growing mycobacterial saprophytes and slow-growing mycobacterial pathogens show an overall trend of genome size reduction. A possible explanation for the genome downsizing of pathogenic mycobacteria could be that adaptation to a pathogenic lifestyle that includes the exploitation of resources of a host organism might make certain gene functions redundant and subject to gene loss. This feature is most

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Mycobacterium tuberculosis evolutionary pathogenesis & its putative impact on drug development  visible in the only 3.2-Mb genome of the obligate intracellular pathogen M. leprae that has undergone a dramatic reductive evolution due to gene decay and gene loss, which is responsible for the fact that M. leprae, despite its discovery by Armauer Hansen more than 140 years ago, can still not be cultured on axenic growth media [10] . The situation of genome reduction is less pronounced for M. tuberculosis, which harbors a 4.4 Mb genome and has retained the ability to grow on axenic culture media [11] , potentially reflecting the extraordinary faculty of this human pathogen to adapt to numerous intra- and extra-cellular environments encountered during its interplay with the host. The particularity of M. tuberculosis in comparison with many facultative mycobacterial pathogens is that it is an obligatory pathogen whose transmission to new hosts occurs from patients who have developed active disease, implicating lung tissue necrosis, cavity formation and coughingup of infectious bacilli into the immediate environment as tiny droplets. This infection strategy seems to be highly efficient as it is estimated that approximately a third of the human population is infected with M. tuberculosis, from which 5–10% develop active disease. M. tuberculosis is responsible for 9 million cases of active TB and 1.3 million deaths per year [26] . This situation suggests that by selection, the pathogen has developed a balance for using the human host for its own proliferation and global distribution during a long-lasting coevolution [23,27,28] . This situation poses enormous challenges to scientists in order to unravel the factors that are responsible for the evolutionary success of M. tuberculosis. Knowledge on the molecular features that contribute to pathogenicity, persistence and efficient spreading of M. tuberculosis among humans will provide important input for the development of alternative strategies in TB control. Genomics & identification of the essential genes of M. tuberculosis Within the more than 130 years since the discovery of M. tuberculosis as the causative microorganism responsible for human TB by Robert Koch, numerous scientific advances have been made that help to cope with this major pathogen. However, despite this progress, M. tuberculosis still holds many unresolved secrets and much work remains to be done in order to translate the basic findings from recent research into novel strategies against the pathogen [12] . After

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Review

several anti-TB drugs were developed in the mid20th century, there has been a long gap in which no new molecules to be used in the treatment of TB were developed. One of the reasons for that could have been that TB was believed to be a disease of the past, which would disappear in modern societies due to improved social conditions, vaccination and already-existing treatment regimens. However, it is now clear that this was not the case for developing countries and also certain population groups in industrialized countries. The synergy of TB with the HIV epidemic, the phenomenon of multidrug resistance, massive global population growth and a worsening of social conditions in many countries dramatically changed the situation, making TB a continuing global health threat even at the beginning of the 21st century [26] . This fact has also lead to a strongly renewed research interest that is likely to be continued in the future. In this context, the accomplishment and publication of the genome sequence of the widely used M. tuberculosis H37Rv strain [11] was one key step that allowed the scientific community to gain deeper insight into the overall organization of this pathogen and to design numerous follow-up strategies in a wide range of postgenomic applications. For scientists interested in increasing the arsenal of new compounds and drugs against TB and its causative organism, the knowledge of the complete gene pool of the pathogen has helped considerably with the identification and investigation of potentially vulnerable targets of M. tuberculosis. The genome information served as a starting point for identifying which among the 4000 genes in the M. tuberculosis genome were essential for the growth and/or survival of the pathogen. The genome-wide use of highdensity transposon mutagenesis in combination with the microarray-based identification of insertion sites revealed a minimal gene set of 614 genes required for optimal in vitro growth of M. tuberculosis in culture broth [29] . This technique, named transposon site hybridization (TraSH), was subsequently also adapted to other biological settings, such as infection models in mice, where a set of 194 genes implicated in growth under in vivo conditions was identified [30] . Among them, numerous genes encoding proteins involved in the pathogenicity of the tubercle bacilli can be found that are also discussed in a later section. Most recently, saturation transposon mutagenesis of M. tuberculosis

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Gordonia aichiensis M. abscessus 5.1 Mb M. chelonae M. hodleri M. neoaurum M. diernhoferi M. frederiksbergense

Outgroup

M. triviale M. heidelbergense M. simiae M. genavense M. lentiflavum M. kubicae M. palustre M. interjectum M. intermedium M. scrofulaceum M. lacus

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2450 (2587, 2524, 3810)

48 (52, 55)

M. fallax M. chitae M. manitobense M. flavescens 7.3 Mb M. duvalii M. tuscia 6.2 Mb M. vaccae M. austroafricanum 6.5 Mb M. vanbaalenii M. aurum M. novocastrense M. acapulcensis 5.9 Mb M. sp. MCS M. sp. KMS 6.3 Mb M. sp. JLS 6.0 Mb M. monacense M. doricum M. smegmatis 7.0 Mb M. goodii M. moriokaense 4.8 Mb M. thermoresistibile M. pulveris M. elephantis M. confluentis 5.7 Mb M. phlei M. brumae M. holsaticum

Long helix 18

616 (699, 683)

M. tuberculosis 624 (653)

M. gadium M. farcinogenes† M. senegalense M. alvei 6.9 Mb M. septicum M. chubuense M. chlorophenolicum M. peregrinum M. wolinskyi M. obuense M. aichiense M. gilvum 6.0 Mb M. parafortuitum M. sphagni 6.3 Mb M. fortuitum M. komossense

M. marinum 1417 (1468)

785 (815, 1353)

M. smegmatis 379 (484)

Rapid growers Slow growers

Extended helix 18

M. cookii M. celatum M. terrae M. nonchromogenicum M. hiberniae M. shimoidei M. heckeshornense M. botniense M. tuberculosis 4.4 Mb M. xenopi M. africanum M. canettii 4.3–4.5 Mb M. tuberculosis complex M. bovis M. microti M. shottsii 6.7 Mb M. marinum M. ulcerans 5.8 Mb M. gordonae M. szulgai M. asiaticum M. malmoense M. gastri 6.4 Mb M. kansasii M. haemophilum 3.3 Mb M. leprae 5.4 Mb M. avium M. avium complex M. paratuberculosis 4.8 Mb M. intracellulare 5.3 Mb

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Mycobacterium tuberculosis evolutionary pathogenesis & its putative impact on drug development 

Review

Figure 2. Phylogenetic organization and gene content of members of the genus Mycobacterium (see facing page). (A) Phylogenetic tree based on the consensus of the 230 most parsimonious trees of the 16S rRNA DNA sequences of 80 species of the genus Mycobacterium with the sequence of the species Gordonia aichiensis as the outgroup. Sequenced genomes are highlighted in yellow and underlined species are considered to be pathogens.  The division between fast- and slow-growing species is indicated by a dotted line. Genome sizes are indicated according to information from the Gold Genome online database [25]. (B) Venn diagram showing orthologous coding sequences among three mycobacterial species as determined by BLASTClust analysis. Numbers in parentheses include paralogous coding sequences. † Mycobacterium farcinogenesis a slow-growing Mycobacterium. (A) Adapted with permission from [22]; (B) Adapted with permission from [13].

grown under different in vitro conditions was combined with next-generation sequencing (NGS) [31] , which allowed a set of 774 genes to be identified, of which 451 overlapped with the initial TraSH-based screen. In addition, this work permitted differences in the essential gene set to be linked to the use of cholesterol instead of glycerol as a carbon source. Identification of the bacterial genes required for cholesterol utilization in M. tuberculosis allowed the widespread metabolic changes to be predicted that are associated with the adaptation of the bacterium to this carbon source and refined the understanding of bacterial physiology [31] . Potential roles of essential genes as drug targets The identification of a set of essential genes for M. tuberculosis has greatly enhanced the ability to define new, potentially vulnerable drug targets of the pathogen. In combination with the advances in crystallography and the increasing availability of structural information on mycobacterial proteins, in silico drug screening should be a promising way to find new active molecules against M. tuberculosis. However, this approach must also be taken with caution, as binding of a given compound to an essential target protein under experimental condition often does not translate to actual growth inhibition of the bacterium in subsequent MIC screens. PknB, a serine–threonine protein kinase of M. tuberculosis, represents a typical example of such a situation. This protein, which is part of the 11 eukaryotic-like serine–threonine protein kinases of M. tuberculosis [11] , is listed as an essential protein in the M. tuberculosis TraSH and NGS datasets [29,31] . The essentiality of this protein has also been suggested by independent genetic studies, in which the knockout of the original pknB gene only became possible once a second intact pknB copy was integrated into the M. tuberculosis

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genome [32] . Similarly, the essentiality of PknB was also demonstrated by gene knockdown experiments, which showed a dramatic impairment of bacterial viability in liquid culture upon depletion of the kinase [33] . Nevertheless, potential inhibitors that showed affinity to PknB were active only in the higher micromolar MIC range [34,35] , which makes their further development into potential anti-TB drugs rather difficult and/or unlikely. For proteins involved in the regulation of essential cell processes, very small amounts might already be sufficient, so that it is very difficult for an inhibitor to fully block the function of the protein. Apart from potential quantitative particularities, many other factors may impact on the final inhibition activity of a compound on the bacterium. For M. tuberculosis, the complex, lipid-rich cell wall composition, which in addition is protected by a polysaccharide-based capsule [15,18,19] , certainly complicates permeability. In addition, the potential effect of efflux pumps and protein binding in the assay media may also play a role in the evaluation of the final antimicrobial activity of a compound. Thus, according to most recent trends in anti-TB drug development, whole-cell screening of compound libraries has re-emerged as a frequently used technique to find new active compounds against M. tuberculosis for which the drug target can then be identified in follow-up studies. This approach is of great importance for enlarging the arsenal of presently used first-line (isoniazid, rifampicin, ethambutol and pyrazinamide) and second-line (streptomycin, fluoroquinolones, para-aminosalicylic acid and the injectable agents amikacin, kanamycin or capreomycin) anti-TB drug regimens (Tables 1 & 2) [36] that may all be subject to resistance mechanisms in extensively and/or totally resistant M. tuberculosis strains [37] . Screening and target identification studies allowed the diarylquinoline drug TMC207 to

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Review  Le Chevalier, Cascioferro, Majlessi, Herrmann & Brosch Table 1. First- and second-line anti-TB drugs used in standard treatment regiments. Anti-TB drug

Principle target(s)

Isoniazid Rifampicin Ethambutol Pyrazinamide Streptomycin Fluoroquinolones Para-aminosalicylic acid

InhA; KatG (activation) RpoB  EmbB RpsA; PncA (activation) RpsL GyrA Dihydropteroate synthase; folate metabolism

Injectable agents Amikacin, kanamycin or capreomycin Ethionamide Cycloserine

30S ribosomal subunit or interbridge InhA; EthA (activation) d-alanine racemase and ligase 

Data taken from [36,38].

be discovered, for example, for which resistant mutants showed single-nucleotide polymorphisms in the gene atpE encoding the C chain of the ATP synthase of M. tuberculosis [39] . The activity of TMC207 on multidrug-resistant M. tuberculosis was confirmed in clinical trials [40] , and in December 2012, the drug (also known as bedaquiline or Sirturo™ [Janssen Therapeutics, NJ, USA]) was approved by the US FDA as a new treatment for multidrug-resistant TB that can be used as an alternative when other drugs fail. Other recently discovered active compounds against M. tuberculosis with potential for further development are the benzothiazinones, which are extremely efficient at blocking the growth of M. tuberculosis under in vitro conditions and are also active in vivo in mouse models [41] . Benzothiazinones target DprE1,

which is involved in the synthesis of arabinans, essential components of the mycobacterial cell wall. DprE1 seems to be a highly vulnerable target, as recently shown for a range of compounds with different chemical scaffolds [38,42] . For example, DprE1 was found as the target of dinitrobenzamide derivatives (DNB1), a class of compounds that were identified by high-content screening to interfere with the replication of M. tuberculosis within macrophages [43] . The employed screening technique, which is based on automated, simultaneous imaging of fluorescent bacteria and host macrophages exposed to different representatives of chemical compound libraries, is a particularly attractive and powerful method for identifying molecules that show activity against intracellular M. tuberculosis but little or no toxicity towards host cells [43] . The

Table 2. Recommended anti-TB drug regimens; including details of the initial phase and continuation phase. Type of TB cases

Total duration of Duration of treatment initial phase (months) (months)

Drug combination during initial phase

Duration of continued phase (months)

Drug combination during continued phase

New smear- or culturepositive cases New culture-negative cases Resistance (or intolerance) to isoniazid  Resistance to isoniazid and rifampicin

6

2

4

4

2

6

6

–

Isoniazid and rifampicin Isoniazid and rifampicin –

12–18

12–18

–

–

Resistance to all first-line drugs

24

24

Isoniazid, rifampicin, pyrazinamide and ethambutol Isoniazid, rifampicin, pyrazinamide and ethambutol Rifampicin, pyrazinamide and ethambutol Pyrazinamide, ethambutol, fluoroquinolones and streptomycin (or another injectable agent) One injectable agent + three drugs from the following group: ethionamide, cycloserine, fluoroquinolones or para-aminosalicylic acid

–

–

2

Data taken from [36,38].

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Mycobacterium tuberculosis evolutionary pathogenesis & its putative impact on drug development  same high-content screening approach was also the starting point for the discovery of Q203, a potent candidate drug that targets qcrB, encoding for an ubiquinol–cytochrome C reductase of the respiration chain of M. tuberculosis [44] . Another vulnerable drug target that is hit by different classes of small-molecule inhibitors is MmpL3, an essential mycobacterial membrane protein that harbors 11 transmembrane domains and is crucial for the transport of cell wall constituents [45] . Probably the most advanced compound in terms of clinical development that was found to target MmpL3 is the diamine SQ109, a drug candidate that is in Phase II of clinical testing [46] . Another molecule that is in clinical studies is PA-824, a nitroimidazole compound [47] , which targets a deazaflavin-dependent nitroreductase [48] . Most recently, thiophene compounds were identified that kill M. tuberculosis by the previously uncharacterized mechanism of inhibition of the polyketide synthase Pks13 [49] , which is an essential enzyme that catalyzes the last condensation step of mycolic acid biosynthesis [50] . In addition, a series of fluoroquinalones were studied in preclinical and clinical settings, with a particular focus on gatifloxacin and moxifloxacin, in order to evaluate whether they can improve the activity of the standard drug regimen when substituted for ethambutol and thereby might help to shorten the duration of treatment for fully drug-susceptible TB [51] . As another example of the clinical evaluation of new drugs, the addition of linezolid into the regimen against extensively drug-resistant forms led to strongly improved prognosis of the treatment outcomes and culture conversion in patients with severe, chronic, extensively drug-resistant TB [52] , which gives hope that other oxazolidinone-based drugs currently under development might also be effective [53] . Recent research has also elucidated the targets and putative modes of action of some anti-TB drugs that have been known for a long time. One striking example is the identification of the enoyl reductase InhA as the drug target of the natural compound pyridomycin [54] , which bridges the NADH- and substrate-binding pockets of the enzyme and thus inhibits InhA in a different way than the first-line drug isoniazid, which is a prodrug and needs activation by KatG in order to form the active isonicotinic acyl–NADH complex. As a result, pyridomycin is also active on the most frequently encountered

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isoniazid-resistant M. tuberculosis strains, which opens new perspectives for drug development [55] . Another old drug that targets InhA is ethionamide, a thiocarbamide-containing compound that is activated by the mycobacterial monooxygenase EthA, whose production is controlled by the transcriptional repressor EthR. A recently applied strategy that is based on inhibiting EthR shows promise for improving the therapeutic index of thiocarbamide derivatives and for overcoming resistance [56] . Finally, for two other ‘old’ drugs, pyrazinamide and para-aminosalicylic acid, new mechanisms of action were recently identified. For pyrazinamide, activity directed against persistent forms of M. tuberculosis was discerned, which involves the inhibition of the process of trans-translation by ribosomal protein S1, which is essential for freeing scarce ribosomes in nonreplicating organisms [57] . For paraaminosalicylic acid, it was found that it acted as a prodrug, which poisons folate-dependent pathways not only by serving as a replacement substrate for dihydropteroate synthase, but also by the generation of toxic byproducts created by the enzymes of these pathways [58] . Mycobacterial secretion systems & their potential utility as drug targets Screening of different classes of compounds with antitubercular activity combined with sequence analysis and recombineering has revealed some new potential drug targets of M. tuberculosis, such as the membrane protein EccB3, which represents a novel candidate not targeted by currently existing anti-TB drugs [59] . The EccB3 protein is a conserved component of the ESX-3 secretion system, which represents one of the five type VII secretion systems of M. tuberculosis [60] . In contrast to ESX-1, which is not essential for in vitro growth of M. tuberculosis but is necessary for multiplication and survival of M. tuberculosis in the host cell [61] , ESX-3 is a secretion system whose genes are essential for the in vitro growth of M. tuberculosis in standard culture media [31] due to their role in mycobactin-mediated iron acquisition and zinc uptake [62,63] . The aforementioned finding that the ESX-3 system can be targeted by small-compound inhibitors is an encouragement for further research and the development of inhibitors that might inactivate components of the secretion systems of M. tuberculosis. Apart from the ESX-3 system, among the type VII secretion systems, there is also the ESX-5 system, which has recently been shown to

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Review  Le Chevalier, Cascioferro, Majlessi, Herrmann & Brosch contain genes (e.g., eccC5 ) that are essential for the in vitro growth of M. tuberculosis [31,64] , in addition to genes that are involved in the virulence and export of PE/PPE antigens (Figure 3) [65,66] . Besides the type VII secretion systems, which represent specialized secretion systems of mycobacteria and related actinobacteria, M. tuberculosis also uses various other secretion pathways for protein export [67] (i.e., the SecA1-mediated general secretory pathway [68] , the alternative SecA2-operated pathway [69] and a twin-arginine translocation (TAT) system [70,71]), which might also serve as targets for new drugs [72] . The mycobacterial TAT system, for example, is constituted by a protein complex that includes TatA (Rv2094c) and TatC (Rv2093c), which are essential for the in vitro growth of M. tuberculosis [29,31,71] and are thought to be localized in the plasma membrane (Figure 3) . A third protein called TatB (Rv1224), which is encoded elsewhere in the genome, also seems to be essential for the function of the TAT apparatus in M. tuberculosis [71] , although in TAT systems of other bacterial species, this does not always seem to be the case [73] . Finally, based on sequence homology, a fourth protein, TatD (Rv1008), a nonessential protein with supposed DNase activity, is also a putative component of the TAT protein export system in M. tuberculosis. The proteins secreted by the TAT system are characterized by the presence of the twin arginine (RR) motif in the N-terminal signal sequence [71,73,74] . In contrast to other secretion pathways of M. tuberculosis, the TAT system exports the proteins in their folded state. Among the various proteins with a TAT signal sequence motif, some have been described as being implicated in pathogenesis, such as the phospholipase C proteins (i.e., PlcA–D) [75] and resistance to β-lactam antibiotics, such as BlaC, representing a class A β-lactamase [74] . Overall, it is clear that many of the proteins secreted by the various secretion pathways of M. tuberculosis play important roles in the survival and growth of the bacterium inside the host. Hence, specific protein export is a very important feature for pathogenic bacteria, as it enables the pathogen to withstand the defence mechanisms mounted by the host cells and/or exploit cellular functions for the benefit of the pathogen. Although the secreted proteins are often not essential for the in vitro growth of the organisms in culture broth, their in vivo growth essentiality might be exploited as an

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in vivo drug target for chemical compounds that could target these proteins during infection. As such, research on the potential virulence factors of pathogenic mycobacteria combined with elucidating their mechanisms of action might uncover new, unconventional targets for the development of new classes of antimycobacterial compounds that might act under in vivo conditions in synergy with more conventional bactericidal drugs. Selected virulence factors of M. tuberculosis & their potential role for alternative intervention strategies As a result of the significant advances in mycobacterial molecular genetics and genomics in recent years, a number of M. tuberculosis genes that intervene in host–pathogen interactions have been identified. The first such mycobacterial virulence genes were identified by the use of pioneering gene knockout techniques [76,77] that were later combined with signature-tagged mutagenesis [78,79] , resulting in a first list of candidate genes involved in the pathogenicity of M. tuberculosis. Among the genes identified by this approach were several with insertions in a 70-kb chromosomal segment that encodes proteins involved in the synthesis and transport of phthiocerol dimycocerosate, which corresponds to specific, extractable lipids in the outer layers of the mycobacterial cell envelope that play an important role in virulence. The number of potential virulence genes was later extended by the use of the TraSH technique adapted to mouse infection, which predicts that, in addition to the 15% of genes that are essential for in vitro growth, another 5% are involved in in vivo growth [30] . Among the approximately 200 listed genes are also several that encode proteins that belong to macromolecular components or secreted proteins of the aforementioned major secretion systems of M. tuberculosis, which might be particularly interesting as potential drug targets, as their encoded proteins are supposed to be localized in the cell envelope and thus more easily accessible by putative inhibitors. As a first example, the gene secA2, which codes for a main constituent of the alternative SecA2-operated pathway, is part of this list. This pathway was shown to export SodA and KatG, which are both involved in the detoxification of reactive oxygen intermediates produced by the host cell as part of the oxidative attack within the phagosome [69,80] . Small-molecule inhibitors

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Mycobacterium tuberculosis evolutionary pathogenesis & its putative impact on drug development 

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6c 5c 4c 61 61 61 rv3 rv3 rv3

A

pA pC pD es es es 64

8 rv3

ESX-1

AAA+ATPase

1 pE spFspGspH eccA 1 e e e

es

ESAT-6 CFP-10

FtsK/SpoIIIE

cB 1

ec

a1

cC

ec

Cb

c ec

1

35 68 xB A pl pe ppe es esx es

cD 1

ec

pK

pJ

pL spB e

es

es

es

2

1

79 rv1 cC

cB 5

ec

ec

8

TAT

cE 1 ycP m

ec

8

78 rv1

ESX-5

3c

88

rv3

00 rv1 D tat

43 86 25 18 6 p1 v17 ppe pe pe2 y r c p

9 7 4 cP 5 e2 pe1sxeMsxN 179 cD 5 my e c v r e

cA 5

cE 5

ec

ec

pp

4 22 rv1 rv2094c-93c C A tat tat

B tat

B

E E S S X X

P P P E E Outer membrane

?†

?† Arabinogalactan MycP

EccB

Peptidoglycan

TatC

TatA

TatB RR

Inner membrane

EccD

Substrate

TatD

EccE

EccC E E S S X X

P E

P P E

EccA

Figure 3. Working model of two selected secretion pathways (twin-arginine translocation and ESX) that are thought to be involved in the pathogenicity of Mycobacterium tuberculosis. (A) The genomic loci of two of the five ESX systems (ESX-1 and ESX-5), as well as the components of the TAT system. The gene nomenclature corresponds to that of the reference strain Mycobacterium tuberculosis H37Rv. (B) A model of the cell envelope of M. tuberculosis with the putative locations of selected proteins from the TAT and the ESX systems. † The exact shape and mechanism by which the proteins are transported through the outer membrane is still unknown. RR: Twin arginine; TAT: Twin-arginine translocation. (A) Data taken from [11,60,64,67]; (B) Data taken from [60,67].

that would inhibit these oxidative stress defense mechanisms of M. tuberculosis might strongly impact on the fitness and transmissibility of

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the pathogen, as suggested by virulence studies on KatG-deletion mutants in the context of isoniazid resistance [81,82] .

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Review  Le Chevalier, Cascioferro, Majlessi, Herrmann & Brosch The list of mutants with in vivo growth defects also points to multiple genes implicated in the ESX-1 type VII secretion pathway [30] . The ESX-1 secretion system was identified as a primary factor determining the host–pathogen interactions of tubercle bacilli by several independent and complementary approaches (reviewed in [61]). Indeed, deletion of part of the ESX-1-encoding chromosomal region, named region of difference 1 (RD1), from Mycobacterium bovis BCG, which still represents the only anti-TB vaccine currently used on a global scale, is one of the main reasons for the loss of virulence of this attenuated live vaccine. Similarly, the ESX-1 system is also truncated in Mycobacterium microti strains, of which several were used as live-attenuated vaccines in the 1960s [61] . Moreover, the presence of the 6-kDa early secreted antigenic target ESAT-6 and its protein partner CFP-10, which are both secreted by ESX-1 in M. tuberculosis, but are absent from M. bovis BCG and M. microti-based vaccines, represents the basis for the differential potential of the next-generation IFN-γ release assays that have substantially refined TB diagnosis [83] . ESX-1 might thus serve as a potential target for the development of molecules that could interfere with the process of ESX-1-mediated host–pathogen interactions. Despite intense research within the last decade since its discovery, the exact function of the ESX-1 system is only partially known. However, recent pathogenicity-related research has shown that the intracellular behavior of wild-type M. tuberculosis is profoundly different from ESX-1-deficient variants of M. tuberculosis (ΔRD1) or other ESX1-deleted tubercle bacilli, such as BCG. The main differences observed during the infection of macrophages are linked to the finding that ESX-1-proficient strains are able to rupture the phagosomal membrane and obtain access to the host cell cytosol at later stages of the infection, whereas strains carrying a partial or interrupted ESX-1 system remain locked in the phagovacuole [84,85] . Hence, the ability of M. tuberculosis to break the phagosomal membrane and access the cytosol seems to be tightly linked to secretion ESAT-6 and CFP-10. The observed activity seems to be specific for ESAT-6 of M. tuberculosis [86,87] that, under acidic pH conditions, may undergo significant conformational changes, which is not the case for orthologous proteins from the nonpathogenic M. smegmatis species [88] . The ESX-1-dependent ability of inducing

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phagosomal breakage seems to be a key factor during the infection of host cells by M. tuberculosis, as only strains with intact ESX-1 secretion can cause cell death [85,89,90] and have enhanced cell-to-cell spread [86,91] . The cytosolic contact of ESX-1-proficient strains also determines the autophagic flux [92,93] , and may promote crosspresentation of mycobacterial antigens by the cytosolic MHC class I-processing machinery (i.e., the proteasome). The differences in cytosolic contact and induction of host cell necrosis between ESX-1-containing M. tuberculosis and naturally ESX-1-deleted BCG are also relevant for NLRP3 inflammasome activation, which leads to IL-1β secretion [94–96] , the generation of type I interferons [97] and impacts induction of CD8 + T-cell responses [98] . The identification of small-molecule inhibitors specifically targeting the ESX-1 secretion machinery or its secreted proteins may represent a suitable alternative way to neutralize the virulence factors of M. tuberculosis [99,100] . Without being bactericidal themselves, these virulence inhibitors could accentuate the innate and adaptive immune responses of the host on bacteria that have become devoid of their ESX-1-linked intracellular defense strategies. Such a strategy, which might work best in combination with conventional, bactericidal, anti-TB drugs, could also help with avoiding the generation of drug resistance due to the action of the immune system on potential escape mutants. Inhibition of the ESX-1 system might also be achieved by targeting the regulation process of ESAT-6/CFP10 secretion, which is linked to the ESX-1-associated proteins EspA, EspC and EspD. The espACD locus, located in a different region of the M. tuberculosis genome than the ESX-1 core components, is important for ESX-1 functions because EspA, EspC and EspD are secreted in an ESAT-6/CFP-10-codependent fashion [101,102] . The expression of EspACD is regulated by a mechanism that involves the virulence regulator EspR [103] , which was recently described as a nucleoid-associated protein [104] , and the two-component regulators MprAB and PhoPR [105,106] . PhoPR is well known for its implication in the virulence of M. tuberculosis [107] and its large regulon that accounts for more than 40 positively and 70 negatively regulated genes [108] . Although the exact regulation cascade of PhoPR, MprAB and EspR in association with EspACD remains unclear [109] , the strong ESX-1-mediated implication of the espACD

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Mycobacterium tuberculosis evolutionary pathogenesis & its putative impact on drug development  operon in the virulence of M. tuberculosis makes these regulators potential drug targets. In analogy with the ESX-1 system, the ESX-5 secretion system might also represent a potential drug target, as it is simultaneously involved with the in vitro viability and in vivo replication of M. tuberculosis [65,66,110] . It was recently shown that disruption of certain ESX-5 core components, such as the predicted transmembrane channel protein EccD5 or the membrane-bound ATPase EccC5, affects the growth of M. tuberculosis on solid medium, yielding small colony morphotypes of mutant colonies in comparison with the wild-type strain [64–65,110] . ESX-5 was also shown to be essential for mycobacterial cell wall integrity, which is further confirmed by the enhanced detergent and hydrophilic antibiotic sensitivities of ESX-5-knockout strains [65,110] . The function of the ESX-5 system in M. tuberculosis appears to be mainly linked to the secretion of Esx and PE/PPE proteins, with the latter corresponding to two large protein families in M. tuberculosis that are named and classified after a characteristic Pro–Glu or Pro–Glu–Glu motif at the N-terminus class, as well as amino acid sequence similarity [11,111] . The functions of these immunogenic proteins [66,112] remain largely unknown, but it is clear now that some of them play a role in pathogenicity. For example, upon the deletion of the genomic section encoding PPE25-PE19 within the ESX-5 locus, the virulence of the resulting M. tuberculosis mutant was strongly diminished in immunocompetent and immunodeficient mice, whereas the complemented strain regained virulence [65,66] . Some of these proteins contain potent T-cell epitopes with or without cross-reactivity with other PE/PPE proteins [66] and might thus show good potential for inclusion into subunit vaccine combinations that might be used in combination with specific drug therapies as a form of immunotherapy, which is analogous to what has been tested in other bacterial species [113] . Conclusion Mycobacteria are very widespread in different environments, including soil and aqueous microbiota, where contact with other microorganisms is dense. From an evolutionary perspective, it seems plausible that factors that contribute today to mycobacterial pathogenesis might have evolved during the long interplay of mycobacteria with other microorganisms, including protozoa. Today’s virulence factors might constitute

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Review

ancestral invasion and defense mechanisms that were transformed by pathogenic mycobacteria into tools that are now used for intracellular survival in cells of vertebrate hosts. These genes involved in pathogenicity, together with the core genes involved in the essential functions of mycobacteria, may constitute promising new targets for the development of specific antimycobacterial molecules and compounds that can act in synergy with other available strategies. Besides the treatment regimens already in place or in development, high-content screening approaches of compounds and/or cellular particularities [43,114] that can simultaneously evaluate the viability of host phagocytes and the bacterial growth characteristics thus show great promise for the identification of appropriate candidates. Comparative genome information from various mycobacterial species and insights into the evolutionary traits of the concerned genes and gene products will be of further help for validation purposes. Future perspective In order to predict future trends, it might be useful to look back some years and evaluate whether the current situation of research and development could have been predicted. Research within the last 20 years since TB was declared a global emergency by the WHO in 1993 [26] has contributed to the enormous gain of knowledge regarding the causative agent of TB and its interactions with its host. Apart from the fact that some of these recent advances of mycobacterial research and technology have already been translated into applications with direct impact on diagnosis (e.g., IFN-γ release assays) or treatment (e.g., bedaquiline), the situation concerning TB research has profoundly changed in recent years concerning our knowledge regarding the evolution, genomics and genetics of M. tuberculosis, the disease-causing processes related to host–pathogen interactions and the discovery of new molecules, immunogens and drug targets. For example, today it is very difficult to imagine biological and medical research without access to genome data and all of the data produced by follow-up strategies, such as genome-wide transposon insertion screens, transcriptome analyses and structural biology approaches, among others. The broad impact of the genome sequence for the mycobacterial research community is reflected in the fact that the publication presenting the first complete genome sequence of M. tuberculosis

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Review  Le Chevalier, Cascioferro, Majlessi, Herrmann & Brosch (H37Rv) [11] represents the most cited article in the field of TB research at more than 4000 citations. The use of genome sequencing has been refined in recent years, as NGS approaches have allowed the cost of genome sequencing to be dramatically reduced. The reduced costs and technical advances opened the NGS technologies up for use in the sequencing of the genomes of many different M. tuberculosis isolates, thereby allowing the phylogenetic reconstruction of the global M. tuberculosis strains to be refined [28] . NGS also showed that epidemiological tracing of

M. tuberculosis-caused outbreaks could be carried out in much greater detail than with the previously used molecular typing methods [115,116] , a finding that could revolutionize epidemiological studies of M. tuberculosis outbreaks in the near future. Recent research on virulence factors and the fate of M. tuberculosis in the phagosome has elucidated many of the key features of mycobacterial host–pathogen interactions. While M. tuberculosis was considered until very recently to be a pathogen that, during the infection of

EXECUTIVE SUMMARY Evolution of mycobacterial pathogenicity ●●

Mycobacterium tuberculosis is characterized by a cell envelope that contains an inner membrane, an outer membrane (mycomebrane) and a capsule. Despite being categorized as a high-GC-content, Gram-positive bacterium, the mycobacterial cell wall functionally resembles that of Gram-negative bacteria.

●●

M. tuberculosis shares a core genome of 2500 genes with nontuberculous mycobacteria. More than 600 genes seem to be specific to the tubercle bacillus.

●●

Comparison of M. tuberculosis with Mycobacterium canettii, known as the nonclonal tubercle bacilli, shows that M. tuberculosis has gained some genes after its evolutionary branching.

Potential roles of essential genes as drug targets ●●

High-density transposon analysis showed that approximately 15% of the genes of M. tuberculosis are essential for optimal growth in broth culture. The gene products of these essential genes represent potential drug targets.

●●

Several of these genes have been recently identified by phenotypic compound screening and target identification as vulnerable targets of M. tuberculosis.

Mycobacterial secretion systems & their potential utility as drug targets ●●

M. tuberculosis encodes major secretion systems that allow the transport of biomolecules across its complex cell envelope.

●●

Some of these systems, such as the SecA1, the twin-arginine translocation system and the ESX-3 and ESX-5 systems, are essential for in vitro growth. The SecA2, ESX-1 and ESX-5 systems were shown to be required for the pathogenicity of M. tuberculosis and represent potential virulence drug targets.

Selected virulence factors of M. tuberculosis & their potential roles in alternative intervention strategies ●●

Analogously to the genes that are essential for in vitro growth, approximately 5% of M. tuberculosis genes were

identified as being essential for in vivo growth (survival in the mouse model) by saturation transposon screening. ●●

In addition, several genes that code for proteins involved in pathogenicity have been predicted by comparative genomics of virulent and attenuated tubercle bacilli and gene-knockout studies.

Conclusion & future perspective ●●

Predictions for the next 20 years, based on the advances of mycobacterial research achieved within the last

two decades, promise we might be able to identify the Achilles’ heel of M. tuberculosis and thereby allow new treatment strategies to be implemented. ●●

Targeting virulence genes could become a strong alternative strategy of treatment that relies on the focused action of the immune system.

●●

It is probable that treatments against the pathogenicity factors of M. tuberculosis might have to be combined with drugs that act against essential targets.

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Mycobacterium tuberculosis evolutionary pathogenesis & its putative impact on drug development  phagocytic cells, resides exclusively in the phagovacuole, technical advances in cryoelectron microscopy and single-cell assays based on fluorescence imaging suggested that M. tuberculosis breaks the phagosomal membrane and accesses the cytosol of the host cell at later stages of infection [84–85,89] . Future research will certainly further refine these observations and link them to the survival strategy of M. tuberculosis, with central importance being given for the induction of host-cell death and bacterial spread. Future therapeutic strategies that would prevent the rupture of M. tuberculosis-containing phagovacuoles could have a strong impact on the outcome of infection. In order to identify new molecules with such activity, phenotypic cell-based assays, which use automated confocal fluorescence microscopy for the high-throughput screening of chemicals that interfere with the replication of M. tuberculosis within macrophages [43] , could be used. An adaptation of this method, which was successfully employed to identify acyl-trehalosecontaining glycolipids involved in phagosome remodeling [114] , might well be used for finding factors that might interfere with the induction of phagosomal rupture and related cellular processes. As mentioned above, this method has the great advantage of simultaneously observing the behavior of the bacterial invader and the defense strategies of the host cell, which is thus a very promising technology for future applications. Looking at the advances in biomedical research of the last few years, which would have been difficult to foresee 20 years ago, the outlook for the future may only be very vague. In order to cope with the global problem of TB, combination strategies might be most advantageous. The trend in human medicine is to individualize treatment, and this might also apply for the prevention and treatment of TB cases. TB is a highly complex disease, with the complexity arising from the varied immunological and nutritional status of the individual, the type of M. tuberculosis strains circulating in the community, the presence of drug resistance and the social environment, References

1

Magee JG, Ward AC. Genus I. Mycobacterium. In: Bergey’s Manual of Systematic Bacteriology, The Actinobacteria. Goodfellow M, Kämpfer P, Busse HJ et al. (Eds). Springer, NY, USA, 312–375 (2012).

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among other factors. In this context, it might be best to combine treatment with immunotherapies (i.e., immunization strategies that would add an additional layer of immune defense to the action of conventional or novel anti-TB drugs). However, care has to be taken in order to avoid inducing a so-called Koch phenomenon, which corresponds to the development of necrotic lesions as a result of hypersensitivity to products of the tubercle bacillus. In addition, the use of molecules that would act against the pathogenicity factors of M. tuberculosis might be a very useful addition to such combinatorial treatment strategies. The last 20 years of TB research have unravelled numerous new details regarding the pathogen and its interactions with the host that are beginning to find practical applications for patients. Apart from increasing the social standards of the populations that are most affected by high infection rates with M. tuberculosis, it is to be hoped that the next 20 years of TB research will reveal even more secrets of the TB pathogen that can be used in the continued fight against TB. We should not make the mistake of the 1970s again, which would be to underestimate the adaptability of this pathogen, whose evolution is directly linked with human evolution. High-quality TB research will have to continue to cope with today’s situation and find novel innovative solutions for future trends. Financial & competing interests disclosure We acknowledge the support by the Fondation pour la Recherche Médicale FRM grant no. DEQ20130326471, the European Community’s (EC) Framework Programme 7 grant MM4TB 260872 and grant 09 BLAN 0400 01 from the Agence Nationale pour la Recherche (France). F Le Chevalier is supported by the French Region Ile-deFrance (Domaine d’Intérêt Majeur Maladies Infectieuses et Emergentes) PhD program. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

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et al. Mycobacterial Esx-3 is required for mycobactin-mediated iron acquisition. Proc. Natl Acad. Sci. USA 106(44), 18792–18797 (2009). 63 Serafini A, Boldrin F, Palu G, Manganelli R.

Characterization of a Mycobacterium tuberculosis ESX-3 conditional mutant: essentiality and rescue by iron and zinc. J. Bacteriol. 191(20), 6340–6344 (2009). 64 Di Luca M, Bottai D, Batoni G et al. The

ESX-5 associated eccB-eccC Locus is essential for Mycobacterium tuberculosis viability. PLoS ONE 7(12), e52059 (2012). 65 Bottai D, Di Luca M, Majlessi L et al.

Disruption of the ESX-5 system of Mycobacterium tuberculosis causes loss of PPE protein secretion, reduction of cell wall integrity and strong attenuation. Mol. Microbiol. 83(6), 1195–1209 (2012). • Important study that evaluated the impact of the ESX-5 secretion system of M. tuberculosis on protein transport and pathogenicity. 66 Sayes F, Sun L, Di Luca M et al. Strong

Immunogenicity and cross-reactivity of Mycobacterium tuberculosis ESX-5 type VII secretion-encoded PE-PPE proteins predicts vaccine potential. Cell Host Microbe 11(4), 352–363 (2012). • Important study that identifies the immunogenic epitopes of PE and PPE proteins within and outside of the ESX-5 secretion system. 67 Van Der Woude AD, Luirink J, Bitter W.

Getting across the cell envelope: mycobacterial protein secretion. Curr. Top. Microbiol. Immunol. 374, 109–134 (2013). 68 Du Plessis DJ, Nouwen N, Driessen AJ.

The Sec translocase. Biochim. Biophys. Acta 1808(3), 851–865 (2011).

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Review  Le Chevalier, Cascioferro, Majlessi, Herrmann & Brosch 69 Feltcher ME, Gibbons HS, Ligon LS,

Braunstein M. Protein export by the mycobacterial SecA2 system is determined by the preprotein mature domain. J. Bacteriol. 195(4), 672–681 (2013).

the secretion of superoxide dismutase A and in the virulence of Mycobacterium tuberculosis. Mol. Microbiol. 48(2), 453–464 (2003). katG mutations on the virulence of Mycobacterium tuberculosis and the implication for transmission in humans. Infect. Immun. 70(9), 4955–4960 (2002).

72 Feltcher ME, Sullivan JT, Braunstein M.

Protein export systems of Mycobacterium tuberculosis: novel targets for drug development? Future Microbiol. 5(10), 1581–1597 (2010). 73 Berks BC, Sargent F, Palmer T. The Tat

protein export pathway. Mol. Microbiol. 35(2), 260–274 (2000).

Impact of bacterial genetics on the transmission of isoniazid-resistant Mycobacterium tuberculosis. PLoS Pathog. 2(6), e61 (2006). 83 Thillai M, Pollock K, Pareek M, Lalvani A.

Interferon-gamma release assays for tuberculosis: current and future applications. Expert Rev. Respir. Med. 8(1), 67–78 (2014).

75 Raynaud C, Guilhot C, Rauzier J et al.

Phospholipases C are involved in the virulence of Mycobacterium tuberculosis. Mol. Microbiol. 45(1), 203–217 (2002). 76 Pelicic V, Jackson M, Reyrat JM,

Jacobs WR Jr, Gicquel B, Guilhot C. Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis. Proc. Natl Acad. Sci. USA 94(20), 10955–10960 (1997). 77 Bardarov S, Kriakov J, Carriere C et al.

Conditionally replicating mycobacteriophages: a system for transposon delivery to Mycobacterium tuberculosis. Proc. Natl Acad. Sci. USA 94, 10961–10966 (1997). 78 Camacho LR, Ensergueix D, Perez E,

Gicquel B, Guilhot C. Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged transposon mutagenesis. Mol. Microbiol. 34, 257–267 (1999). 79 Cox JS, Chen B, McNeil M,

Jacobs WR Jr. Complex lipid determines tissue-specific replication of Mycobacterium tuberculosis in mice. Nature 402(6757), 79–83 (1999). 80 Braunstein M, Espinosa BJ, Chan J,

Belisle JT, Jacobs WR Jr. SecA2 functions in

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ESX-1 dependent impairment of autophagic flux by Mycobacterium tuberculosis in human dendritic cells. Autophagy 8(9), 1357–1370 (2012). • Important study that reports the cell biological consequences of ESX-1-mediated cytosolic contact of M. tuberculosis. 93 Watson RO, Manzanillo PS, Cox JS.

Extracellular M. tuberculosis DNA targets bacteria for autophagy by activating the host DNA-sensing pathway. Cell 150(4), 803–815 (2012). 

84 Van Der Wel N, Hava D, Houben D et al.

M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells. Cell 129, 1287–1298 (2007). • Landmark study that revises a long-kept scheme of mycobacterial pathogenicity. 85 Simeone R, Bobard A, Lippmann J et al.

Phagosomal rupture by Mycobacterium tuberculosis results in toxicity and host cell death. PLoS Pathog. 8(2), e1002507 (2012).

74 Mcdonough JA, Mccann JR, Tekippe EM,

Silverman JS, Rigel NW, Braunstein M. Identification of functional Tat signal sequences in Mycobacterium tuberculosis proteins. J. Bacteriol. 190(19), 6428–6438 (2008).

92 Romagnoli A, Etna MP, Giacomini E et al.

82 Gagneux S, Burgos MV, Deriemer K et al.

71 Saint-Joanis B, Demangel C, Jackson M et al.

Inactivation of Rv2525c, a substrate of the twin arginine translocation (Tat) system of Mycobacterium tuberculosis, increases beta-lactam susceptibility and virulence. J. Bacteriol. 188(18), 6669–6679 (2006).

Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis. Mol. Microbiol. 51(2), 359–370 (2004).

81 Pym AS, Saint-Joanis B, Cole ST. Effect of

70 Mcdonough JA, Hacker KE, Flores AR,

Pavelka MS, Jr., Braunstein M. The twin-arginine translocation pathway of Mycobacterium smegmatis is functional and required for the export of mycobacterial beta-lactamases. J. Bacteriol. 187(22), 7667–7679 (2005).

91 Guinn KI, Hickey MJ, Mathur SK et al.

•

Important study that confirms and largely extends the hypothesis of phagosomal breakage due to ESX-1-proficient M. tuberculosis and recombinant BCG strains.

86 Hsu T, Hingley-Wilson SM, Chen B et al.

The primary mechanism of attenuation of bacillus Calmette–Guerin is a loss of secreted lytic function required for invasion of lung interstitial tissue. Proc. Natl Acad. Sci. USA 100(21), 12420–12425 (2003). 87 De Jonge MI, Pehau-Arnaudet G, Fretz MM

et al. ESAT-6 from Mycobacterium tuberculosis dissociates from its putative chaperone CFP-10 under acidic conditions and exhibits membrane-lysing activity. J. Bacteriol. 189, 6028–6034 (2007). 88 De Leon J, Jiang G, Ma Y, Rubin E, Fortune S,

Sun J. Mycobacterium tuberculosis ESAT-6 exhibits a unique membrane-interacting activity that is not found in its ortholog from non-pathogenic Mycobacterium smegmatis. J. Biol. Chem. 287(53), 44184–44191 (2012). 89 Houben D, Demangel C, Van Ingen J et al.

ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria. Cell. Microbiol. 14(8), 1287–1298 (2012). 90 Aguilo J, Alonso H, Uranga S et al.

ESX-1-induced apoptosis is involved in cell-to-cell spread of Mycobacterium tuberculosis. Cell. Microbiol. 15(12), 1994–2005 (2013).

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Important study that shows the consequences of mycobacterial DNA release into the cytosol.

94 Mishra BB, Moura-Alves P, Sonawane A et al.

Mycobacterium tuberculosis protein ESAT-6 is a potent activator of the NLRP3/ASC inflammasome. Cell. Microbiol. 12(8), 1046–1063 (2010). 95 Wong KW, Jacobs WR Jr. Critical role for

NLRP3 in necrotic death triggered by Mycobacterium tuberculosis. Cell. Microbiol. 13(9), 1371–1384 (2011). 96 Dorhoi A, Nouailles G, Jorg S et al.

Activation of the NLRP3 inflammasome by Mycobacterium tuberculosis is uncoupled from susceptibility to active tuberculosis. Eur. J. Immunol. 42(2), 374–384 (2012). 97 Stanley SA, Johndrow JE, Manzanillo P,

Cox JS. The type I IFN response to infection with Mycobacterium tuberculosis requires ESX-1-mediated secretion and contributes to pathogenesis. J. Immunol. 178(5), 3143–3152 (2007). 98 Ryan AA, Nambiar JK, Wozniak TM et al.

Antigen load governs the differential priming of CD8 T cells in response to the bacille Calmette Guerin vaccine or Mycobacterium tuberculosis infection. J. Immunol. 182(11), 7172–7177 (2009). 99 Chen JM, Pojer F, Blasco B, Cole ST.

Towards anti-virulence drugs targeting ESX-1 mediated pathogenesis of Mycobacterium tuberculosis. Drug Discov. Today Dis. Mechan. 7(1), e25–e31 (2010). 100 Bottai D, Serafini A, Cascioferro A, Brosch R,

Manganelli R. Targeting type VII/ESX secretion systems for development of novel antimycobacterial drugs. Curr. Pharm. Des. 20(27), 4346–4356 (2013). 101 Fortune SM, Jaeger A, Sarracino DA et al.

Mutually dependent secretion of proteins

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Mycobacterium tuberculosis evolutionary pathogenesis & its putative impact on drug development  required for mycobacterial virulence. Proc. Natl Acad. Sci. USA 102(30), 10676–10681 (2005). 102 Macgurn JA, Raghavan S, Stanley SA, Cox JS.

A non-RD1 gene cluster is required for Snm secretion in Mycobacterium tuberculosis. Mol. Microbiol. 57(6), 1653–1663 (2005). 103 Raghavan S, Manzanillo P, Chan K,

Dovey C, Cox J. Secreted transcription factor controls Mycobacterium tuberculosis virulence. Nature 454(7205), 717–721 (2008). 104 Blasco B, Chen JM, Hartkoorn R et al.

Virulence regulator EspR of Mycobacterium tuberculosis is a nucleoid-associated protein. PLoS Pathog. 8(3), e1002621 (2012). • Very interesting study that decribes the mechanism of the virulence regulator EspR, which regulates the secretion of several important antigens and virulence factors of M. tuberculosis. 105 Frigui W, Bottai D, Majlessi L et al. Control

of M. tuberculosis ESAT-6 secretion and specific T cell recognition by PhoP. PLoS Pathog. 4(2), e33 (2008). 106 Pang X, Samten B, Cao G et al. MprAB

regulates the espA operon in Mycobacterium tuberculosis and modulates ESX-1 function and host cytokine response. J. Bacteriol. 195(1), 66–75 (2013). 107 Perez E, Samper S, Bordas Y, Guilhot C,

Gicquel B, Martin C. An essential role for

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phoP in Mycobacterium tuberculosis virulence. Mol. Microbiol. 41(1), 179–187 (2001). 108 Walters SB, Dubnau E, Kolesnikova I,

Laval F, Daffe M, Smith I. The Mycobacterium tuberculosis PhoPR two-component system regulates genes essential for virulence and complex lipid biosynthesis. Mol. Microbiol. 60(2), 312–330 (2006). 109 Hunt DM, Sweeney NP, Mori L et al.

Long-range transcriptional control of an operon necessary for virulence-critical ESX-1 secretion in Mycobacterium tuberculosis. J. Bacteriol. 194(9), 2307–2320 (2012). 110 Houben EN, Bestebroer J, Ummels R et al.

Composition of the type VII secretion system membrane complex. Mol. Microbiol. 86(2), 472–484 (2012). • Highly interesting study that focuses on the copurification of core proteins of type VII secretion machineries. 111 Bottai D, Brosch R. Mycobacterial PE, PPE and

ESX clusters: novel insights into the secretion of these most unusual protein families. Mol. Microbiol. 73(3), 325–328 (2009). 112 Demangel C, Brodin P, Cockle PJ et al. Cell

envelope protein PPE68 contributes to Mycobacterium tuberculosis RD1 immunogenicity independently of a 10-kilodalton culture filtrate protein and ESAT-6. Infect. Immun. 72(4), 2170–2176 (2004).

Review

113 Propst KL, Troyer RM, Kellihan LM,

Schweizer HP, Dow SW. Immunotherapy markedly increases the effectiveness of antimicrobial therapy for treatment of Burkholderia pseudomallei infection. Antimicrob. Agents Chemother. 54(5), 1785–1792 (2010). 114 Brodin P, Poquet Y, Levillain F et al. High

content phenotypic cell-based visual screen identifies Mycobacterium tuberculosis acyltrehalose-containing glycolipids involved in phagosome remodeling. PLoS Pathog. 6(9), e1001100 (2010). • Highly interesting study that shows how high-throughput cell-based screening may be used to identify potential pathogenicity factors. The same technology might be useful for identifying compounds that can interfere with important host–pathogen interaction steps. 115 Roetzer A, Diel R, Kohl TA et al. Whole

genome sequencing versus traditional genotyping for investigation of a Mycobacterium tuberculosis outbreak: a longitudinal molecular epidemiological study. PLoS Med. 10(2), e1001387 (2013). 116 Bryant JM, Harris SR, Parkhill J et al.

Whole-genome sequencing to establish relapse or re-infection with Mycobacterium tuberculosis: a retrospective observational study. Lancet Respir. Med. 1(10), 786–792 (2013).

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