Mycobacterium tuberculosis: drug resistance, virulence and possible solutions

Danilenko V.N.; Zaychikova M.V.; Dyakov I.N.; Shur K.V.; Maslov D.A.

MYCOBACTERIUM TUBERCULOSIS-. DRUG RESISTANCE, VIRULENCE AND POSSIBLE SOLUTIONS

Danilenko VN1>2 ^ Zaychikova MV1, Dyakov IN3, Shur KV1, Maslov DA1

1 Laboratory of Bacterial Genetics, Department Genetics and Biotechnology, Vavilov Institute of General Genetics, Moscow

2 BIOAN Research Center for Biotechnology of Antibiotics, Moscow

3 Laboratory of Immunoglobulin Biosynthesis, Mechnikov Research Institute of Vaccine and Sera, Moscow

In spite of successful measures taken to reduce mortality associated with tuberculosis, this disease is still widely spread. In some Russian regions the number of patients with tuberculosis is no short of the epidemic level. The long-term use of antibiotics, changes in the composition of the human microbiota and a few other factors have contributed to the emergence of drug-resistant and hypervirulent sublineages of Mycobacterium tuberculosis. Insufficient fundamental knowledge of mechanisms underlying the emergence and evolution of M. tuberculosis clones simultaneously resistant to a wide spectrum of antibiotics and exhibiting increased virulence complicates the situation and necessitates a new strategy to combat the disease. The key concepts of this strategy are «superorganism», «microbiota» and «resistome». The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains should be addressed in the context of the «superorganism»; among its components are the human body, its microbiota (specifically, the bacteria that affect the immune status), and M. tuberculosis itself. Clinically studied phenotypes and genotypes of MDR/XDR strains are a result of clonal variability that M. tuberculosis develops as part of this «superorganism». Therefore, it is important to focus on the development of vaccines, adjuvants and probiotics with selective immunomodulating and antioxidant properties.

Keywords: tuberculosis, Mycobacterium tuberculosis, drug resistance, adjuvants, vaccines, cross-drug resistance

Funding: this work was supported by the Project of the Ministry of Education and Science of the Russian Federation «The role of region-specific polymorphisms of virulence genes in the formation of drug resistance of Mycobacterium tuberculosis» (Project ID RFMEFI61317X0068).

gg Correspondence should be addressed: Valery N. Danilenko Gubkina 3, Moscow, 119991; valerid@vigg.ru

Received: 05.06.2018 Accepted: 10.07.2018

DOI: 10.24075/brsmu.2018.038

MYCOBACTERIUM TUBERCULOSIS. ПРОБЛЕМЫ ЛЕКАРСТВЕННОЙ УСТОЙЧИВОСТИ, ВИРУЛЕНТНОСТИ И ПОДХОДЫ К ИХ РЕШЕНИЮ

В. Н. Даниленко1*2 М. В. Зайчикова1, И. Н. Дьяков3, К. В. Шур1, Д. А. Маслов1

1 Лаборатория генетики микроорганизмов, отдел генетических основ биотехнологии, Институт общей генетики имени Н. И. Вавилова, Москва

2 Научно-исследовательский центр биотехнологии антибиотиков «БИОАН», Москва

3 Лаборатория биосинтеза иммуноглобулинов, Научно-исследовательский институт вакцин и сывороток имени И. И. Мечникова, Москва

Несмотря на достигнутые успехи мероприятий, направленных на снижение смертности от туберкулеза, данное заболевание по прежнему крайне распространено, а в некоторых регионах России численность больных достигает показателей, характерных для уровня эпидемии. Многолетнее широкое применение антибиотиков, изменение состава микробиоты человека и ряд других факторов привели к появлению лекарственноустойчивых и высоковирулентных сублиний Mycobacterium tuberculosis. Недостаточность уровня и объема фундаментальных знаний о механизмах возникновения и формирования клонов M. tuberculosis, одновременно устойчивых ко многим антибиотикам и обладающих повышенной патогенностью, усложняет проблему и требует разработки новой концепции борьбы с туберкулезом. Ключевые понятия этой концепции — «суперорганизм», «микробиота» и «резистом». Возникновение форм с множественной (МЛУ) и широкой (ШЛУ) лекарственной устойчивостью следует рассматривать в контексте их формирования в составе некоторого суперорганизма, элементами которого являются собственно организм человека, его микробиота (в том числе влияющая на иммунный статус) и M. tuberculosis. Клинически тестируемые фенотипы и генотипы штаммов МЛУ/ШЛУ формируются на основе клональной изменчивости M. tuberculosis в «суперорганизме». Поэтому при разработке противотуберкулезных препаратов следует обращать особое внимание на создание вакцин, адъювантов и пробиотиков с селективными иммуномодулирующими и антиоксидантными свойствами.

Ключевые слова: туберкулез, Mycobacterium tuberculosis, лекарственная устойчивость, адъюванты, вакцины, перекрестная лекарственная устойчивость

Финансирование: работа была выполнена в рамках проекта Министерства образования и науки Российской Федерации №RFMEFI61317X0068 — «Роль регион-специфичных полиморфизмов генов вирулентности в формировании лекарственной устойчивости Mycobacterium tuberculosis».

Для корреспонденции: Валерий Николаевич Даниленко ул. Губкина, д. 3, г Москва, 119991; valerld@vigg.ru

Статья получена: 05.06.2018 Статья принята к печати: 10.07.2018

DOI: 10.24075/vrgmu.2018.038

In 2017 the Russian government adopted a strategy to prevent the spread of antimicrobial resistance in the Russian Federation by 2030. One of the goals set by the strategy is to study the mechanisms underlying the emergence of antimicrobial resistance and to develop novel antimicrobial medications, alternative methods, technologies and means of prevention, diagnosis and treatment of infectious diseases in humans, animals and plants.

According to the 2016 report by the World Health Organization, that year tuberculosis reached the incidence of 10.4 million new cases and killed 1.8 million people becoming the leading cause of death associated with infection [1]. Mycobacterium tuberculosis is the causative agent of tuberculosis. The emergence and spread of its multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains are the central challenges in the battle against this disease [2-4]. Statistically, 4% of new and 21% of previously treated cases are multidrug-resistant. In Russia these numbers are 22% and 53%, respectively. To survive, mycobacteria can evolve new mechanisms of resistance in response to any currently known drug. They are also naturally resistant to antibiotics, being equipped with a large arsenal of genes and genetic systems that make up the resistome. Proposed in 2006, the resistome concept refers to the set of antibiotic resistance determinants, including resistance genes that are intrinsic to a certain bacterial strain, organism or ecosystem [5, 6]. The resistome of M. tuberculosis comprises genes coding for different protein classes, such as transporters, proteins that modify the targets or chemical structure of pharmaceutical drugs, transcription factors involved in stress response, and some others.

Another alarming trend is the emergence of previously unknown hypervirulent M. tuberculosis sublineages [7-9]. In vitro and in vivo studies carried out in macrophage and mouse models, respectively, have established an association between virulence and a genotype the pathogen belongs to [10]. Increased virulence is mostly observed in the Beijing genotype (lineage). Its epidemiological significance cannot be overestimated as it continues to spread globally and tends to frequently evolve into MDR forms [11, 12]. The Beijing strains are genetically heterogenous branching off into a few sublineages. Although the high frequency of increasingly virulent and drug-resistant forms is generally typical for the entire Beijing family, it still varies among its sublineages [13, 14]. Moreover, the clinically significant characteristics of these bacteria can vary among the strains representing the same sublineage.

Over the past decades, the study of mechanisms underlying the emergence of MDR/XDR strains of M. tuberculosis, the discovery of antibiotics capable of killing these strains and the development of genetically engineered vaccines and adjuvants to prevent and treat the disease have helped the researchers to identify a few important problems [15]. We cannot develop a novel effective drug unless we understand molecular and genetic mechanisms underlying the emergence and evolution of multiple drug resistance and virulence.

Drug resistance and development of novel antituberculosis antibiotics

Bacteria are not limited to acquired drug resistance. They are also naturally, though not so strongly, resistant to antibiotics. When M. tuberculosis cells are exposed to an antibacterial agent, the pathogen activates its transcription factors that regulate the expression of genes responsible for the modification of the drug or its target and activation of reverse transport systems that pump the drug or its derivatives out of

the bacterial cell. Genes underpinning the mechanisms that ensure natural resistance to antibiotics are targeted by a variety of biological factors including antibiotics, which affects their expression and therefore reduces susceptibility to drugs.

The use of antibiotics for treating co-infections in patients with tuberculosis or their absorption with food can contribute to increasing drug resistance of M. tuberculosis.

In 2015 there were over 580,000 patients infected with MDR and XDR tuberculosis strains worldwide. Their dramatic spread was driven by the long-term use of the same old medications. It was not until recently that bedaquiline, the first new antituberculosis drug in 40 years, was introduced into clinical practice [16].

In this light, development of novel antituberculous drugs is becoming a task of paramount importance. These pharmaceutical agents are expected to satisfy a number of requirements, such as high antimicrobial activity against both drug-sensitive and MDR strains of M. tuberculosis and excellent specificity to a new biological target. At present, development of novel antituberculosis drugs that have a potential to overcome the phenomenon of drug resistance and/ or to reduce the length of treatment is carried out by the leading pharmaceutical companies and research groups all over the world, including Lilly TB Drug Discovery Initiative, GSK, Roche, Sanofi, TB Alliance, Colorado State University, and some others (http://www.newtbdrugs.org).

In Russia, research in this field was stimulated by the Pharma-2020 federal program. For example, Vavilov Institute of General Genetics, Moscow, has been conducting a series of preclinical trials in collaboration with medicinal chemists from state-funded and commercial research institutions, such as Postovsky Institute of Organic Synthesis, the Ural Branch of RAS; Gause Institute of New Antibiotics; Novosibirsk Institute of Organic Chemistry, the Siberian Branch of RAS; Zelinsky Institute of Organic Chemistry; BIOAN Research Center, and New Science Technologies Ltd. The tested drugs belong to new classes of medical compounds, such as derivatives of usnic acid [17], substituted azolo(1,2,4,5)tetrazines [18], aminopyridines and aminopyrimidines[19], and aminopurine derivatives [20].

The advent of the postgenomic era witnessed two approaches to the discovery of novel antituberculosis drugs: target-to-drug and drug-to-target [21-23].

Unfortunately, the first approach did not fully live up to the expectations. Many drug candidates with good inhibiting properties exhibited against the target enzyme in vitro either were not active against M. tuberculosis in vitro due to the low permeability of the bacterial cell wall or were ineffective in in vivo models because the target was no longer vitally important for the bacteria under those conditions [22, 24].

Yet there are a few successful experiments worth mentioning. In one of them, a compound termed BDM31343 was identified capable of inhibiting EtHR, the EthA repressor which, in turn, activated ethionamide [25]. This compound was shown to increase susceptibility of mycobacteria to ethionamide enhancing its effect threefold in mouse models [26].

Because the target-to-drug approach proved to be less than effective, researchers turned to a more traditional drug-to-target search strategy based on whole-cell screening [24]. All drugs currently used to treat tuberculosis, including bedaquiline, pretomanid, delamanid, Q203, SQ-109, and BTZ043, were discovered using this approach [27].

The drug-to-target search strategy often involves high-throughput screening against M. tuberculosis H37Rv cultures and related M. bovis BCG and M. smegmatis model strains

[24, 28]. The libraries of chemical compounds used in such experiments are enormously huge. For example, GSK researchers consecutively screened a total of 2 million compounds against M. bovis BCG and M. tuberculosis H37Rv to select 7 low-toxic drug candidates exhibiting high activity and capable of diffusing through the cell membrane [29].

The drug-to-target approach entails the need for whole-genome sequencing of antibiotic-resistant mutants in order to identify potential biotargets and for further research aimed at confirming the activity of selected drug candidates against those targets [24].

The discovery of drugs capable of killing persistent forms of M. tuberculosis remains a global challenge. So far, pyrazinamide appears to be the most effective antibiotic against persistent M. tuberculosis [30]. Resistance to pyrazinamide can significantly worsen clinical prognosis, especially in patients with MDR tuberculosis [31, 32].

Development of antituberculosis vaccines

Although vaccination against tuberculosis is advocated everywhere, the incidence of the disease remains abnormally high. This can be explained by the low efficacy of the BCG vaccine used for global immunization, which varies between 0% and 80% depending on the individual's age, immune status, area of residence, etc. [33]. Among other reasons reducing the efficacy of the vaccine is the genetic diversity of the pathogen. It is hypothesized that resistance to vaccination demonstrated by the ubiquitous Beijing strains may explain their evolutional success [11]. Considering that, creation of novel vaccines against tuberculosis should be a top-priority task.

Development of such vaccines has taken two paths. The first is to use the attenuated pathogen itself. For this purpose, deletion mutants of M. tuberculosis are being engineered. Among the knocked-out genes are those coding for virulence factors, such as Mce (mammalian cell entry) proteins facilitating pathogen invasion; PPE proteins; proteins participating in lipid synthesis; sigma factors; two-component systems, and some others.

The second approach is to compose a subunit vaccine containing genetically engineered pathogen antigens [34, 35]. Advantageously, such vaccines are highly specific, have a low allergenic potential, are easy to fabricate, cost-effective, and convenient to store and transport [36].

Candidate proteins for next-generation vaccines include secretory proteins of the Ag85 complex that interact with T cells; TB10.4 (rv0288); Hsp65; PE and PPE proteins. The greatest promise is held by the protein components of the ESAT6 and CFP secretion systems [36].

However, in spite of the considerable interest in this field, genetically engineered vaccines did not live up to the expectations. The main drawback of such vaccines is their low immunogenicity.

The key challenge in the development of genetically engineered vaccines is the selection of optimal antigens [36]. Here, strong antigenic potential is exhibited by the structural elements conferring pathogenicity, of which M. tuberculosis has over 300; some of them have already been segregated to design a subunit vaccine [37]. Many of these genes typically have a single nucleotide polymorphism resulting in an amino acid substitution, which affects the structure of the protein modulating its antigenic activity. At present, the intraspecies diversity of M. tuberculosis is unfairly overlooked in the production of genetically engineered vaccines, which are usually based on a sequence of the standard laboratory strain H37Rv. If cultured for too long, the M. bovis strain used for BCG

production can develop mutations (a natural consequence of its microevolution) reducing the efficacy of the vaccine [38]. It is possible that the antigenic activity of proteins is not identical in different M. tuberculosis strains.

Another promising area of research is related to the development of a candidate mucosal vaccine against tuberculosis that induces the sustained local mucosal immune response. The importance of the local immunity against tuberculosis has been demonstrated in a number of works. It has been shown that intranasal administration of protective IgA, pretreatment of virulent M. tuberculosis with protective IgA and intranasal administration of M. bovis BCG trigger a sustained immune response to M. tuberculosis infection. [39-42]. The mucosal vaccine administered alone or in combination with its subcutaneous form could offer a solution to the problems accompanying BCG vaccination.

It should be noted, though, that so far none of the mentioned vaccines have been introduced into clinical practice. Again, the drawback of such vaccines is their low immunogenicity necessitating the use of adjuvants.

Prospects for the development of antituberculosis vaccine adjuvants based on probiotic strains

An adjuvant is a compound with non-specific activity that enhances the immune response to antigens administered in combination with adjuvants [43]. Of all commonly used adjuvants, aluminum hydroxide and aluminum phosphate are the most remarkable [44]. However, the boosting effect of these compounds is not always sufficient. Other substances that can serve as adjuvants include synthetic polyoxidonium and chitosan, a naturally obtained polysaccharide. Bacterial cell components are also tested for their adjuvant properties, specifically those that contain pathogen-associated molecular patterns (PAMP) triggering the immune response. A few works have already described the adjuvant effects of lactic acid bacteria [45], bacterial cell wall components [46, 47], the fibronectin-binding protein 1 of Streptococcus pyogenes [48], surface flagellins [49], etc.

Some strains of probiotic bacteria, bifidobacteria in particular, can trigger production of Th17 and Th1 cytokines [50] that play an important role in the induction of the mucosal immune response against tuberculosis [39]. Administered intranasally, probiotics can exert their vaccine-boosting effect, inducing protective immunity against virulent strains of M. tuberculosis. Commensal bifidobacteria and lactobacilli are capable of stimulating the mechanisms of protective immunity, regulating the secretion of both pro- and anti-inflammatory cytokines. As a rule, in vitro studies of the immunomodulating activity of bacterial strains employ intestinal cell lines (Caco-2, HT-29) or immunocytes (EC-6, THP-1). Similar in vivo experiments are carried out in lab animals (healthy or with compromised immunity, gnotobiotic or those with experimentally induced infections or non-infectious pathologies) [51, 52].

It should be noted that different strains of bifidobacteria and lactobacilli, as well as their components, have different immunomodulating effects in terms of intensity [53-55]. Lactobacilli and bifidobacteria have already demonstrated their adjuvant effects in the vaccines against viruses [56, 57], streptococci [58], and allergies [48, 59]. Intranasal lactobacilli boost local mucosal immunity and modulate systemic mechanisms of the immune defense, increasing resistance to the respiratory syncytial [56, 57, 60] and influenza viruses. These findings allow us to conclude that intranasally administered probiotics can act as adjuvants to a vaccine,

Mycobacterium tuberculosis (the hypothetical ancestral)

The Beijing-ancestral sublineage (ancient)

I

The Beijing-modern sublineage (modern)

The B0/W-148 sublineage

i

7.000 years ago

Emergence of the Beijing-ancestral lineage on the territory of North China

1.000 years ago Formation of the Beijing-modern sublineage in China

* "X

% The spread of the Beijing-ancestral in South Africa following migration from China in the late 18th- early 19th century

100 years ago

The Beijing-modern sublineage appears on the territory of Russia, brought there by the migrants

50 years ago (the era of antibiotics)

The B0/W sublineage branches off from the Beijing-modern sublineage in the Russian Federation

Present day

Globally observed changes in the immune status of the world population resulting from HIV, chronic conditions and stress

Fig. 1. Evolution of the Beijing lineages. Wide use of antibiotics In the recent decades has provided selective advantage to the B0/W-14 strain characterized by a high level of drug resistance

effectively inducing the protective immune response against M. tuberculosis in the mucosa.

CONCLUSION

Throughout their history, humans have been colonized by latent and active M. tuberculosis [61]. The Beijing strains that emerged on the territory of modern China about 7, 000 years ago and have widely spread across the world since then are a live example of the ongoing evolution of the pathogen that still forms new sublineages, such as B0/W-148 (Fig. 1) [62, 63].

It is known that susceptibility to tuberculosis is affected by the level of gene expression in T cells [64]. In this light, the problem of drug resistance and increased virulence and the discovery of a new generation of antituberculosis drugs should be addressed in the context of the "superorganism" concept. The antibiotic-based treatment of tuberculosis affects not only the pathogen, but the host as well, altering the microbiota composition and, therefore, compromising the immunity, which is known to be directly affected by the gut microbiota. Antibiotics interfere with the functions of the central and peripheral nervous systems of the host; other systems and

organs may also be affected. The unregulated use of antibiotics in agriculture leads to the formation of cross-resistance to drugs in bacteria. Besides, antibiotic-based therapies can "wake" the latent tuberculosis infection.

To sum up, the major factor that has been stimulating the positive selection of drug-resistant virulent forms of M. tuberculosis over the past 60 years is the uncontrolled use of antibiotics. Other factors include the wide spread of immunity-compromising diseases, such as HIV, type 2 diabetes mellitus, hepatitis B, etc. Diet and migration stimulated by globalization lead to the shifts in the gut microbiota composition, which in turn make their contribution to the problem. The genetic diversity of M. tuberculosis shaped by single nucleotide polymorphisms in the genes responsible for virulence, natural resistance to drugs and persistence, IS elements and possibly CRISPR-cas systems also affect the adaptation of the pathogen to the host [65, 66].

Advances in epidemiology, molecular genetics, comparative genomics, proteomics and systemic biology have improved our understanding of the multifactorial nature of tuberculosis revealing the need for a tailored approach to the treatment of this disease.

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