Citation: 

Singh P, Tufariello J, Wattam AR, Gillis TP, Jacobs Jr WR. 3 April 2018, posting date. Genomics insights into the biology and evolution of leprosy bacilli. In Scollard DM, Gillis TP (ed), International textbook of leprosy. www.internationaltextbookofleprosy.org.

Funding: 
No funding has been declared.
Competing Interests: 
No competing interests have been stated.

Introduction

General introduction

Leprosy is a chronic granulomatous disease that affects the peripheral nerves, skin, and eyes. It was strongly believed to be a hereditary disease until 1873, when a young Norwegian physician, Gerhard Armauer Hansen, demonstrated the causative bacterium of the disease. The pathogen, known as Mycobacterium leprae, has since defied all efforts to cultivate it in any artificial medium, thereby limiting traditional microbiological investigations. The availability of a very effective multi-drug therapy (MDT; see Chapter 2.6; Chapter 5.2) for over three decades has drastically reduced the prevalence of the disease; however, it has also facilitated the notion held by the general public and health policymakers that it is a disease of the past. This notion is clearly not the case, as new case detection rates of leprosy have stubbornly remained at or near 200,000 cases annually over the past decade [1]. The persistent rate of new cases indicates that transmission has not been completely interrupted by MDT and that a fuller understanding of transmission (see Chapter 1.2) is required to fashion strategies aimed at reaching zero transmission of M. leprae and eventually eliminating leprosy. Genomics has contributed several important clues and molecular tools for sensitively and accurately detecting M. leprae, monitoring its transmission dynamics, and determining its susceptibility to anti-leprosy drugs.

While attempts to cultivate M. leprae in vitro have been unproductive, Shepard’s report of limited multiplication of M. leprae in the foot pads of mice [2] (see Chapter 10.3) and Kirchheimer and Storrs’ subsequent successful experimental propagation of M. leprae in armadillos (see Chapter 10.2) in 1971 allowed the recovery of huge quantities of M. leprae [3] for in-depth study. Armadillos have proven to be an excellent animal model for leprosy, as leprosy in armadillos closely resembles leprosy in humans, including neurological involvement and the spectrum of immunological responses to infection [4], [5], [6], [7], [8]. The availability of M. leprae purified from armadillo tissues enabled the construction of libraries for whole genome sequencing [9], [10]. The first of these sequencing projects was completed using the M. leprae TN strain [11], which was originally isolated from a multibacillary (MB) patient from Tamil Nadu, India, and subsequently propagated in armadillos.

The TN genome provided evidence that M. leprae exhibited exceptional reductive evolution, with a record number of pseudogenes in comparison with any known bacterium. Genomic analysis of M. leprae has explained why some drugs work against M. leprae and others do not based upon the presence or absence of their intact genomic/proteomic targets [12]. Genomic analysis has also suggested some explanations for the extremely slow growth rate of M. leprae, which may be related to its very long incubation period prior to clinical disease. Finally, genomic analysis has provided possible explanations for past cultivation failures, as several key metabolic and stress-response proteins and enzymes either were missing or were present in the form of pseudogenes. Using these genomic insights, it has become possible to design more logical and informed experiments to improve our understanding of the unique biology and pathogenesis of M. leprae.

Diverse clinical spectrum

Leprosy manifests a clinical (see Chapter 2.1; Chapter 2.4) and immunological (see Chapter 6.1; Chapter 6.2) spectrum. At one end of this spectrum are the tuberculoid leprosy (TT) cases with strong cell-mediated immunity (CMI) to M. leprae antigens, scanty or undetectable bacilli, and little to no antibodies to M. leprae antigens. The disease at the other end of the spectrum, referred to as lepromatous leprosy (LL), is characterized by numerous acid-fast bacilli and a strong antibody response to M. leprae antigens. There are also three borderline, often unstable, clinical forms (borderline lepromatous [BL], mid-borderline [BB], and borderline tuberculoid [BT]) of leprosy. Sudden changes in host immunity can cause inflammatory responses, collectively known as reactions. All of these clinical manifestations have been ascribed to host immunologic factors with little or no emphasis on the etiological agent’s virulence and pathogenesis.

M. leprae strains from around the world present strikingly low levels of genetic diversity [13]. However, the M. leprae strains that have been studied and characterized in detail have all been isolated from MB cases at the LL end of the spectrum. Whether pathological variants of M. leprae exist remains unknown, but their existence has been implied by the observation that certain M. leprae genotypes show frame-shift inactivation of the transcriptional regulator gene ML0825c [14]. The ortholog of this regulator gene in M. tuberculosis rv2358 contributes to the zinc-dependent repression of several genes, including virulence-associated secretion system genes esxG and esxH [15], [16]. Thus, further genomic investigations into the differential pathogenesis of various M. leprae strains and the adequate representation of M. leprae genotypes from the tuberculoid end of the disease spectrum may prove informative.

Discovery of M. lepromatosis

In addition to the clinical presentations and reactions mentioned above, a particular form of leprosy known as diffuse lepromatous leprosy (DLL) has been described. Cases of DLL have been reported since 1852, primarily in Mexico and the Caribbean countries [17]. DLL cases lack dermal nodules and are often characterized by the diffuse infiltration of skin by histiocytes and acid-fast bacilli, giving the skin a spotted appearance referred to as “pure and primitive diffuse lepromatosis (PPDL)”. Some of these cases develop acute skin reactions known as “erythema necroticans”, also called “Lucio’s Phenomenon” [18]. Originally thought to be geographically restricted to Mexico, Costa Rica, and the Caribbean region, there have been reports of DLL in Singapore, Canada [19], [20], Brazil [21], [22], [23], India [24], [25], [26], Iran [27], Malaysia [28], and Myanmar [21]. Such cases often involve diffuse mycobacterial invasion of endothelial cells surrounding small blood vessels, which leads to a progressive blockade of blood and oxygen supply. The blockade results in initially cyanotic lesions that gradually become necrotic (see Chapter 2.4).

M. leprae has been considered the exclusive causative agent of all types of leprosy, including DLL and Lucio’s phenomenon. However, despite enormous numbers of acid-fast bacilli in DLL and Lucio’s phenomenon cases, some have been found to be negative for M. leprae DNA by PCR, creating confusion about the diagnosis and etiology of this condition. This confusion became evident upon identification of a new mycobacterial species, M. lepromatosis, in two Mexican patients with DLL [19]. The samples were negative for the M. leprae-specific repetitive sequence, RLEP, indicating the absence of M. leprae. Cloning of a few selected genes (rpoB, 16S rDNA, gyrA, etc.) [19], [29] from the bacteria present in infected tissues revealed that their sequences exhibited marked nucleotide differences (only ~90% sequence similarity) from corresponding M. leprae sequences. Phylogenetically, these sequences clustered tightly with those of M. leprae when compared to other mycobacterial species [29].

The first independent confirmation of this species in a DLL case was reported in 2011 in an archived specimen from a Mexican patient from Monterrey. During drug resistance surveillance studies, the smear-positive sample was consistently found to be negative for M. leprae using RLEP, folP1, and gyrA templates. rpoB testing, however, produced a faintly positive reaction and DNA sequencing of the PCR product showed perfect alignment with the M. lepromatosis rpoB sequences [30] reported by Han et al. [19], [29]. Recognition of this agent as a new species causing a leprosy-like condition remained unclear [31] due to both the limited pathological evidence available and the reports of coinfection with M. leprae in several cases [32].

Subsequently, the DNA of M. lepromatosis was enriched (by removing human DNA using biotinylated baits and by capturing the pathogen DNA using probes spanning the M. leprae genome on an array) directly from a patient skin biopsy (coded as Mx1-22A). This enrichment enabled a genome-wide analysis of the bacterium, making it the first unsequenced mycobacterial genome to be deduced using this approach. The de novo assembly of these sequences yielded several contigs. Alignment of the M. lepromatosis genome assembly with the M. leprae TN genome showed near perfect colinearity and synteny with almost identical genome size, G+C content, and gene/pseudogene content. In spite of the similarities between M. leprae and M. lepromatosis, a deep evolutionary divergence is evident, supporting the claim that they are separate species. This conclusion stems from the fact that, in contrast to M. leprae isolates from around the world that exhibit only a few hundred SNPs (single nucleotide polymorphisms), the M. lepromatosis genome differs from the M. leprae strains at over a quarter of a million bases (i.e., >275,000 SNPs between M. leprae and M. lepromatosis). With a substitution rate of 7.67−9substitutions per site per year, the most recent common ancestor (TMRCA) for M. leprae and M. lepromatosis has been estimated to be 13.9 million years ago. Despite such a deep evolutionary divergence, both species share remarkable similarities in their biology and, in fact, remained indistinguishable for over a century. Though at present there is a paucity of well-described clinical presentations caused by M. lepromatosis, it is evident that it is not the sole cause of DLL or Lucio’s phenomenon. More detailed investigations using accurate and species-specific methods for studying the distribution of these two organisms and associated clinical presentation in a representative number of cases will therefore be informative.

The most recent identification of an animal reservoir for M. lepromatosis was the red squirrel in Scotland [33]. Subsequent investigations revealed the presence of M. leprae in England (Brownsea Island) and M. lepromatosis in Ireland and on the Isle of Wight in these animals [34]. The whole genome comparison of the red squirrel-derived M. leprae strain exhibited striking similarity with the ancient M. leprae strain from a medieval England human leprosy skeleton [34]. After the 16thcentury, leprosy cases declined in the UK and most of Europe and there have been almost no human leprosy cases identified there since. These observations underscore the significance of the possibility of additional animal reservoirs, even in countries where there have been almost no human cases for the past 3–4 centuries. Accordingly, the search for animal reservoirs needs to be encouraged, particularly in endemic countries where new case detection rates remain stubbornly high despite the implementation of effective chemotherapy.

Genome-Wide Comparison of M. leprae and M. lepromatosis

The M. leprae TN genome, sequenced in 2001 [11], was among the first genomes completed within the Mycobacterium genus, second only to M. tuberculosis H37Rv [35]. Compared to the 4.4 Mb genome of M. tuberculosis, the M. leprae genome, at 3.26 Mb, presented extensive reductive evolution in terms of genome downsizing, loss of coding capacity, and accumulation of a record number of pseudogenes since the time of their divergence from a common ancestor [11]. The M. lepromatosis Mx1-22A genome also exhibited remarkable similarities with the M. leprae genome in terms of genome downsizing and reductive evolution. Only half of the genome in M. leprae and in M. lepromatosis codes for functional genes (compared to over 90% in M. tuberculosis), while the rest mainly harbors pseudogenes and gene remnants whose intact counterparts are present in M. tuberculosis. As in M. leprae, the M. lepromatosis genome exhibits evidence of metabolic streamlining and loss of redundancy with shrinkage of all functional categories. All of these similarities point to the shared evolutionary history of M. leprae and M. lepromatosis. In addition, the displaced and inverted position of the prolyl tRNA synthetase gene (proS) in M. leprae and M. lepromatosis, in comparison to that of the prokaryotic type proS gene in M. tuberculosis, points to their eukaryotic origin through a recent horizontal transfer from a eukaryotic host [11]. It is interesting that the proS gene of M. leprae and M. lepromatosis shows only a 27% amino acid identity with M. tuberculosis but a 44.5% amino acid identity with parts of the human proS gene. The M. haemophilum proS exhibits over 91% amino acid identity with the corresponding sequences in M. leprae and M. lepromatosis, and thus points to its acquisition from a eukaryotic source in M. haemophilum as well.

A summary of the genome features of M. leprae and M. lepromatosis is presented in Table 1 and contrasted with other closely related mycobacterial pathogens.

TABLE 1 Salient features of M. leprae and M. lepromatosis genomes and their comparison with other mycobacterial pathogens

 

M.
leprae

TN

M. lepromatosis
Mx1-22A

M. haemophilum
DSM44634

M. tuberculosis
H37Rv

M.
marinum

M

M.
ulcerans

Agy99

Reference

[11]

[36]

[37]

[35]

[38]

[39]

Genome size (Chromosomal DNA Mb)

3.27

~3.21§

4.23

4.41

6.64

5.63

Extrachromo-somal DNA (Plasmid)

None

None

NoneΨ

None

1#
pMM23

1##
pMUM001

G+C content (%)

57.79

57.89

63.9

65.6

66.08

65.72

Protein-coding sequences (CDS)

1614

1477

3750

3,998

5426

4,288

Pseudogenes

1306*

1334

226

30

57

771

Gene density (bp/gene)

2036

2171

1052

1103

….

1,313

Average gene length (bp)

1009

~1044

948

1006

1,097

961

Protein coding percentage

49.6

~49

93.1

91.2

90%

73.2%

tRNAs

45

45

45

45

46

45

rRNA genes

3

3

3

3

3

3

rRNA operon

1

1

1

1

1

1

* Initial annotation reported 1116 pseudogenes. Upon the availability of other mycobacterial genomes for comparison, another 177 pseudogenes were subsequently identified [40].

§The 3,206,741 bases are in the form of 126 contigs; later on another draft genome was also announced [41].

ΨNone detected in the WGS project, though the possibility of low copy number plasmid is not fully ruled out.

#pMM23 is a 23.317 kb plasmid and possesses 29 genes and a G+C content of 67.86 %.

##pMUM001 is a 174.155 kb plasmid and possesses 81 genes and a G+C content of 62.77%.

Note: Due to different releases of various databases and genome (re)annotations, some of the features listed above may vary slightly in different sources.

Comparison of Gene and Pseudogene Content

The M. lepromatosis gene and pseudogene content is strikingly similar to that in M. leprae (Table 1). This similarity points to a shared evolutionary history of reductive evolution. M. leprae and M. lepromatosis share 1349 CDS (coding sequences) and 1016 pseudogenes. In addition to this shared pool of CDS, M. leprae possesses an additional 132 CDS whose counterparts exist as pseudogenes in M. lepromatosis. However, a majority of them are annotated as conserved hypothetical proteins, with a few exceptions. The exceptions are ilvX, proA, cysE, and cysK, all of which are required for amino acid biosynthesis in M. leprae, implying that M. lepromatosis might be relatively more constrained in these processes than M. leprae.

On the other hand, 26 genes present in M. lepromatosis have been found as pseudogenes in M. leprae. However, most of this contrasting genomic content (sequences which are annotated as CDS in one species but as a pseudogene in the other) is comprised of conserved hypothetical proteins. Since diverging from their most recent common ancestor, both M. leprae and M. lepromatosis have continued to lose additional DNA from their genomes. Losses have occurred from different regions and both organisms have accumulated “species-specific” genes, pseudogenes, and genomic regions [36]. The nucleotide identity between corresponding CDS of both species is ~93%, while the pseudogenes have accumulated more divergence (~82% identity), perhaps due to lower selection pressure.

Reductive Evolution

The process of reductive evolution is evident in several obligate intracellular bacterial pathogens (e.g., Rickettsia, Chlamydia). Reductive evolution has been hypothesized to occur as an acclimatizing survival response to drastic change(s) in ecological niches. Hence, several genes and pathways required for survival in a previous habitat—for example, a free-living ancestor—would no longer be necessary or useful inside a specialized host. Genes whose functions are no longer required may become inactivated over time while newer genes might evolve through duplication, recombination, horizontal gene transfer, or acquisition of plasmid(s), which can facilitate additional functions. This evolution can impart a selective advantage to the recipient microbial cell in coping with new and often hostile conditions inside a host, thereby enabling their successful multiplication and dissemination. An example of this process can be seen in genome features of M. ulcerans that evolved from its aquatic free-living ancestor M. marinum. As shown in Table 1, the number of coding sequences in M. ulcerans decreased by 20% from the 5,426 CDS in its ancestor M. marinum. This decrease was accompanied by an accumulation of over 700 pseudogenes in M. ulcerans from 57 in M. marinum [39].

The M. leprae and M. lepromatosis genomes are extreme examples of reductive evolution seen in the form of genome downsizing and the accumulation of massive numbers of pseudogenes. The resultant metabolic streamlining, particularly the loss of several catabolic functions and redundant pathways, as compared to M. tuberculosis, has shaped M. leprae into a highly specialized, niche-adapted obligate intracellular pathogen [11], [42]. Based on the nucleotide substitution rate in M. leprae pseudogenes, it has been estimated that a massive pseudogenization event took place in the last 20 million years [40], [43]. Based upon genome-wide phylogenetic comparison and Bayesian dating analysis of M. leprae and M. lepromatosis, it appears that the species diverged ~14 million years ago [36]. The pseudogene formation appears to have occurred as a single event, perhaps as a result of a sudden change of lifestyle from a free living to a highly specialized niche inside a host. However, it is evident that this process has continued, albeit at a much slower rate, thereby accounting for the accumulation of ‘specific’ pseudogenes and genomic deletions (present in M. lepromatosis but not in M. leprae and vice versa). This continuing process accounts for nearly 5% of the genome of both organisms being species-specific, most of which, however, is composed of pseudogenes with very few exceptions. For example, the genomic region containing the intact hemN gene in M. lepromatosis is completely absent in the M. leprae genome (see the next section for further discussion). Another example of the continuation of pseudogene formation in more recent evolutionary history can be seen from the genome sequencing of M. leprae clinical isolates, where ten new pseudogenes have been identified in different strains. These new pseudogenes are not present in the M. leprae TN genome but were detected when an additional 16 M. leprae genomes, belonging to diverse phylogeographic origins, were sequenced [13], [14]. It is noteworthy that the age of the most recent common ancestor of these 16 M. leprae strains was determined to be within the last 5000 years [13]. Therefore, the new pseudogenes must have appeared within this time span.

Role of recombinational events

The reduction in M. leprae genome size and loss of synteny in comparison to M. tuberculosis is considered a result of extensive recombinational events between various copies of repetitive elements. These elements belong to four families known as RLEP (present in 37 copies), REPLEP (15 copies), LEPREP (8 copies), and LEPRPT (5 copies) [44]. The corresponding sequences in the M. lepromatosis genome were also found, albeit with a divergence rate ranging from 75–90% at the nucleotide level. It appears that the genome-downsizing was mediated in the most recent common ancestor by these repeats. The repeats, present in higher copy number in the M. leprae genome, have accumulated greater divergence in comparison to the corresponding M. lepromatosis repeats. However, the exact copy number of these repeats in M. lepromatosis is unknown, as the current assembly has several gaps and repeats tend to be at the ends of contigs. The determination of multicopy templates can be useful for a more sensitive detection/enumeration of M. lepromatosis DNA, similar to the RLEP-based assay for M. leprae [45].

Reduction in PE/PPE genes and G+C content

Mycobacterial pathogens possess highly polymorphic proline-glutamate (PE) rich proteins and proline-proline-glutamate (PPE) proteins that are unusually high in G+C content and possibly impart genetic and antigenic variations. The M. tuberculosis genome possesses 167 PE/PPE genes [35]. In contrast, both M. leprae and M. lepromatosis possess fewer than 9 PE/PPE genes, which in part accounts for their lower overall G+C content. It is noteworthy that the average G+C content of genes in M. leprae and M. lepromatosis is ~60%, while the pseudogenes and non-coding regions have a G+C content of ~56.5 and 54.5%, respectively. Hence, another reason for the unusually low G+C content of M. leprae is the accumulation of massive numbers of translationally inert pseudogenes, which perhaps do not have codon usage preference, unlike the active genes [35].

Comparison of Genomic Features of Iron Uptake

The ability to acquire iron within vertebrate hosts is a critical requirement for nearly all bacterial pathogens. Iron uptake by bacteria inside host tissues is a particularly important requirement for obligate intracellular pathogens, for which they have developed different pathways. For the purpose of understanding the potential capacities and limitations of M. leprae to acquire iron from its environment, it is informative to compare M. haemophilum to M. leprae and M. lepromatosis. Among culturable mycobacteria, M. haemophilum has unique requirements for iron supplementation to facilitate its growth. Among sequenced mycobacterial species, it is also the most similar to M. leprae and M. lepromatosis, both by phylogenetic comparison of several genes [19], [46] as well as by comparison of whole genomes [37].

Both M. leprae and M. lepromatosis lack mbt-1 and mbt-2 gene clusters (necessary for the synthesis of mycobactin and carboxymycobactin siderophores) and iron-regulated transporters A (irtA) and irtB (functioning in the uptake of siderophore-bound iron). The ESX-3 secretion system has also been implicated in the acquisition of iron from mycobacterial siderophores [47], [48], [49]. M. leprae and M. lepromatosis lack a readily identifiable rv0286 (ppe4) homologue within the esx-3 locus [11], [36], a deficit which may impact the ability of the ESX-3 secretion system to function in iron acquisition [50]. The absence of these various loci relevant for siderophore-mediated iron uptake is shared by M. haemophilum, an emerging opportunistic pathogen which requires external iron supplementation for its growth on artificial media [37]. Therefore, it is of interest to investigate the genomic repertoire in M. haemophilum that enables iron uptake and utilization from iron (hemin)-supplemented media.

This question has been recently investigated in detail by sequencing the M. haemophilum genome, which revealed that M. haemophilum possesses a homolog (B586_01335) of the M. tuberculosis gene rv0203 [37]. In M. tuberculosis, gene rv0203 encodes a secreted heme-binding protein believed to act in combination with the MmpL11 and MmpL3 inner membrane proteins and constitutes a putative heme acquisition system [51]. While both M. leprae and M. lepromatosis possess intact mmpL11 and mmpL3 with high (~90%) nucleotide identity to those in M. haemophilum, they lack the counterpart of Rv0203 (Figure 1). This lack may result in defects in the capacity to scavenge heme-iron or utilize externally supplemented hemin in media.

FIG8_2_1.png

FIG 1 Comparison of a Putative Heme-Acquisition System in M. leprae (top) and M. tuberculosis.

In M. tuberculosis, rv0203, along with the mycobacterial membrane proteins mmpL11 and mmpL3, constitute a functional iron acquisition system, which is also present in M. haemophilum (not shown). In contrast to M. tuberculosis and M. haemophilum, the genome of M. leprae has retained mmpL11 and mmpL3 (highlighted by ovals) while the corresponding gene for rv0203 (marked by a red square) is missing, presumably making this system non-functional. The genome coordinates are shown.

More recently, a screen for M. tuberculosis transposon mutants resistant to the toxic heme analogue gallium (III)-porphyrin (Ga-PIX) has uncovered proteins required for heme utilization by the bacterium, including the cell surface accessible PPE36 and PPE62 (predicted outer membrane protein) and the periplasmic Rv0265c [52]. In this model, heme uptake across the inner membrane is mediated by a transporter that remains to be identified. The M. haemophilum genome encodes a protein (B586_00960) with substantial homology to Rv0265c (79% identity), while the M. leprae counterpart (ML2548) is annotated as a pseudogene [53]. Homology among PPE proteins can make it challenging to determine the presence or absence of a specific family member. However, M. haemophilum encodes a protein (B586_10660) with 79% identity to PPE36 and in a similar chromosomal context, while a correspondingly close homologue is absent from M. leprae [54]. This absence may be significant regarding the capacity for heme iron acquisition, as a PPE36 deletion mutant of M. tuberculosis failed to grow in a minimal medium with hemin as the sole iron source [52]. Both M. leprae and M. lepromatosis possess a homologue of mhuD (mycobacterial heme utilization, degrader) which encodes a heme-degrading enzyme important for the cytoplasmic release of iron from heme [55], [56]. The homologue shares over 90% amino acid identity with its counterpart in M. haemophilum.

In summary, M. leprae and M. lepromatosis appear to lack components of putative mycobacterial heme iron acquisition pathways, which are present in M. haemophilum. Of further note, M. leprae has lost the genomic region containing hemN, a putative component of the heme biosynthetic pathway that is present in both M. lepromatosis and M. haemophilum. The loss of hemN from the M. leprae genome took place after M. leprae and M. lepromatosis diverged from their ancestor. The unique lack of hemN implies that M. leprae may be even more limited in heme production than M. lepromatosis. However, it should also be noted that, despite its annotation, it remains questionable whether hemN actually plays a role in mycobacterial heme biosynthesis [57], [58].

Surprisingly, despite their small genome sizes and the effects of reductive evolution, M. lepromatosis and M. leprae both possess two copies of the Nramp family gene (natural resistance-associated macrophage protein, mntH/nramp), similar to M. haemophilum (B586_01840 and _06145), which perhaps points to a similar role in their in vivo niche. In contrast, M. tuberculosis possesses only a single copy [35]. Nramp homologues are pH-dependent divalent cation transporters that are candidate iron uptake factors [37].

In addition to the above comparison of the iron-uptake capabilities of M. leprae and M. lepromatosis with M. haemophilum, it is noteworthy that the latter’s genome is much bigger and possesses several other genes/pathways, a few of which might contribute additional metabolic capabilities that can enable its in vitro cultivation in iron-supplemented media. For instance, the presence of ferrous iron transport proteins encoded by feoA and feoB enable the transport of ferrous ion across cytoplasmic membrane [59], [60]. feoA and feoB are present in several other non-tuberculous mycobacteria (M. kansasii, M. marinum, M. genavense, and M. xenopi) but missing in M. tuberculosis, M. leprae, and M. lepromatosis. Additionally, the M. haemophilum genome possesses another locus (B586_00145 to _00155) with homology to the ATP binding cassette (ABC) transport system of iron/siderophore/heme/vitamin B12. This system is important for iron uptake in other bacteria [61] but is missing from the genomes of M. tuberculosis, M. leprae, and M. lepromatosis.

Biology and Clinical Management

Neuropathogenicity potential

Leprosy is essentially a disease of nerves and, hence, neuropathogenicity is the defining hallmark of the etiological agent. In this regard, the neurotropism (see Chapter 9.1; Chapter 9.2) of M. leprae has been attributed to the presence of laminin-binding protein (encoded by ML1683c) and the terminal trisaccharide moiety of phenolic glycolipid (PGL-1) [62], [63]. Both are bacterial cell wall components. The genes for these adhesion systems are intact in the M. lepromatosis genome and are highly conserved [36]. Hence M. lepromatosis’s tropism for nerves has a genomic basis, although experimental evidence from clinical and histopathological specimens will provide a more definitive answer.

Molecular diagnostics

Leprosy has been described as a great imitator [64] due to its diverse and confusing clinical presentations (see Chapter 2.1). This diversity makes leprosy diagnosis a challenge, especially for physicians with limited experience in diagnosing and managing the disease. The biggest difficulty in the areas where leprosy is extremely rare is the lack of clinical suspicion due to unfamiliarity among physicians. Declining leprosy expertise among physicians can cause diagnostic delays, thus contributing to serious neurological consequences [65], [66], [67]. Using sensitive and specific diagnostic tests in cases in which clinical symptoms are suggestive of leprosy can help minimize this problem.

Applications of PCR-based diagnostics

The identification of the M. leprae-specific repetitive genomic sequence RLEP [45], [68] has made M. leprae detection more sensitive and specific, as it is present in 37 copies in the genome. Therefore, RLEP-based PCR has been a valuable tool in leprosy diagnosis and research, especially for paucibacillary (PB) and environmental [69] or formalin fixed paraffin-embedded (FFPE) [70] specimens as well as ancient skeletal samples (see Chapter 11.1), in which DNA is often highly fragmented and quantities are minimal [14], [71], [72], [73], [74], [75], [76]. Likewise, a similar multicopy target for M. lepromatosis would be valuable, particularly for such difficult samples. However, until multicopy loci are identified, specific genomic regions, for example hemN, are good targets for the sensitive and accurate detection of M. lepromatosis, especially in cases of co-infection with M. leprae. Notably, the mere presence of M. leprae or M. lepromatosis DNA in a patient sample does not constitute a diagnosis of leprosy (see Chapter 7.2), as a significant percentage of healthy household contacts show M. leprae DNA positivity in their nasal secretions [77], [78], [79]. Hence, the results of PCR tests are to be interpreted in conjunction with other clinical or histopathological evidence (see Chapter 2.4).

Drug targets and Molecular Drug Susceptibility Testing (MDST)

The development of newer anti-tuberculosis drugs raises the question of whether they can also be effective against M. leprae (and M. lepromatosis). In the absence of culture-based phenotypic tests, genomics offers helpful clues. For instance, the genomic target of the experimental drugs bedaquiline, benzothiazinones, and Q203 are intact in M. leprae and M. lepromatosis. In contrast, the genomic targets for the action of nitroimidazole prodrugs, PA824, TBA354, and delamanid, are missing.

The Mouse Foot Pad (MFP) assay has been the gold standard phenotypic test for the determination of anti-leprosy drug resistance (see Chapter 5.2; Chapter 5.3). However, the routine use of the MFP assay is impractical [80], [81]. Thanks to the assay’s excellent correlation with the resistance-associated mutations in the drug resistance-determining regions (DRDRs) of folp1 (dapsone), rpoB (rifampin), and gyrA (fluoroquinolones) [82], [83], [84], [85], [86], [87], [88], [89], [90], [91], [92], [93], drug resistance trends are now being monitored successfully using molecular tests that target resistance-associated mutations [94]. The comparison of corresponding genomic regions in M. lepromatosis reveals drug susceptibility-related sequences, which is reassuring as it indicates that the same drugs will likely be effective for both leprosy pathogens. Successful treatment of M. lepromatosis-infected patients with standard MDT has been reported [95].

Molecular epidemiology

Single nucleotide polymorphism (SNP) typing

Even before the advancements of detailed sequence comparisons, it was noted that M. leprae strains from diverse geographical origins possess minimal genomic diversity [68], [96] and, hence, discrimination of one strain from another was challenging. Comparative genomics of a limited number of M. leprae isolates has helped in identifying phylogeographically informative SNPs [97] for strain typing. Upon survey of these SNPs in isolates from diverse geographical origins, a SNP-typing scheme was developed that places M. leprae strains from all over the world into four major SNP-types, identified as SNP Types 1 through 4. These four SNP-types were further resolved into 16 subtypes (1A through 4P) upon whole genome sequencing and comparison with another M. leprae strain (Br4923 from Brazil) [14]. The SNP genotypes generally exhibit geographical association, as represented in Table 2. The SNP-genotyping scheme has been very helpful in studying the global dissemination of leprosy, along with ancient human migrations, and has also helped in studying the evolutionary history of M. leprae and M. lepromatosis [13], [36]. In addition, the scheme has been successfully used for molecular epidemiological investigations into the focal transmission of M. leprae [98], [99] and the identification of M. leprae genotypes in environmental samples from the surroundings of leprosy patients [100]. Though SNPs provide a reliable phylogenetic association between M. leprae strains from geographically distant locations, they provide limited resolution for studying short-range transmission due to their relative stability [14], [97].

TABLE 2 Geographical association of various M. lepraegenotypes

SNP-Type

SNP-Subtype


Countries where these M. leprae genotypes have been detected

1

A

Bangladesh, French West Indies, India, Indonesia, Japan, Korea, New Caledonia, Nepal, Philippines, Thailand, USA*

1

B

Bangladesh, French West Indies, India

1

C

Bangladesh, India, New Caledonia, Nepal, USA*

1

D

India (most prevalent genotype), Japan, Madagascar, Malawi, Nepal, Pakistan, Philippines, Venezuela, USA*

2

E

Malawi

2

F

Iran, Europe (ancient DNA from Denmark, Sweden, UK), Turkey

2

G

New Caledonia, Nepal

2

H

Ethiopia

3

I

Bolivia, Brazil, Europe (ancient DNA from Sweden, Denmark, UK), Morocco, Mexico, Uruguay, USA**, Venezuela

3

J

New Caledonia

3

K

China, Indonesia, Japan, Korea, New Caledonia, Philippines, Turkey (modern and ancient DNA), USA*

3

L

New Caledonia, USA*

3

M

French West Indies, Europe (ancient DNA from Hungary)

4

N

Benin, Brazil, Guinea, Ivory Coast, French West Indies, Mali, Morocco, Senegal, Venezuela

4

O

Ivory Coast, Mali, Senegal, Venezuela

4

P

Benin, Brazil, Mali, USA*, Venezuela

This table is based upon the literature and other recent findings, which together represent >1000 strains from all over the world. However, it should be noted that further diversity of M. leprae strains in an area may be revealed when a representative number of strains are analyzed by SNP-genotyping to detect the genotypes present in a lower frequency.

* In USA, nearly 2/3 of the cases are found in patients with immigration histories from leprosy-endemic countries and often represent the genotypes prevalent in their countries of origin.

** All M. leprae strains found in wild armadillos and most USA patients with no history of foreign residence were found to be type 3I and shared striking levels of identity upon their detailed genomic analysis using SNPs, VNTR, and whole genome sequencing.

Variable number tandem repeats (VNTR)-typing

VNTRs are stretches of 2–50 nucleotide long repeats present in variable copy numbers.These VNTRs are highly variable and evolve much more rapidly than SNPs due to slipped-strand mispairing, which makes the probability of mutations (insertion and deletion of repeat copies) up to 100,000 times more frequent as compared to SNPs [101], [102]. Though rapid evolution makes them ideal for increased resolution, the phylogenetic information is lost in the descendent population. Several VNTR loci have been identified in the M. leprae genome [103], [104] and have been used for VNTR-typing aimed at the discrimination of M. leprae isolates in leprosy endemic regions [70], [105], [106], [107], [108], [109], [110]. However, some VNTR loci have been reported to be hyper-variable, as they vary among samples from different lesions of a single patient, thereby making the VNTR inappropriate for molecular epidemiological investigation [111].

Combined application of SNPs and VNTRs: A synergistic approach

When SNP and VNTR-typing are combined, the limitations of both are overcome. Therefore, a combined application of SNPs with selected VNTRs (showing a reliable level of allelic diversity) was developed, providing much higher resolution. At the same time, the predominance of SNP markers (for example, 84 informative SNPs in comparison to a battery of 10 VNTRs) helps in retaining the phylogeographic association while still achieving an optimal level of strain differentiation. Phylogeographic association and strain differentiation are two important components of a genotyping method. It was the power of this combined use of SNPs (identified by whole genome sequencing of M. leprae strains from the same geographic area) and VNTRs that confirmed the role of armadillos in perpetuating zoonotic leprosy in the southern United States [112], [113], which had long been suspected [114], [115], [116], [117], [118], [119], [120] but was never confirmed earlier.

Potential of WGS for routine clinical applications and current challenges

Whole genome sequencing (WGS) directly from archived patient biopsies and skeletal samples has demonstrated its value and feasibility as an “all-in-one” approach to investigate pathogenesis, molecular drug susceptibility testing, molecular epidemiology, and phylogeny-related questions. In addition, the preparation of sequencing-ready libraries converts M. leprae genomic DNA into a valuable “renewable” resource for further investigations into the pathogen as well as into the host. While it is tempting to advocate for the routine application of WGS in the clinical setting, it first must be made cost- and effort-effective. With the current pace of improvements in library preparation and enrichment methods, WGS could become a realistic proposition in the foreseeable future. WGS also allows for investigations into M. leprae strains from the PB portion of the clinical spectrum of leprosy. In the latest application of this approach, the phylogenomics of 154 M. leprae strains from 25 countries were investigated, revealing novel insights into the evolution of drug resistance in M. leprae [121].

Another potential application of WGS is in the discrimination of relapse versus reinfection. It is important to differentiate whether the second episode of the disease is attributable to endogenous reactivation of persisting M. leprae bacilli from the first episode or the result of an exogenous reinfection with another strain. This determination requires genome-scale comparison of M. leprae from the first and any subsequent episodes of the disease. The comparison can be done using archived clinical samples from the various episodes. This approach, which was successfully used in a recent study, proved that some of the relapses could be attributed to exogenous reinfection while others could be due to endogenous reactivation [122]. The availability of this information could be helpful in determining the optimal duration of therapy for PB and MB cases.

Clues into origin and evolution

Based upon the genome-wide comparison of M. leprae from around the world, including DNA from 10–13thcentury leprosy skeletons from Europe (see Chapter 11.1), the M. leprae genotype 3K strains were found to be the closest to M. lepromatosis, The type 3K samples have been reported from China, Iran, Indonesia, Japan, Korea, New Caledonia, Philippines, Turkey [13], [14], [36], and U.S. patients originating from Guam and Micronesia [112], [113]. The wide geographic distribution of this genotype and the insufficient sampling for whole genome comparison studies precludes an accurate estimate of the tentative origin of leprosy bacilli. However, the 16 M. leprae genomes exhibit only ~800 SNPs, while the comparison with M. lepromatosis reveals over 275,000 SNPs, indicating that M. lepromatosis has deep evolutionary divergence from M. leprae [36] ranging in the time frame of several million years. By comparison, it is noteworthy that the species belonging to the M. tuberculosis complex have evolved from their most recent common ancestor in only the last 7000 years [123].

Concluding Remarks

Genomics has opened up several avenues for the study of M. leprae pathogenesis, metabolism, and transmission as well as for understanding the origin, evolution, and global dissemination of the disease. Useful genomic insights into the biology of M. leprae and M. lepromatosis have enabled the development of sensitive assays for the accurate detection of these pathogens and have also helped in their clinical management, for example, by monitoring anti-leprosy drug resistance trends. In addition, the comparative genomics of various mycobacterial pathogens (M. leprae, M. lepromatosis, M. haemophilum, M. ulcerans, M. marinum, etc.) has been helpful for an improved understanding of the process of mycobacterial evolution and niche adaptation.

Acknowledgements

Dr. Pushpendra Singh is grateful to the following funding agencies/organizations for supporting his research: the University Grants Commission Faculty Recharge Programme; The Ramalingaswami Re-entry Fellowship (Department of Biotechnology, Government of India); The Maharaja Sayajirao University of Baroda, Vadodara, India; the Turing Foundation and the Leprosy Research Initiative, The Netherlands; the effect:hope (Leprosy Mission Canada), and the Seeding Labs Boston, USA. Dr. JoAnn Tufariello and Dr. William R Jacobs, Jr., would also like to acknowledge the generous support of effect:hope (Leprosy Mission Canada) on behalf of the Research to Stop Neglected Tropical Disease Transmission (R2STOP) Initiative. Dr. Jacobs would also like to acknowledge the support provided by NIH grant AI026170. Ms Priya Paithankar and Ms Purna Dwivedi’s help in compiling and formatting the reference list is also appreciated.

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borderline tuberculoid leprosy
paucibacillary leprosy
tuberculoid leprosy
lepromatous leprosy
multi-drug therapy
MDT
PB
RR
MB
LL
BL
BT
BB
TT