5 курс / Пульмонология и фтизиатрия / Clinical_Tuberculosis_Friedman_Lloyd_N_,_Dedicoat

INTRODUCTION: THE GLOBAL PROBLEM AND TB IN INFECTIOUS DISEASE HISTORY

Tuberculosis (TB) has been a leading cause of death throughout human history. The World Health Organization reported over 1.6 million deaths from TB and over 10 million new cases of TB in 2017, making TB the single leading cause of death from an infectious agent today.1 The TB problem has worsened in the last 50 years, with the emergence of the HIV epidemic, which has caused increased transmission, increased incidence, and the emergence of drug resistance. Mycobacterium tuberculosis strains have evolved that are resistant to the two front-line TB drugs—Isoniazid (INH) and Rifampicin. These strains are called multi-drug-resistant M. tuberculosis (MDR).2 Ominously, extensively drug-resistant strains (XDR) have also evolved that are resistant to as many as 10 TB drugs.3 This emergence of resistance is surprising as TB is a disease for which control programs are employed around the world:

(1) rapid diagnostic tests, (2) a vaccine, BCG (bacilli Calmette and Guerine), and (3) a sterilizing chemotherapy. If so many solutions are available, then why is TB still a major global health threat and economic burden?

The question of whether TB was caused by a transmissible agent or was merely a manifestation of an inherited disposition was a controversial subject in the nineteenth century, but the 1868 demonstration by Jean Antoine Villemin that inoculation of tuberculous materials from humans and cattle into rabbits elicited characteristic granulomatous lesions swung the argument strongly in favor of an infectious cause.4 The matter was finally and irrefutably settled on March 24, 1882 when Robert Koch described a series of meticulous studies in which he had not only isolated the causative bacilli but also rigorously demonstrated causality by

fulfilling criteria laid out in his well-known postulate.5,6 In that 1882 paper, Robert Koch wrote: “To prove that tuberculosis was caused by the invasion and multiplication of bacilli, it was necessary to: (1) isolate the bacilli; (2) grow them in pure culture, and

(3) administer them to an animal to produce the same moribund condition.”3,4

While others had hypothesized that many diseases were caused by microbes, the inability to grow these microbes in pure cultures prevented stringent testing of this hypothesis. Robert Koch, like Louis Pasteur, developed methods to grow pure cultures of microbes, providing a new tool to visualize and characterize these organisms. Jean Antoine Villemin performed the first TB animal experiment when he showed the transfer of tubercles from patients with TB caused the disease in rabbits.4,7 As a surgeon, Villemin could see the differences between the tubercles in TB patients compared to the tumors he observed in patients with lung cancers. Brilliantly, he demonstrated the transfer of cancerous lesions to rabbits caused no disease whereas transfer of tubercle lesions always caused TB. Villemin concluded TB was not a cancer, but rather was caused by an infectious agent.7 Robert Koch actually first fulfilled the conditions of the Henle−Koch theory with the anthrax bacillus and published this work in 1876.8 By obtaining fluid from a cow’s eye, he had a sterile fluid he could inoculate with anthrax bacillus in vitro to obtain pure cultures of the pathogen. He went on to show that these pure cultures reproducibly caused disease in rabbits and mice, thus fulfilling the three conditions of Koch’s postulate to prove that the anthrax bacillus was the etiologic agent of the disease. The same approach enabled Robert Koch to demonstrate that TB was caused by a bacillus. For growing the tubercle bacilli, Koch needed larger amounts of growth media and so he decided to use sera from cows.8 After drawing blood and allowing the red cells to settle, he sterilized the sera by repeated cycles of heating to 65°C and cooling. At the end of the

seventh cycle, he would allow the serum to solidify in a slanted tube. A gelatinous surface would form, which upon inoculation, enabled the growth of tubercle bacilli. Koch used these pure cultures of tubercle bacilli to infect rabbits, mice, guinea pigs, cows, and cats; all developed TB-like disease. For the first time in history, we knew TB was caused by an infectious agent. For his foundational work, Robert Koch received the Nobel Prize in 1905.

GENE TRANSFER IN M. TUBERCULOSIS

Gregor Mendel published his work in 1866 establishing the concept of discreet units of hereditary (genes) caused by characteristics (phenotypes in plants).9 Interestingly, the molecular basis for a gene was not determined until 80 years later and this was achieved by an experiment selecting for a virulent bacterium in a mouse.10

Phenotypes are characteristics of an organism that result from a genotype and its environment. For bacteria, common phenotypes refer to characteristics such as virulence, drug-resistance, and dye-staining properties or colony morphologies. Molecular Koch’s postulate was a concept first proposed by Stanley Falkow for establishing causality of a phenotype.11 A paraphrased version of Koch’s postulate would be: To prove that a phenotype such as drug resistance of an organism is caused by a specific genotype, it was necessary to: (1) isolate a mutant exhibiting drug resistance, (2) clone the genotype from the drug-resistant mutant, and (3) transfer the cloned genotype to a parental strain and demonstrate the acquisition of drug resistance. This is the essence of genetics.

Sixty years after Koch discovered the tubercle bacillus and before DNA was known to encode genetic material, molecular genetics began with Beadle and Tatum in 194112 where mutants of Neurospora crassa were generated by irradiation and plated on complex media. These mutants, referred to as auxotrophic mutants, required single nutrients such as pyridoxine, thiamine, or para-amino benzoic acid and later amino acids. In completing the requirement of gene transfer, Beadle and Tatum fulfilled the third condition of Molecular Koch’s postulate and discovered, for the first time, that one gene encoded one enzyme. In fact, the discovery of the molecular nature transforming principle (an isolated fraction of a virulent cell extract of Streptococcus pneumoniae) allowed Avery, MacLeod, and McCarty to discover in 1941 that DNA was the genetic material.13

In contrast to Neurospora, which was a diploid organism that underwent meiosis, transfer of DNA directly into Streptococcus was called transformation. The question as to whether bacteria had genes was answered convincingly when Salvador Luria and Max Delbruck used phages (bacterial viruses) as selecting agents, providing evidence that Charles Darwin’s survival of the fittest premise existed even for bacteria.14 Gene transfer studies exploded when Joshua Lederberg and Edward Tatum demonstrated in 1946 that Escherichia coli could transfer genes from cell to cell (conjugation) using double auxotrophic mutants.15 E. coli would become the model organism for molecular genetics for the next 30 years. The lingering debate as to whether DNA, not protein, was the genetic material was convincingly proved by Hershey and Chase’s experiment with phages, bacteria, and radiolabeled DNA

Figure 3.1  Scanning electron micrograph of TM4-based shuttle phasmids attached to M. tuberculosis. The TM4-based shuttle phasmids provided a facile system for delivering foreign DNA into M. tuberculosis. These phages have been used to: (1) systematically develop transformation for mycobacteria, (2) deliver transposons for transposon mutant libraries, (3) move mutated alleles into M. tuberculosis strains (specialized transduction), and (4) deliver reporter genes such as firefly luciferase to rapidly assess drug susceptibilities.

or protein.16 Notably, Norton Zinder, while trying to repeat the Lederberg experiment for Salmonella, discovered phages could transfer genes to other bacterial cells—a process he named transduction.17 Thus transformation, conjugation, and transduction became the key tools for the successful fulfillment of the third condition of Molecular Koch’s postulate. The slow growth and virulence precluded successful gene transfer in M. tuberculosis until 1987.18 Using the first chimeric mycobacteriophage−E. coli shuttle vector, termed a shuttle phasmid—it became possible to transfer genes in M. tuberculosis (Figure 3.1). Nearly 100 years later, plasmid transformation, efficient transposon mutagenesis systems, specialized transduction, and reporter mycobacteriophages have enabled the acquisition of many phenotypes of M. tuberculosis, opening the doors for new therapeutic approaches (see reviews19–21).

THE GENOME OF M. TUBERCULOSIS

A landmark in TB research occurred with the publication of the genome sequence of M. tuberculosis in 199822 on a widely used reference strain, H37Rv. No extrachromosomal genetic elements were detected. The genome of M. bovis has also been sequenced and shows more than 99.95% similarity with that of M. tuberculosis although it is very slightly smaller, with 4,345,492 base pairs.23 In contrast, the genome of M. leprae,24 with 3.27 million base pairs, is considerably smaller than that of M. tuberculosis, and it differs from the latter in that many of its genes, around a half, are defective and non-functional, explaining why this organism has never been cultivated in vitro and is an obligate intracellular pathogen.

The chemical structure of the genome of M. tuberculosis is remarkably uniform with a high guanine + cytosine content

(65.6%) throughout, indicating that it has evolved with minimal incorporation of DNA from extraneous sources. Other notable differences between this genome and those of other bacteria have been determined. In particular, M. tuberculosis has a very large number of genes coding for enzymes involved in lipid metabolism, around 250 compared to only 50 in E. coli. All known lipid biosynthesis pathways encountered elsewhere in nature, as well as several unique ones, are detectable in the mycobacteria. Most of these enzymes are involved in the synthesis of the extremely complex lipid-rich mycobacterial cell walls.

The mycobacterial genome is also unique in containing a large number of genes, up to 10% of the total coding potential, that code for two unrelated families, Pro-Glu and Pro-Pro-Glu, of acidic, glycine-rich proteins that contribute to the diversity in antigenic structure and virulence. Because they undergo frequent genetic remodeling by various mechanisms, they may have contributed substantially to the evolution of the M. tuberculosis complex and adaptation of its various members to different hosts.25 Despite much research in the decade since their discovery, the function of the PE/PPE group of proteins remains poorly understood, although they induce or modulate a range of innate and acquired immune reactions.26

Four main types of genomic differences among strains within the M. tuberculosis complex have been described: those involving single-nucleotide variations (single-nucleotide polymorphisms, or SNPs),27 those involving several sequential nucleotides (longsequence polymorphisms, or LSPs), minisatellites, and micro­ satellites. Although there are 1075 SNP differences between M. tuberculosis H37Rv and a recent clinical isolate, and 2437 between H37Rv and the sequenced strain of M. bovis, these differences are small in relation to the four million or more nucleotide pairs in the genomes of these strains. The LSPs are much fewer in number than the SNPs in the M. tuberculosis complex and include 20 well-defined regions of difference (RD) that are described here.

The function of bacterial minisatellites is unknown, but their analogues in eukaryotic genomes contribute to genetic diversity by mediating chromosome recombination during meiosis.28 In the mycobacteria, they often occur as tandem repeats in the regions between functional genes and some are designated mycobacterial interspersed repetitive units (MIRUs). Minisatellites are utilized in a typing system known as variable number tandem repeat (VNTR) analysis, as described in Chapter 5. By insertion or deletion, microsatellites, also known as single sequence repeats, cause reversible frame-shift mutations at a relatively high rate and impart a genetic “plasticity” to the genomes of pathogens, enabling them to adapt readily to different hosts.29,30

The genome of M. tuberculosis contains mobile units of DNA, informally known as “jumping genes” that contribute to genetic variation and evolution. These mobile elements include a class termed insertion sequences (ISs) of which 56 different types, grouped in several families, are present in the genome of strain H37Rv. Most, but not all, strains of M. tuberculosis contain copies, usually numbering from 4 to 14, of the insertion sequence IS6110.31 With some exceptions, strains of M. bovis contain fewer copies of IS6110 than M. tuberculosis, with, as described earlier, daughter strains of BCG containing either one or two copies. The

considerable variation in the numbers and position of copies of IS6110 between strains forms the basis of the restriction fragmentlength polymorphism (RFLP) typing system used in epidemiological purposes.

The genome of members of M. tuberculosis contains a complex termed the direct repeat (DR) locus consisting of repetitive 36 base-pair units of DNA separated by non-repetitive 34–41 basepair spacer oligonucleotides. The DR region of M. tuberculosis is an example of a region present in all bacterial genomes and is termed clustered regularly interspaced short palindromic repeats (CRISPR). The function of this region is unknown, but it may be the bacterial analogue of the centromere found in eukaryote chromosomes. There are numerous possible combinations of spacer oligonucleotides, and these are very stable, providing a highly discriminative typing scheme known as spacer oligonucleotide typing, or “spoligotyping”,32 as described in Chapter 5. There is evidence that variation in the DR locus is due to mutational events in the spacer oligonucleotides, including their disruption by translocations of the ISs.33 Such mutational events occur at a very slow rate and serve as an evolutionary “clock.”

Since the year 2000, there has been intense interest in a class of small, non-coding, RNA molecules (microRNAs) that regulate cellular metabolism by binding to messenger RNA, thereby blocking transcription in the ribosome and “silencing” gene expression. Interest has principally focused on microRNAs in eukaryotic, including human, cells as possible therapeutic agents, but analogous molecules, sRNAs, are present in bacteria. Many sRNAs have been detected in M. tuberculosis, and their expression varies considerably according to growth conditions, particularly between bacilli in exponential growth and stationary phases of cultivation and between those extracted from infected lungs and those grown in vitro.34 Therefore, sRNAs may play a key role in the adaptation of M. tuberculosis to a wide range of in vivo and in vitro environments.

The nomenclature of the causative organisms of human and mammalian TB is not entirely logical. As described here, analysis of their genomes has shown that these bacilli, grouped in the M. tuberculosis complex, are very closely related, with less than 0.1% genomic difference between them; thus, they clearly belong to what should be regarded as a single species.

The great majority of strains of M. tuberculosis produce rough colonies on solid media (Figure 3.1), but one rare variant, termed the Canetti type and also but unofficially M. canetti, produces smooth colonies due to large amounts of lipopolysaccharides on their cell surfaces.35,36 This variant, almost all strains of which have been isolated in the Horn of Africa, appears to be a primitive “living fossil” form of M. tuberculosis (see the following section). Human-to-human transmission has not been confirmed, and epidemiological findings suggest that human infection is acquired from animate or non-animate non-human sources.37

Strains of M. bovis differ from M. tuberculosis in several respects including resistance to the anti-TB agent pyrazinamide. However, isolates susceptible to this agent, though in most other respects similar to M. bovis, have been isolated from animals, principally goats, in Spain and Germany. Because there are genomic differences between these strains and M. bovis, they have been

allocated to the separate species M. caprae.38 Occasional strains of otherwise typical M. bovis are also susceptible to pyrazinamide.39 TB caused by M. caprae has also been reported in humans, notably in Germany where one-third of 166 strains of human origin initially identified as M. bovis, isolated between 1999 and 2001 from patients principally living in south Germany, were found to be M. caprae.40 The patients were in the same elderly age range as those infected with M. bovis, suggesting that disease due to both

types represents reactivation of old infections.

An important “man-made” variant of M. bovis is the BCG vaccine that was derived, by 230 sub-cultivations on a potato-bile medium between the years 1908 and 1921, from a strain (“Lait Nocard”) isolated from a case of bovine mastitis. Some currently available daughter strains of BCG, including the Brazilian, Japanese, Romanian, Russian, and Swedish strains, were issued by the Institut Pasteur before 1932. These differ from those daughter strains issued after this date in their cell wall structure, their active secretion of an antigenic protein, MPB70, and having two copies, rather than one, of the insertion sequence IS6110 (see the following section).41

A group of tubercle bacilli principally isolated from humans in equatorial Africa and in migrants from that region have properties intermediate between M. tuberculosis and M. bovis and have been given the separate species name M. africanum. Originally, two geographical variants of this species were described: Type I strains, principally from West Africa, resembling M. bovis, and Type II, mainly found in East Africa, resembling M. tuberculosis.42 More recently, it has been suggested that Type I strains should be subdivided into West African Types 1 and 2 of M. africanum and Type II strains reclassified as the Uganda genotype of M. tuberculosis.43 Around half the cases of human TB in West Africa are caused by M. africanum, and there is a tendency for patients to be older, more frequently infected with HIV, and more malnourished than those with disease caused by classical M. tuberculosis, indicating that the former is of lower virulence.42

In addition to M. bovis, there are strains that clearly belong to the M. tuberculosis complex with unique distinguishing properties that cause disease in various animals, with humans as rare secondary hosts. The first to be described was M. microti, so-named because it was first isolated from the vole, Microtus agrestis, and originally termed the vole tubercle bacillus. It was regarded as being attenuated in humans, having the same order of virulence as BCG vaccine, and was evaluated as a vaccine for human use in comparison with BCG in clinical trials,44 but there have been several reports of human TB due to M. microti in immunocompetent and immunocompromised patients in recent years.45 A very similar organism has been isolated from the dassie, or rock hyrax, and the llama.

Strains with sufficient genomic differences to justify the separate species name M. pinnepedii have been isolated from tuberculous lesions in free and captive seals and sea lions in Australia, New Zealand, South America, and the Netherlands, and transmission to animal keepers has been confirmed by tuberculin skin testing and interferon-gamma release assays.46,47 A further cluster termed M. mungi has been isolated from the banded mongoose (Mungos mungo) in Botswana.48 Strains with distinct features have been isolated from tuberculous lesions in other animals including oryx, water buffaloes, and cats but have not been given separate species names.

KNOWLEDGE OF M. TUBERCULOSIS

PHENOTYPES, GENOTYPES, AND CLINICAL IMPLICATIONS

Before the development of gene transfer we did not know: (1) acidfastness is regulated by a signal transduction pathway; (2) the primary attenuation of BCG is a loss of a Type VII secretion system;

(3) M. tuberculosis lives an autarkic lifestyle; (4) the targets of isoniazid, its mechanisms of action, and its mechanism of resistance;

(5) the targets for other TB-specific drugs; and (6) persistence is mediated by a unique expression profile and can be reversed by stimulating respiration. These unknown facts seemed surprising for the pathogen that Koch demonstrated to be the cause of an infectious disease. A brief summary follows of how genetics provides the critical mutants for elucidating the facts.

Acid-fast staining is regulated by a signal transduction pathway

Microscopic analysis of clinical samples remains one of the fastest way to diagnose infections. The Gram stain, developed by Christian Gram, remains a standard test for distinguishing Gram-positive organisms (such as Staphylococci or Streptococci) from Gram-negative organisms (such E. coli or Pseudomonas). M. tuberculosis and the leprosy bacillus stain acid-fast positive. Dyes can be removed from the cell walls of Gram-negative bacteria when treated with alcohol, whereas Gram-positive organisms retain the dye. During the process of investigating the role of specific genes of mycolic acid biosynthesis, it was discovered that a mutant of M. tuberculosis that had the kasB gene deleted no longer stained acid fast. The kasB gene encodes a keto−acyl synthase enzyme that mediates the addition of the last six carbons on the mycolic acid chain increasing the length from 76 carbons to 82 carbons. Surprisingly, the elimination of this enzyme makes the M. tuberculosis cells lose their acid-fast staining property.49 Importantly, a year after Koch discovered the tubercle bacillus, another group found M. tuberculosis strains that did not stain acid-fast positive still caused TB. This was called Koch’s paradox.50 Interestingly, this kasB deletion has been shown to infect and kill mice lacking T and B cells (SCID mice), but is unable to kill immunocompetent isogenic parents. Surprisingly, the enzyme activity of the KasB keto−acyl synthase is turned off when two of the threonines located within the amino acid sequence of the protein are phosphorylated, demonstrating that this enzyme activity is regulated by a signal transduction pathway of a serine/threonine kinase.51 Unlike most bacteria, M. tuberculosis possess 11 of these serine/threonine kinases that are well-known to regulate different pathways in eukaryotic cells. This enzyme regulation activity provides an explanation for Koch’s paradox as it demonstrates there exist strains of M. tuberculosis that can infect, multiply in hosts, and are not virulent unless the patient’s immune system is compromised. Moreover, the acid-fast negative bacteria represent a variation of M. tuberculosis that can persist in the host. Finally, since acid-fast staining represents the method by which M. tuberculosis cells are detected, subpopulations of acid-fast M. tuberculosis cells may represent an unidentified and unappreciated set of

M. tuberculosis cells representing a significant, unidentified set of organisms in TB patients. The development of a reagent such as a monoclonal antibody that selectively identifies these acid-fast- negative organisms might be a useful tool for characterizing these persistent forms of the organism.

The primary attenuation of BCG is a loss of a type VII secretion system

BCG (bacilli Calmette and Guerine) is a TB vaccine still given to children at birth or within the first two weeks of birth in most developing countries. While it has not eliminated TB, numerous studies demonstrate BCG reduces severe forms of TB in children and hence WHO recommends its administration in countries where TB is highly present.52 The strain was originally isolated from a bovine tubercle bacillus in 1904 and demonstrated by Drs. Calmette and Guerin to be highly attenuated in animals. In 1921, Dr. Calmette administered the strain orally to a child whose mother had died of TB.53 The child never developed TB and by 1928, the League of Nations had recommended it be given to all children at birth. The efficacy results with BCG have proven to be variable with over 70% protection in some trials and no protection in others. These differences have been postulated to be due to: (1) genetic differences in BCG isolates, (2) human population differences, and/or (3) differences in exposure to environmental mycobacteria.54

Before genome sequences and gene transfer, there was no way to know the molecular genetic basis for the attenuation of BCG. The lab of Ken Stover was the first to discover genome differences between BCG and M. tuberculosis using genomic subtractive hybridization methodology.55 This elegant study clearly showed many genomic deletions amongst various BCG strains but also reported one deletion, the RD1 deletion, was common to all BCG strains. The availability of microarrays from the original M. tuberculosis genome sequence demonstrated significant accumulations of various deletions of BCG strains that had been passaged in independent labs over the years.56 It is noteworthy to point out that this group was unable to restore full virulence to BCG by transferring in the deleted genes. In fact, virulence in SCID mice was restored when a cosmid spanning RD1 was introduced into BCG.57 Precise deletions of only the RD1 region in virulent M. tuberculosis and M. bovis showed that this deletion was sufficient for a high degree of attenuation.58 The RD1 region has been extensively studied and found to be involved in generating connections between the phagolysosomal vacuole in which M. tuberculosis resides and the cytoplasm of the infected cell.59,60 The clinical implications are numerous. First, the deletion of the genes encoding ESAT-6 and CFP-10 explains why the QuantiFERON test can distinguish BCG vaccination from M. tuberculosis infection. Second, the attenuation of BCG is related to its ability to cause lysis of lung pneumocytes or its connection of phagosomal vacuole to the cytoplasm of M. tuberculosis cells providing tools for probing the cell biology of macrophages and dendritic cells. Lastly, the precise deletion of RD1 from M. tuberculosis or M. bovis failed to improve, or only slightly improved, vaccine derivative efficacy. Therefore, the hypothesis became that even if the original BCG was found, its

protectiveness was not going to be improved in the current human population. The world needs a better TB vaccine that elicits a different protective immune response than BCG.

The autarkic lifestyle of

M. tuberculosis

By studying the biology of the auxotrophic mutants of M. tuberculosis, it is clear that the tubercle bacillus, unlike many other intracellular pathogens, has evolved to have an autarkic or self-suf- ficient lifestyle. Other intracellular pathogens, such as Francisella or Legionella, are naturally occurring amino acid auxotrophs. Whereas, M. tuberculosis and M. leprae sequences reveal intact genes to synthesize all 20 amino acids,22,24 M. tuberculosis fails to be able to multiply in mice if it is unable to make leucine,10 methionine,61 or arginine.62 Plants, fungi, and most bacteria have the ability to make all 20 amino acids. Legionella cannot make arginine, methionine, cysteine, leucine, valine, isoleucine, phenylalanine, and tyrosine. Interestingly, leucine starvation for M. tuberculosis is bacteriostatic whereas starvation for methionine or arginine is rapidly bactericidal thereby suggesting the enzymes to make these amino acids might be excellent drug targets.

The target of Isoniazid

Isoniazid was discovered in 195263,64 as an analogue of nicotinamide, which was serendipitously discovered to kill M. tuberculosis while it was used as a remedy to treat the ill effects of cancer therapies.65 During the synthesis of one of these synthetic molecules, namely γ-pyridyaldehyde thiosemicarbazone, an intermediate was discovered to have surprisingly strong activity against TB. This intermediate was called isonicotinic acid hydrazide or INH.63,64 The combination of Streptomycin, p-amino salicylic, and Isoniazid was the first combination therapy that led to sterilizing chemotherapy.

Although researchers had found this wunderkind of a drug, a major hurdle still remained: how exactly did it work? The answer to this question lingered over TB research for the next 50 years.

For five decades following the discovery of INH, many researchers attempted to determine its mechanism of action. The specificity to act only on mycobacteria and the lack of gene transfer in mycobacteria made it impossible to discover the target of INH and thereby elucidate its precise mechanism of action. A drug target is a component of a cell (usually an enzyme or a protein component of a complex cellular machine) to which a drug binds and inhibits, and this inhibition leads to the death of the cell. The mapping and sequencing of genes conferring drug resistance allows for the elucidation of the mechanism of drug action and drug resistance.

For example, streptomycin—the first drug discovered to have bactericidal activity against M. tuberculosis—was also found to be active against E. coli. By isolating mutants and mapping the locations of their resistance alleles, researchers concluded that resistance was due to a ribosomal protein (S12) of the small subunit designated rpsL.66 Consistent with this conclusion, the recent elucidation of the three-dimensional structure of the ribosome with streptomycin shows the binding to streptomycin to the S12 protein.67

Without gene transfer, early published reports on INH mechanisms of action were associated with inhibition of DNA biosynthesis, inhibition of NAD glycosylase, and disruption of cell walls (reviewed in Vilcheze and Jacobs Jr.2). Frank Winder was the first to find evidence that INH inhibited mycolic acid biosynthesis.68 Importantly, Kuni Takayama, showed that the death of M. tuberculosis cells correlated with the inhibition of mycolic acid biosynthesis.69 Moreover, Takayama demonstrated that the inhibition of mycolic acid biosynthesis resulted in an accumulation of a monounsaturated fatty acid allowing him to conclude that the specific target had to be: (1) a desaturase, (2) a cyclopropane, or

(3) an enzyme component of the fatty acid elongation pathway.70 The question of the target was further complicated by the fact that most of INH-resistant mutants that had been isolated were found to correlate with a loss of a catalase peroxidase activity.71 A resolution of the mechanism of action of INH would not be resolved until gene transfers could be performed in mycobacteria.

To prove that a phenotype (INH-resistance) was mediated by a specific genotype (a specific DNA sequenced allele), it was necessary to: (1) isolate a mutant that was INH-resistant; (2) clone the mutant allele; and (3) transfer the allele and demonstrate it ­conferred INH-resistance to the parental strain. While this set of three conditions seemed simple enough, performing allelic exchanges in M. tuberculosis chromosome had never been demonstrated. The easiest way to clone a gene was to clone it on a plasmid, but plasmid transformation would have to wait until a plasmid transformable M. smegmatis mutant mc2155 was

developed.72,73 Using mc2155, INH-resistant mutants were isolated that either had a loss of function (a loss of the INH activator activity74) or a gain of INH-resistance in a gene named inhA.75 The loss of function did indeed correlate with a loss of a katG-catalase- peroxidase activity, which suggested that the catalase peroxidase was an activator that activated INH. The InhA encodes a NADHdependent enoyl-reductase (Figure 3.2), which is involved in elongating the mycolic acid chain from C18 to a C50 or C56 (reviewed in Vilcheze and Jacobs Jr.2). Surprisingly, X-ray crystallographic analyses revealed the actual inhibitor of InhA to be an INH-NAD adduct.76 Importantly, the genetic data found in M. smegmatis was confirmed with M. tuberculosis.77–79

Thus, we know that that INH, when activated by the katG- encoded catalase peroxidase, forms an adduct with NAD that inhibits the enoyl reductase of mycolic acid synthase. As predicted for a drug target, a mutation within the structural gene that confers resistance would display reduced binding of the drug. This was observed for the INH-NAD adduct.79 In addition to structural mutations, target proteins can also confer resistance by target overexpression thereby titrating the inhibitor.51

Persistence: The single greatest impediment to TB control

Persistence was defined by Walsh McDermott as the capacity of the tubercle bacillus to survive sterilization in mouse tissues.80 Numerous clinical trials had demonstrated that curing

TB required long therapies as too short a therapy would lead to reactivated TB with M. tuberculosis strains that are fully drug sensitive. These studies demonstrated that TB has the ability to persist during treatment.81 In order to better understand this phenomenon of persistence, a highly reproducible in vitro model was developed for M. tuberculosis and INH.2,82,83 For this model, if 106 exponentially growing M. tuberculosis cells are freshly inoculated into media with or without INH and samples are taken daily for the next 28 days, 99%–99.9% of the INH-treated cells are killed in the first 3 days, but the remaining cells are not sterilized (Figure 3.3). The 0.1%–1% survivors are INH-sensitive when regrown. These represent a subpopulation of M. tuberculosis cells that are expressing phenotypic resistance. By analyzing the mRNA

Figure 3.3  In vitro model revealing Isoniazid persisters and their sterilization when INH is combined with vitamin C. Exponentially growing M. tuberculosis cells are mixed with or without INH and viable colony forming units are quantitated at various days postaddition as can be seen by day 3, 99%–99.9% of the cells are killed but 0.1%–1% of cells express phenotypic INH resistance and eventually lead to the emergence of genetically resistant INH cells. The addition of vitamin C with INH leads to the rapid sterilization of the

M. tuberculosis culture.

Concluding remarks  47

expression pattern microarrays, we discovered that these INHpersister cells had down-regulated many of the normal growth genes involved in cell division or respiration but were upregulated for stress responses.84 These INH-tolerant cells can be visualized using a dual reporter mycobacteriophage (Figure 3.4), which expresses an RFP fused to a highly upregulated dnaK promoter but will not express a GFP that is fused to a strong phage promoter.84 Importantly, we have discovered that in the setting of INH administration, the addition of vitamin C or N-acetyl cysteine results in sterilization of the cultures and this correlates with increased respiration.82

CONCLUDING REMARKS

Although TB was one of the first diseases proven to be caused by an infectious agent over 135 years ago, it remains the single leading infectious cause of death in the world today. Success in controlling many other infectious diseases has come about with rapid sterilizing chemotherapies and/or effective vaccines. Moreover, the ability to observe gene transfer by conjugation, transformation, or transduction has allowed for the discovery of drug-resis- tance genes, virulence genes, and many genes involved in evading host defenses. This knowledge has led to improved therapies for many other pathogens.

The characteristic slow growth of M. tuberculosis and its virulence have slowed down its research. However, the ability to perform gene transfer in M. tuberculosis and other mycobacteria has provided many new ways to understand the tubercle bacillus and its biology. Moreover, the availability of thousands of genomic sequences of M. tuberculosis and efficient means for generating transposon mutagenesis and targeted gene disruptions will most

certainly lead to many new chemotherapeutic and immunotherapeutic interventions.

It is likely that the single greatest impediment to TB control is the ability of M. tuberculosis to persist when assaulted by bactericidal drugs or immune effectors. The phenomenon of persistence is particularly relevant for this slowly replicating organism and has been difficult to study. There are now ways to study M. tuberculosis persistence with precisely defined genomes and reproducible methodologies. The slow growth of the tubercle bacillus still makes the analysis of this phenomenon challenging. Resistance is not mediated by specific singular genes, as can be observed for drugresistant mutants but rather, a complex genetic array of expression patterns that confer phenotypic resistance. By understanding the mechanisms that M. tuberculosis uses to enter into this persistence state, novel strategies can be developed to treat this deadly disease.

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