Molecular mechanism of Clofazimine resistance in tuberculosis

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INTRODUCTION
Tuberculosis (TB) is the most crucial bacterial infectious disease and public health concern worldwide caused by Mycobacterium tuberculosis that naturally infects lungs and other organs, such as the kidney, spine, or brain.
The increase of multidrug-resistant TB (MDR-TB)/ extensively drug-resistant tuberculosis (XDR-TB), with resistance to isoniazid and rifampin, poses major clinical challenges to treatment with present available anti-TB drugs. The crisis is enhanced by the increasing emergence of extensively drug-resistant (XDR) strains of M. tuberculosis, which cause inability to treat diseases effectively with subsisting elements (Zumla et al., 2013). According to the latest World Health Organization (WHO) report, there were an estimated 10 million people infected with TB in 2018 and 1.5 million deaths were imputed to the disease. One million children infected with the disease, including 251000 persons who died were reported among HIV-infected persons (WHO, 2019). However, even more disturbing is the emergence of drug resistance. In 2017, there were an estimated 450,000 cases of MDR-TB and 170,000 deaths were as a result. The treatment outcome of MDR-TB and XDR-TB patients is often poor and unsuccessful in the absence of an optimal number of active drugs (Belachew T et al., 2022;Seung KJ et al., 2015). MDR-TB is a disease caused by bacterial strains of M. tuberculosis that are resistant to at least rifampicin and isoniazid, two main drugs in the treatment of the disease. Depending on the present situation, it is widely acknowledged that we need to develop new beneficial drug combination for TB and that these new regimens should be tested at the preclinical stage, rather than testing a series of single drugs separately, wherein compensate the TB drug development pipeline more effectively . In addition to being MDR are also resistant to any antibiotics that are commonly used to treat a variety of illnesses or fluoroquinolone and to at least one of the most injectable second-line drugs: Clofazimine, kanamycin, capreomycin or amikacin (Gandhi et al., 2006). Moreover, clofazimine which was currently exhibited to reduce the duration of TB in a mouse model of TB (Tyagi et al., 2015;Ivan et al., 2019). Early identification of all forms of drug resistance in TB is a main factor to reduce and contain the spread of these resistant strains. A better knowledge of the mechanisms of different anti-TB drugs and the development of drug resistance will allow identifying new drug targets and better findings to detect drug resistance. This present investigation will review the background, diagnosis, mode of action and resistance mechanisms of the main anti-TB drugs as well as Clofazimine drugs on recent details described with anti-TB activity. New tuberculosis drug which is used for leprosy and multidrugresistant tuberculosis prevention (Olayanju et al., 2018), is also active against Masoud Abessi and shows synergistic action when given together with amikacin (Singh et al., 2014;Shen et al., 2010).
Clofazimine is a significant antimicrobial force which has activity in vitro against mycobacteria. It is a riminophenazine drug which was initiated as an anti-TB agent in the 1950s, (Barry et al., 1957) but is also mostly used to treat leprosy (Browne et al., 1962). Although this drug has showed good reaction against M. tuberculosis in vitro, it was not used in the treatment of pulmonary TB MDR-TB, there has recently been interest in the use of clofazimine to treat MDR-TB (Zumla et al., 2013) and its use might shorten MDR-TB treatment to 9 months. Clofazimine, commonly known as B663, which was first synthesized in 1954 by a group study of scientists (Barry et al., 1957) at Trinity College, Dublin, was originally intended as an anti-tuberculosis drug but was substantiated ineffective. In 1959, a researcher named Y. T. Chang destines its essential effectiveness against leprosy. Clofazimine was approved in the United States in 1986 for medical purpose use (Dagron, 2016). It is on the World Health Organization's List of Essential Medicines, the most functionalize and safe medicines needed in a health system in the world (WHO, 2015). Inconsistent results in animal models hindered its development for tuberculosis, but it was licensed for treatment of leprosy in 1969 (Reddy et al., 1999). Clofazimine has a biological shelf-life of about 70 days. Common side effects include abdominal pain, itchiness, dry skin diarrhea and change in color of the skin of human ( Van et al., 1982). This may also cause swelling of the lining of gastrointestinal problem, increased blood sugar level, and sensitivity to the sun. Autopsies performed on those who have died while on clofazimine show crystal-like aggregates in the intestinal mucosa, liver, spleen, and lymph nodes (Baik and Rosania, 2011). It is a drug suggested to work by interfering with DNA. The immunosuppressive important action of clofazimine was immediately observed when applied in animal model. Macrophages were first studied to be inhibited due to the stabilization of lysosomal membrane by clofazimine. Clofazimine also showed a dosage-dependent inhibition of neutrophil motility, lymphocyte, transformation (Gartner et al., 1982), mitogen-induced PBMC proliferation (  and complement-mediated solubilization of preformed immune complexes in vitro condition (Kashyap et al., 1992). Clofazimine has also been sporadically reported with some success in other autoimmune diseases such as psoriasis, Miescher's granulomatous s cheiliti (Chuaprapaisilp and Piamphongsant, 1978). In recent years, as the global prevalence of drug-resistant TB has increased, researchers have sought to reevaluate the chemotherapeutic role of CFZ as an antituberculosis drug. Tuberculosis (TB) infected early humans in Africa 70,000 years ago and was carried worldwide by human out-of-Africa migrations, according to new research investigation into the genome sequences of M. tuberculosis, the bacteria responsible for the serious disease. Tuberculosis (TB) remains a leading cause of morbidity and mortality in the world health condition (Podmore and Burrows, 1986). Despite years of research, no vaccine currently exists that protects reliably against pulmonary TB in adults, which is the most transmissible form of the disease on condition to another (WHO, 2019). History suggests that TB appeared about 70 000 years ago and has remained in a sporadic state up to the 18th century. Last of all, it became epidemic during the industrial revolution, owing to the increased population density and the unfavorable living conditions (Copin et al., 2014). Epidemiological data and the work by Comas et al. (2013) on the comparison of mitochondrial DNA from humans and from MTBC race suggest that the different race might have specifically adjusted to the different human populations.

MODE OF ACTION OF CLOFAZIME
The mechanism(s) of antimicrobial action of CFZ ( Figure  1) is not well understood. However, it has been exposed that the initial action site of this antibiotic appears to be the outer site of the membrane. In this mechanism, the mycobacterial respiratory chain and ion transporters are the putative targets and CFZ objects by inhibiting these targets (Lange et al., 2019). Another study (Arbiser and Moschella, 1995) suggests that the mechanism of action was primarily thought that CFZ works by binding to the guanine bases of bacterial DNA, thereby inhibiting bacterial proliferations. However this theory has been replaced by the belief that it acts through outcome on intracellular redox cycling (Gopal et al., 2013) and membrane destabilization condition (Gopal et al., 2013) ( Figure 2). Some previous studies investigated that CFZ which selectively binds to guanine base of DNA, and it is possible that this drug has a selective performance on DNA functions target microorganism in M. tuberculosis (Oliva et al., 2004). Also, CFZ can increase the outcome of bacterial phospholipase A2 and release lysophospholipids, the enzymatic hydrolysis products which are mostly toxic to M. tuberculosis, leading to underpin the anti-mycobacterial effect of this drug (Parker et al., 2007). Through increasing reactive oxidant species, CFZ is a drug which may act to enhance killing of antibiotic tolerant M. tuberculosis persisted organisms (Grant et al., 2012). In addition other antimicrobial action studies suggested that, the drug has other pharmacological activities such as prooxidative, antiinflammatory and other immune-pharmacological properties (Reddy et al., 1999). Synergistic effects of interferon gamma and CFZ, as observed by Parak and Wadee (1991) may play an important role to the antituberculosis effect of the drug. CFZ system reverses the inhibitory effect of M. tuberculosis; these are derived factors on phagocyte intracellular killing mechanisms which may also exploit to enhance M. tuberculosis killing (Wadee et al., 1988). Whereas study on the phenazine molecules is auto-oxidizable compounds, they could work as artificial electron acceptors molecule. Consequently, respiratory system oxidizes CFZ instead of NADH, because of a reduction in the amount of ATP available for all type of active cellular processes (Murugesan et al., 2018), have also shown a potential synergistic effect of CFZ with pyrazinamide(PZA) (Tasneen et al., 2011) and with clarithromycin (CLM) (Lu et al., 2010) in M. tuberculosis killing. Due to highly lipophilicity characters and redox potential of CFZ, the anti-mycobacterial action of this drug is followed by an oxidation of reduced CFZ, leading to the production of reactive oxygen species. Ammerman et al. (2016) have investigated that CFZ has time delayed anti-M. The tuberculosis action was due to its mechanism of action. This team have focused that although in the first week of administration, CFZ did not show any bactericidal activity at any concentration neither in vitro nor in vivo condition, it showed concentrationdependent antimicrobial action during the second week which expose both in vitro and in vivo condition (Ammerman, et al., 2016).

MOLECULAR MECHANISM OF CLOFAZIMINE
The antileprosy drug clofazimine is also of highly interest for the treatment of multidrug-resistant tuberculosis. Recent study, due to the spread of multidrug-resistant M. tuberculosis strains there has been renovated interest in the use of clofazimine for treatment of multidrug-resistant tuberculosis disease (Dalcolmo et al., 2017). It is a phenazine dye and is regarded to act by interfering with DNA (Kaufman, 2013). Clofazimine is an antibiotic which has been clearly elucidated in M. tuberculosis to be a prodrug; which is reduced by NADH dehydrogenase (Ndh2), and then upon spontaneous reoxidation by O2, to release reactive oxygen species (ROS) (Murugesan et al., 2018). As there is no specific target state for ROS, resistance to clofazimine is rare. However, the mechanisms of resistance to clofazimine in M. tuberculosis are not still well characterized. rv0678 is a regulatory gene which is clearly an emergent genetic locus involved in such resistance, as it is generally found in both laboratory selective and clinically isolated mutants of M. tuberculosis (Almeida et al., 2016;Hartkoorn et al., 2014). Another study also found clofazimine resistant mutants isolated in-vitro analysis from M. tuberculosis H37Rv and found various new mutations in the rv0678 gene that had not been previously studied (Zhang et al., 2015) (Tables 1 and 2). In addition, new mutations were found in two new genes, rv1979c and rv2535c, which are importantly associated with clofazimine resistance in M. tuberculosis. InDels that cause frame shift and nonsense or point mutations that result in stop codons were responsible for large proportion (70%) of all mutations in rv0678 (Zhang et al., 2015). BLAST which was used to find homologous involvement traits proteins with more than 40% identities and 90% query coverage for Rv1979c, Rv2535c and Rv0678 (Altschul et al., 1990).
On the contrary, functional uniformity between homologous trait proteins is less predictable, which Pearson (2013) investigated to infer functional similarity by orthologues.
Recently, some studies reported that mutations in rv0678 and pepQ regulate the significant mechanisms of clinical resistance to bedaquiline and clofazimine (Hameed et al., 2018;Islam et al., 2017). Rv0678 is well agreed upon as a transcriptional repressor proteins that binds to a sequence located in the intergenic region between this gene locus and the neighboringmmpL5 and mmpS5gene (Hartkoorn et al., 2014). Clofazimine in vitro mutants due to mutation in the Rv0678 regulatory gene turn out for up regulation of the MmpL5 efflux pump gene. In a previous study (Milano et al., 2009) many mutations in Rv0678 were found that led to de-repression of mmpL5 gene. The MmpL protein family has the suppositional function of transporting lipids, and an essential proportion of these membrane related proteins are conserved within the genome of Mycobacterium leprae. This makes sense because all the mutations that abolish the repressor activity of Rv0678 will cause the overexpression of efflux pump MmpL5, which in turn causes clofazimine resistance. The crystal structure of Rv0678 demonstrated that it forms a dimer and binds to a 2-stearoylglycerol ligand (Radhakrishnan et al., 2014). This finding suggests that Rv0678 may be subject to regulation by metabolic products of the bacteria, which may react as the efflux activity of MmpL5 and susceptibility to clofazimine. The mutation information identified in this study will be important for designing new molecular tests for rapid detection of clofazimine resistance and its cross resistance to bedaquiline in future, as it is more commonly used for treating TB and MDR-TB. However, this study provides new insight into mechanisms of resistance to clofazimine by identifying two new genes (rv1979c and rv2535c) (Table 1), which was previously not well known to be associated with clofazimine resistance. Consequently these mycobacterial proteins possibly have tributary roles in the virulence and drug      resistance of the pathogens in difficult situation. Since Rv0678 is a transcriptional repressor protein of MmpS5 and MmpL5, mutations in this gene lead to an increased expression level of this efflux pump (Milano et al., 2009). Mutations of Rv1979c were found in in vitro selected clofazimine-resistant isolates without Rv0678 mutations. It is of interest to note that the clofazimine-resistant strain previously studied here (Phelan et al., 2016;Xu et al., 2017) 011873, had a mutation in Rv1979c and did not display cross-resistance to bedaquiline. Zhang et al. (2015) also found that another orthologue of Rv1979c is Mb2001c (in Mycobacterium bovis), but it is absent in Bacillus Calmette-Guerin (BCG) due to deletion effect, and its role in altering clofazimine susceptibility in BCG is thus unknown. The molecular function of Rv1979c is believed to be that of amino acid transmembrane transporter with permease activity. The role of Rv1979c is annotated as a possible conserved permease that might be involved in amino acid transport. It is possible that Rv1979c is involved in clofazimine transport or uptake that alters the physiology of the bacteria (Zhang et al., 2015). Although the molecular function of Rv253c has not been fully characterized, it is likely to be that of a peptidase activity. He found that only one mutant except the common rv0678 mutation had a T1052C mutation in a new gene, rv1979c (Table 2), due to the amino acid change V351A. Rv1979c is a putative permease that might be attached in amino acid transport. It is possible that Rv1979c is associated with clofazimine transport or uptake, either directly or indirectly, to alter the physiology of the bacteria so as to be less susceptible to the effect of clofazimine. In addition, it is of interest to note that Rv1979c is localized in the RD2 region and its deletion has been shown to be involved in virulence alleviation in BCG (Kozak et al., 2011). It remains is to be tested whether resistance to clofazimine alters the virulence of M. tuberculosis. It is intriguing that he also found a stop codon mutation at E89 in a second new gene, rv2535c, associated with clofazimine resistance, inactivating the function of this protein. The exact function of Rv2535c is unknown. Orthologues of Rv2535c include Mb2564c (in M. bovis) and MMAR_2180 (in Mycobacterium marinum).
Other previous study showed the main mechanism of clofazimine resistance in Mycobacterium abscessus. Mutations in MAB_2299c encode the transcriptional repressor of efflux pump are analogous to Rv0678 mutations in causing clofazimine resistance in M. tuberculosis. Other new genes associated with clofazimine resistance in M. abscessus seem to affect transcription or transport functions. Yuan Chen et al. identification of MAB_2299c, MAB_1483, and MAB_0540 mutations in most clofazimine-resistant mutants has implications for rapid molecular discovery of clofazimineresistant M. abscessus . Contrary to what is generally assumed, clofazimine resistance in M. tuberculosis, M. absessus has been reported, though detailed information of the underlying molecular mechanisms is lacking (Ebenezer et al., 2002). Extended term of treatment with clofazimine, especially on a highly remittent basis, say once monthly (as per WHO guidelines on treatment of multibacillary tuberculosis), may increase the propensity for resistance in M. tuberculosis to this riminophenazine. If a person with tuberculosis also has latent infection or subclinical disease due to M. tuberculosis, it would not be hard to take into consideration the possible development of dual bacillary resistance in both mycobacterial species, following clofazimine treatment for the former disease, with involvement of very similar molecular mechanisms. Such a review may explain, at least partially, primary clofazimine resistance in M. tuberculosis. Although clofazimine is not generally included in the regimens for treatment of non-tuberculosis mycobacterial infections, it sometimes finds a space for such treatment under specific settings, with the attendant risk of development of mycobacterial resistance. In addition, while horizontal transfer of genetic materials between M. abscessus, Mycobacterium leprae and M. tuberculosis has not been reported, such a possibility cannot be totally excluded in other mycobacteria, as revealed by the recent research findings concerning genomic islands which showed a wide spectrum of genes, including those annotated as phagic plasmid genes, inclinical system and environmental Mycobacterium avium sub sp. Hominissuis (Sanchini et al., 2016). The relevance of these observations for clofazimine resistance in different mycobacterial species appears; however, future research work is warranted for better descriptive note.

CONCLUSION
This study reviews an extensive number of mutants resistant to clofazimine and found mutations in rv0678 to be the most prominent mechanism of clofazimine resistance. The present findings of mutation properties associated with clofazimine resistance grant valuable information for the next study of new molecular tests for the rapid discovery of clofazimine resistance in the clinical management. Also, the study discovered two some new genes such as rv1979c, rv2535c and also in M. abscessusMAB_2299c, MAB_1483, and MAB_0540 associated with clofazimine resistance. Further researches are needed to identify the nature of rv1979c, rv2535c and also in M. abscessus MAB_2299c, MAB_1483, and MAB_0540 in clofazimine resistance and to clearly understand the whole mechanisms of clofazimine.
The growing outbreak of MDR-and XDR-TB worldwide spread the emergent essential for extra eff ective drugs and also therapeutic state, especially in TB, HIV, and leprosy co-infected people with high mortality rates. Though therapeutic state for DR-TB is still limited, a complementary approach is to repurpose existing antibiotics. Different research that investigated the available warrant, recommends CFZ-containing combinations as an additional promising treatment option for DR-TB, even though the duration of conduct and optimal dimensions of CFZ necessitate further investigations (Figure 1).