Mycobacterium Tuberculosis Evolutionary Pathogenesis and its Putative Impact on Drug DevelopmentFabien Le Chevalier, Alessandro Cascioferro, Laleh Majlessi, Jean Louis Herrmann, Roland Brosch
Future Microbiol. 2014;9(8):969-985.
http://www.medscape.com/viewarticle/833628
Abstract and IntroductionAbstract
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 putativenew drug targets that are essential for mycobacterial growth under in vitro or in vivo conditions.
Introduction
Pathogenic mycobacteria are important causes of human and animal disease. Despite the availability of antibiotics andchemotherapeutic 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 genusMycobacterium 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 asStaphylococcus 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, namedthe mycomembrane, which is specific to mycobacteria and might fulfill a similar barrier function as the outer membrane of Gram-negative bacteria.[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, fast-growing 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 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 polymorphisms 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 ref-12 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. canettiiand M. tuberculosis can now be further investigated for their impact on the different phenotypes of the concerned phylogenetically diverging tubercle bacilli.
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].
Figure 2.
Phylogenetic organization and gene content of members of the genus Mycobacterium . (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 Mycobacteriumwith 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 speciesis 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].
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 explanationfor 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 visible in the only 3.2-Mb genome of the obligateintracellular 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 coughing-up 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 andglobal 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. TuberculosisWithin 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 recentresearch into novel strategies against the pathogen.[12] After several anti-TB drugs were developed in the mid-20th century, there has been a long gap in which no new molecules to be used in the treatment of TBwere 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 usedM. 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 useof high-density 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 discussedin a later section. Most recently, saturation transposon mutagenesis of M. tuberculosis 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 TargetsThe 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 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 theregulation 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 ( & )[36] that may all be subject to resistance mechanisms in extensively and/or totally resistant M. tuberculosis strains.[37]
Table 1. First- and second-line anti-TB drugs used in standard treatment regiments.
Anti-TB drug Principle target(s)
Isoniazid InhA; KatG (activation)
Rifampicin RpoB
Ethambutol EmbB
Pyrazinamide RpsA; PncA (activation)
Streptomycin RpsL
Fluoroquinolones GyrA
Para-aminosalicylic acid Dihydropteroate synthase; folate metabolism
Injectable agents
Amikacin, kanamycin or capreomycin 30S ribosomal subunit or interbridge
Ethionamide InhA; EthA (activation)
Cycloserine D-alanine racemase and ligase
Data taken from [36,38].
Table 2. Recommended anti-TB drug regimens; including details of the initial phase and continuation
phase.
Type of TB cases
Total duration oftreatment
Duration of initial phase
Drug combination during initial phase
Duration of continued
Drug combination during
(months) (months)phase (months)
continued phase
New smear- or culture-positive cases
6 2Isoniazid, rifampicin, pyrazinamide and ethambutol
4Isoniazid and rifampicin
New culture-negative cases
4 2Isoniazid, rifampicin, pyrazinamide and ethambutol
2Isoniazid and rifampicin
Resistance (or intolerance) to isoniazid
6 6rifampicin, pethambutol
– –
Resistance to isoniazid and rifampicin
12–18 12–18
Pyrazinamide, ethambutol, fluoroquinolones and streptomycin (or another injectable agent)
– –
Resistance to all first-linedrugs
24 24
One injectable agent + three drugs from thefollowing group: ethionamide, cycloserine, fluoroquinolones or para-aminosalicylic acid
– –
Data taken from [36,38].
Screening and target identification studies allowed the diarylquinoline drug TMC207 to be discovered, for example, for which resistant mutants showed single-nucleotide polymorphisms in the gene atpE encodingthe 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 (DNref-1), 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 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 asthe 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 aprodrug 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 isoniazid-resistant M. tuberculosisstrains, 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, pyrazinamideand 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 ofthe process of trans-translation by ribosomal protein S1, which is essential for freeing scarce ribosomes in nonreplicating organisms.[57] For para-aminosalicylic 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 TargetsScreening of different classes of compounds with antitubercular activity combined with sequence analysisand 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 essentialfor 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 zincuptake.[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 inactivatecomponents 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 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]
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 oftwo of the five ESX systems (ESX-1 and ESX-5), as well as the components of the TAT system. The genenomenclature corresponds to that of the reference strain Mycobacterium tuberculosis H37Rv. (B) A modelof 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].
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, thisdoes 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 in vivo drug targetfor 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 inthe 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 that would inhibit these oxidative stress defense mechanisms of M. tuberculosis might strongly impact on the fitness and transmissibility of the pathogen, as suggested by virulence studies on KatG-deletion mutants in the context of isoniazid resistance.[81,82]
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 livevaccine. 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 ESX-1-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 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 cross-presentation 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 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, whichis 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. Forexample, 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 thusshow 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]
ConclusionMycobacteria 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 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 PerspectiveIn 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 (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] NGSalso 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 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 importancebeing 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 theoutcome 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-trehalose-containing glycolipids involvedin 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 ofthe 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, 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 moleculesthat 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.
SidebarExecutive 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.
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).
2. Aitken ML, Limaye A, Pottinger P et al. Respiratory outbreak of Mycobacterium abscessus subspecies massiliense in a lung transplant and cystic fibrosis center. Am. J. Respir. Crit. Care Med. 185(2), 231–232 (2012).
3. Pawlik A, Garnier G, Orgeur M et al. Identification and characterization of the genetic changes responsible for the characteristic smooth-to-rough morphotype alterations of clinically persistent Mycobacterium abscessus. Mol. Microbiol.90(3), 612–629 (2013).
4. Bryant JM, Grogono DM, Greaves D et al. Whole-genome sequencing to identify transmission of Mycobacterium abscessus between patients with cystic fibrosis: a retrospective cohort study. Lancet 381(9877), 1551–1560 (2013).
5. Shang S, Gibbs S, Henao-Tamayo M et al. Increased virulence of an epidemic strain of Mycobacterium massiliense in mice. PLoS ONE 6(9), e24726 (2011).
6. Nessar R, Cambau E, Reyrat JM, Murray A, Gicquel B. Mycobacterium abscessus: a new antibiotic nightmare. J. Antimicrob. Chemother. 67(4), 810–818 (2012).
7. Catherinot E, Roux AL, Vibet MA et al. Inhaled therapies, azithromycin and Mycobacterium abscessus in cystic fibrosis patients. Eur. Respir. J. 41(5), 1101–1106 (2013).
8. Maurer FP, Ruegger V, Ritter C, Bloemberg GV, Bottger EC. Acquisition of clarithromycin resistance mutations in the 23S rRNA gene of Mycobacterium abscessus in the presence of inducible erm(41). J. Antimicrob. Chemother. 67(11), 2606–2611 (2012).
9. Springer B, Stockman L, Teschner K, Roberts GD, Bottger EC. Two-laboratory collaborative studyon identification of mycobacteria: molecular versus phenotypic methods. J. Clin. Microbiol. 34(2), 296–303 (1996).
10. Cole ST, Eiglmeier K, Parkhill J et al. Massive gene decay in the leprosy bacillus. Nature 409(6823), 1007–1011 (2001).
11. Cole ST, Brosch R, Parkhill J et al. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393(6685), 537–544 (1998).
12. Gordon SV, Bottai D, Simeone R, Stinear TP, Brosch R. Pathogenicity in the tubercle bacillus: molecular and evolutionary determinants. Bioessays 31(4), 378–388 (2009).
13. Stinear TP, Seemann T, Harrison PF et al. Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis. Genome Res. 18(5), 729–741 (2008).
14. Veyrier FJ, Dufort A, Behr MA. The rise and fall of the Mycobacterium tuberculosis genome. Trends Microbiol. 19(4), 156–161 (2011).
* Reviews the evolutionary processes that could have led to the evolution of pathogenicity of slow-growing mycobacteria, including Mycobacterium tuberculosis.
15. Kaur D, Guerin ME, Skovierova H, Brennan PJ, Jackson M. Chapter 2: biogenesis of the cell wall and other glycoconjugates of Mycobacterium tuberculosis. Adv. Appl. Microbiol. 69, 23–78 (2009).
16. Zuber B, Chami M, Houssin C, Dubochet J, Griffiths G, Daffe M. Direct visualization of the outer membrane of mycobacteria and corynebacteria in their native state. J. Bacteriol. 190(16), 5672–5680 (2008).
17. Hoffmann C, Leis A, Niederweis M, Plitzko JM, Engelhardt H. Disclosure of the mycobacterial outer membrane: cryo-electron tomography and vitreous sections reveal the lipid bilayer structure. Proc. Natl Acad. Sci. USA 105(10), 3963–3967 (2008).
18. Sambou T, Dinadayala P, Stadthagen G et al. Capsular glucan and intracellular glycogen of Mycobacterium tuberculosis: biosynthesis and impact on the persistence in mice. Mol. Microbiol. 70(3), 762–774 (2008).
19. Sani M, Houben EN, Geurtsen J et al. Direct visualization by cryo-EM of the mycobacterial capsular layer: a labile structure containing ESX-1-secreted proteins. PLoS Pathog. 6(3), e1000794 (2010).
20. Jackson M, McNeil MR, Brennan PJ. Progress in targeting cell envelope biogenesis in Mycobacterium tuberculosis.Future Microbiol. 8(7), 855–875 (2013).
* Describes the different components of the mycobacterial cell wall, taking into account the advances of numerous research projects.
21. Ripoll F, Pasek S, Schenowitz C et al. Non mycobacterial virulence genes in the genome of the emerging pathogenMycobacterium abscessus. PLoS ONE 4(6), e5660 (2009).
22. Gey Van Pittius NC, Sampson SL, Lee H, Kim Y, Van Helden PD, Warren RM. Evolution and expansion of theMycobacterium tuberculosis PE and PPE multigene families and their association with the duplication of the ESAT-6 (esx) gene cluster regions. BMC Evol. Biol. 6, 95 (2006).
23. Supply P, Marceau M, Mangenot S et al. Genomic analysis of smooth tubercle bacilli provides insights into ancestry and pathoadaptation of Mycobacterium tuberculosis. Nat. Genet. 45, 172–179 (2013).
* Reports and discusses the ancestral gene pool of tubercle bacilli (Mycobacterium canettii strains) from which M. tuberculosis has emerged and pinpoints genes that seem to have been acquired by M. tuberculosis through horizontal transfer. It remains to be determined whetherthese genes have contributed to the evolutionary success of M. tuberculosis as a very widespread key pathogen.
24. Gopinath K, Moosa A, Mizrahi V, Warner DF. Vitamin B(12) metabolism in Mycobacterium tuberculosis. Future Microbiol. 8(11), 1405–1418 (2013).
25. Genomes Online Database (GOLD). http://genomesonline.org.
26. Dye C, Williams BG. The population dynamics and control of tuberculosis. Science 328(5980), 856–861 (2010).
27. Gutierrez MC, Brisse S, Brosch R et al. Ancient origin and gene mosaicism of the progenitor of Mycobacterium tuberculosis. PLoS Pathog. 1(1), e5 (2005).
28. Comas I, Coscolla M, Luo T et al. Out-of-Africa migration and Neolithic coexpansion of Mycobacterium tuberculosis with modern humans. Nat. Genet. 45(10), 1176–1182 (2013).
29. Sassetti CM, Boyd DH, Rubin EJ. Genes required for mycobacterial growth defined by high density mutagenesis. Mol. Microbiol. 48(1), 77–84 (2003).
30. Sassetti CM, Rubin EJ. Genetic requirements for mycobacterial survival during infection. Proc. Natl Acad. Sci. USA100(22), 12989–12994 (2003).
31. Griffin JE, Gawronski JD, Dejesus MA, Ioerger TR, Akerley BJ, Sassetti CM. High-resolution phenotypic profiling defines genes essential for mycobacterial growth and cholesterol catabolism. PLoS Pathog. 7(9), e1002251 (2011).
* Key study that refines the previous transposon site hybridization-based analyses to the level of essential regions of proteins.
32. Fernandez P, Saint-Joanis B, Barilone N et al. The Ser/Thr protein kinase PknB is essential to sustain mycobacterial growth. J. Bacteriol. 188, 7778–7784 (2006).
33. Forti F, Crosta A, Ghisotti D. Pristinamycin-inducible gene regulation in mycobacteria. J. Biotechnol. 140(3–4), 270–277 (2009).
34. Lougheed KE, Osborne SA, Saxty B et al. Effective inhibitors of the essential kinase PknB and their potential as anti-mycobacterial agents. Tuberculosis (Edinb.) 91(4), 277–286 (2011).
35. Chapman TM, Bouloc N, Buxton RS et al. Substituted aminopyrimidine protein kinase B (PknB) inhibitors show activity against Mycobacterium tuberculosis. Bioorg. Med. Chem. Lett. 22(9), 3349–3353 (2012).
36. Raviglione MC, O'Brien RJ. Tuberculosis. In: Harrison's Principles of Internal Medicine (17th Edition). Fauci AS, Braunwald E, Kasper DL et al. (Eds). McGraw-Hill, Inc., NY, USA, 1006–1020 (2008).
37. Klopper M, Warren RM, Hayes C et al. Emergence and spread of extensively and totally drug-resistant tuberculosis, South Africa. Emerg. Infect. Dis. 19(3), 449–455 (2013).
38. Zumla A, Nahid P, Cole ST. Advances in the development of new tuberculosis drugs and treatment regimens. Nat. Rev. Drug Discov. 12(5), 388–404 (2013).
39. Andries K, Verhasselt P, Guillemont J et al. A diarylquinoline drug active on the ATP synthase of Mycobacterium tuberculosis. Science 307(5707), 223–227 (2005).
* Landmark study presenting the first entirely new anti-TB compound after many years of quiescence, which is now in clinical use for treatment against multiresistant TB.
40. Diacon AH, Pym A, Grobusch M et al. The diarylquinoline TMC207 for multidrug-resistant tuberculosis. N. Engl. J. Med.360(23), 2397–2405 (2009).
41. Makarov V, Manina G, Mikusova K et al. Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis. Science 324(5928), 801–804 (2009).
* Seminal study presenting new, highly active compounds and a vulnerable target that had not been found before. Compounds of the same chemical scaffold are being developed for clinical use.
42. Lechartier B, Rybniker J, Zumla A, Cole ST. Tuberculosis drug discovery in the post-post-genomicera. EMBO Mol. Med.6(2), 158–168 (2014).
43. Christophe T, Jackson M, Jeon HK et al. High content screening identifies decaprenyl-phosphoribose 2' epimerase as a target for intracellular antimycobacterial inhibitors. PLoS Pathog. 5(10), e1000645 (2009).
44. Pethe K, Bifani P, Jang J et al. Discovery of Q203, a potent clinical candidate for the treatment of tuberculosis. Nat. Med. 19(9), 1157–1160 (2013).
45. Grzegorzewicz AE, Pham H, Gundi VA et al. Inhibition of mycolic acid transport across the Mycobacterium tuberculosisplasma membrane. Nat. Chem. Biol. 8(4), 334–341 (2012).
46. Tahlan K, Wilson R, Kastrinsky DB et al. SQ109 targets MmpL3, a membrane transporter of trehalose monomycolate involved in mycolic acid donation to the cell wall core of Mycobacterium tuberculosis. Antimicrob. Agents Chemother.56(4), 1797–1809 (2012).
47. Stover CK, Warrener P, Vandevanter DR et al. A small-molecule nitroimidazopyran drug candidate for the treatment of tuberculosis. Nature 405(6789), 962–966 (2000).
48. Manjunatha U, Boshoff HI, Barry CE. The mechanism of action of PA-824: novel insights from transcriptional profiling.Commun. Integr. Biol. 2(3), 215–218 (2009).
49. Wilson R, Kumar P, Parashar V et al. Antituberculosis thiophenes define a requirement for Pks13 in mycolic acid biosynthesis. Nat. Chem. Biol. 9(8), 499–506 (2013).
50. Portevin D, De Sousa-D'auria C, Houssin C et al. A polyketide synthase catalyzes the last condensation step of mycolic acid biosynthesis in mycobacteria and related organisms. Proc. Natl Acad. Sci. USA 101(1), 314–319 (2004).
51. Grosset JH, Singer TG, Bishai WR. New drugs for the treatment of tuberculosis: hope and reality. Int. J. Tuberc. Lung Dis. 16(8), 1005–1014 (2012).
52. Lee M, Lee J, Carroll MW et al. Linezolid for treatment of chronic extensively drug-resistant tuberculosis. N. Engl. J. Med. 367(16), 1508–1518 (2012).
* Describes the clinical use of a relatively new drug in conjunction with other treatment regimens and the life-saving treatment advantages gained for patients.
53. Williams K, Minkowski A, Amoabeng O et al. Sterilizing activities of novel combinations lacking first- and second-line drugs in a murine model of tuberculosis. Antimicrob. Agents Chemother. 56(6), 3114–3120 (2012).
54. Hartkoorn RC, Pojer F, Read JA et al. Pyridomycin bridges the NADH- and substrate-binding pockets of the enoyl reductase InhA. Nat. Chem. Biol. 10(2), 96–98 (2013).
55. Hartkoorn RC, Sala C, Neres J et al. Towards a new tuberculosis drug: pyridomycin – nature's isoniazid. EMBO Mol. Med. 4(10), 1032–1042 (2012).
* Very carefully executed study on the mechanisms of action of an almost-forgotten anti-TB drug.
56. Willand N, Dirie B, Carette X et al. Synthetic EthR inhibitors boost antituberculous activity of ethionamide. Nat. Med.15(5), 537–544 (2009).
57. Shi W, Zhang X, Jiang X et al. Pyrazinamide inhibits trans-translation in Mycobacterium tuberculosis. Science333(6049), 1630–1632 (2011).
58. Chakraborty S, Gruber T, Barry CE 3rd, Boshoff HI, Rhee KY. Para-aminosalicylic acid acts as an alternative substrate of folate metabolism in Mycobacterium tuberculosis. Science 339(6115), 88–91 (2013).
59. Ioerger TR, O'Malley T, Liao R et al. Identification of new drug targets and resistance mechanisms in Mycobacterium tuberculosis. PLoS ONE 8(9), e75245 (2013).
* Presents a list of new potential drug targets that have recently been identified.
60. Bitter W, Houben EN, Bottai D et al. Systematic genetic nomenclature for type VII secretion systems. PLoS Pathog.5(10), e1000507 (2009).
61. Simeone R, Bottai D, Brosch R. ESX/type VII secretion systems and their role in host–pathogen interaction. Curr. Opin. Microbiol. 12(1), 4–10 (2009).
62. Siegrist MS, Unnikrishnan M, Mcconnell MJ 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 tuberculosisviability. 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).
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).
70. Mcdonough JA, Hacker KE, Flores AR, Pavelka MS, Jr., Braunstein M. The twin-arginine translocation pathway ofMycobacterium smegmatis is functional and required for the export of mycobacterial beta-lactamases. J. Bacteriol.187(22), 7667–7679 (2005).
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).
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).
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).
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 the secretion ofsuperoxide dismutase A and in the virulence of Mycobacterium tuberculosis. Mol. Microbiol. 48(2),453–464 (2003).
81. Pym AS, Saint-Joanis B, Cole ST. Effect of katG mutations on the virulence of Mycobacterium tuberculosis and the implication for transmission in humans. Infect. Immun. 70(9), 4955–4960 (2002).
82. Gagneux S, Burgos MV, Deriemer K et al. Impact of bacterial genetics on the transmission of isoniazid-resistantMycobacterium 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).
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).
* 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).
91. Guinn KI, Hickey MJ, Mathur SK et al. 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).
92. Romagnoli A, Etna MP, Giacomini E et al. 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).
* 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 tuberculosisrequires 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 ofMycobacterium 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 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 tuberculosisvirulence. 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 phoP in Mycobacterium tuberculosisvirulence. 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).
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 phagosomeremodeling. 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 genotypingfor investigation of aMycobacterium 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).
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 andgrant 09 BLAN 0400 01 from the Agence Nationale pour la Recherche (France). F Le Chevalier is supported by the French Region Ile-de-France (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.
Future Microbiol. 2014;9(8):969-985. © 2014 Future Medicine Ltd.
