Adaptive Mechanisms of Mycobacterium tuberculosis: Role of fbiC Mutations in Dormancy and Survival

Abstract

This review examines the impact of F420 biosynthesis protein C (fbiC) mutations in Mycobacterium tuberculosis (Mtb) and their influence on the bacterium’s dormancy mechanisms. The potential role of fbiC mutations and functional impairments in the persistence of Mtb is emphasized. Tuberculosis (TB) bacilli can enter a dormant state with minimal metabolic activity, allowing them to conserve resources and survive in low-nutrient, low-oxygen environments for extended periods. While the fbiC gene contributes to dormancy, Mtb can achieve this state through multiple genetic and metabolic pathways, suggesting that it may still undergo dormancy even with functional impairments in fbiC. In this review, we utilized several scientific databases, including PubMed, Web of Science, and Google Scholar, and set of key search terms including “fbiC gene,” “F420 Biosynthesis,” “Mycobacterium tuberculosis,” “Dormancy,” and “Drug Resistance” to highlight the significance of the fbiC gene in regulating dormancy and explore how Mtb compensates for fbiC dysfunction through various metabolic adaptations.

INTRODUCTION

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), possesses a remarkable ability to enter a dormant state, allowing it to persist in the host for extended periods without triggering active disease.^[1,2] This dormancy is characterized by significantly reduced metabolic activity, enabling the bacterium to survive under unfavorable conditions.^[3,4] Importantly, dormancy is not merely a response to nutrient scarcity; it represents a complex physiological adaptation to various environmental stressors, including nutrient limitations, low oxygen levels, and host immune responses.^[5] Unlike many other pathogens that quickly succumb to adverse conditions, Mtb can enter a nonreplicating but viable state, often referred to as “dormancy” or “latency.” This state allows the bacterium to conserve energy and endure environments that would typically be lethal. The ability to enter dormancy is particularly significant in the human lung, where oxygen levels can fluctuate, especially within granulomas.^[6] These localized immune structures form in response to infection, creating a hypoxic, nutrient-limited environment designed to contain the bacteria. However, Mtb has evolved to thrive under these challenging conditions. In our previous experimental setup, we demonstrated the ability of TB bacilli to alter their metabolic processes, utilize alternative nutrient sources, and undergo morphological changes. Dormant cells typically shift from rod-shaped to smaller, oval, or round forms, significantly reducing in size.^[7] Remarkably, these dormant states can render the bacteria almost devoid of cell walls and unable to replicate, complicating their detection. Generally, the phenotypic changes are correlated with genomic alterations. In this regard, we have published results from whole genome sequencing of dormant and active TB bacilli.^[8] Among the key genetic factors involved in Mtb’s adaptation to dormancy, the FO synthase (fbiC) gene is particularly noteworthy. This gene is crucial for the biosynthesis of coenzyme F420, which plays a vital role in the bacterium’s ability to withstand oxidative stress and manage its redox balance – both essential for survival in hostile environments.^[9] F420 functions as an antioxidant, neutralizing reactive oxygen species produced by host immune cells, particularly macrophages, which attempt to eliminate the bacteria.^[10] The fbiC gene encodes an enzyme responsible for the final steps of F420 biosynthesis.^[11] Mutations or functional impairments in fbiC can disrupt F420 production, potentially compromising Mtb’s ability to cope with oxidative stress. Despite this vulnerability, the bacterium exhibits impressive metabolic adaptability, allowing it to compensate for the loss of fbiC through alternative pathways and genetic mechanisms.^[11] This adaptability enables Mtb to sustain dormancy and survival even in the presence of such mutations. In this review, we aim to highlight the significance of the fbiC gene in regulating dormancy and explore how Mtb compensates for fbiC dysfunction through various metabolic adaptations.

METHODS

Ethical consideration

The review was conducted in accordance with established ethical guidelines for literature reviews and systematic reviews, ensuring methodological rigor and ethical integrity. The review aimed to contribute positively to the scientific community by synthesizing knowledge that could inform future research and improve understanding of Mtb, particularly regarding treatment strategies. The authors took responsibility for the accuracy and reliability of the information presented, ensuring that conclusions were supported by evidence from the literature. The study was approved by the scientific committee at the Institute (IR.SBMU.NRITLD.REC.1403.066).

Type of sampling and reason for selection

To conduct this review, we employed a comprehensive search strategy to gather relevant literature on the fbiC gene and its role in Mtb dormancy and drug resistance. We utilized several scientific databases, including PubMed, Web of Science, and Google Scholar, to ensure broad coverage of the literature. A set of search terms related to the fbiC gene, F420 biosynthesis, Mtb dormancy, and drug resistance was formulated. Key search terms included “fbiC gene,” “F420 Biosynthesis,” “Mycobacterium tuberculosis,” “Dormancy,” and “Drug Resistance.” The reasons for selection were to gather literature that specifically examined the fbiC gene, its mutations, and their impacts on Mtb’s metabolic processes and resistance mechanisms. Purposive sampling allowed us to select studies that provided pertinent insights into these areas, and by targeting peer-reviewed articles, clinical studies, and comprehensive reviews, we ensured that the included studies met a certain standard of scientific rigor and credibility. This focus on quality was essential for drawing reliable conclusions.

Inclusion and exclusion criteria

Criteria were established to filter the search results. Inclusion criteria encompassed peer-reviewed articles, reviews, and clinical studies published in English, focusing on the relationship between fbiC mutations, dormancy, and drug resistance in Mtb. Articles published in the last 10 years were included to ensure relevance and incorporation of the latest findings. Only studies published in English were used to facilitate comprehensive understanding and analysis. Exclusion criteria included articles not directly related to these topics, conference abstracts, and nonpeer-reviewed literature.

RESULTS

Gene positioning and arrangement

Figures 1 and 2 illustrate the positional context of the fbiC gene within the Mtb genome. Figure 1 shows a detailed genomic segment spanning approximately 1,300,000–1,310,500 bp, part of a total sequence length of 4,411,532 bp. This segment reveals the arrangement of several genes, including notable Pro-Pro-Glu motif-containing (PPE) family proteins, such as PPE family protein 17 (Rv1676) and PPE12 (Rv1711). These genes are recognized for their roles in modulating immune responses, underscoring their significance in the pathogenicity of Mtb.^[12] Central to this genomic map is the fbiC gene (Rv1781), which is crucial for coenzyme F420 biosynthesis. This coenzyme is essential for various biochemical processes, including the reduction of substrates in metabolic pathways.^[13]

Figure 1: Genomic organization and sequence analysis of the F420 biosynthesis protein C (fbiC) gene region in Mycobacterium tuberculosis. The figure shows a ~ 10 kb genomic segment (coordinates 1,300,000–1,310,000) containing the fbiC gene and surrounding genes. Yellow arrows at the top indicate forward strand reads, whereas green arrows below indicate reverse strand reads. Gene annotations are shown as arrows, with different colors representing different genes. The fbiC gene (pink) is flanked by several other genes including 2,4-dienoyl-CoA reductase, PE family protein 12, Mycothiol deacetylase, PPE family protein 17, and various Rv-designated genes. A repeat region containing tuberculosis protein 8.4 is noted. The total sequence length is 4,411,532 base pairs. fbiC: F420 biosynthesis protein C, fadH: 2,4-dienoyl-CoA reductase, PE12: PE family protein 12, mshB: Mycothiol deacetylase, PPE17: PPE family protein 17, lipX: Probable lipase/esterase, TB8.4: Tuberculosis protein 8.4, bp: base pairs

Figure 2: Circular genomic map showing the organization of F420 biosynthesis protein C (fbiC) and neighboring genes in Mycobacterium tuberculosis. The circular representation spans 4,411,532 base pairs with genomic coordinates marked along the circle. The genes are represented as boxes with different colors: green for tRNA genes and metabolic genes, gray for Rv-designated genes, and pink/purple for the fbiC cluster including 2,4-dienoyl-CoA reductase, tuberculosis protein 8.4 (TB8.4), and related genes. Internal green bars indicate GC content or genetic features along the genome. The fbiC gene region is shown with its neighboring genes including repeat regions and TB8.4. fbiC: F420 biosynthesis protein C, fadH: 2,4-dienoyl-CoA reductase, TB8.4: Tuberculosis protein 8.4, fdxC: Ferredoxin protein C, tRNA genes (green boxes): ser (SUVX): Serine tRNA genes, leu (TUWX): Leucine tRNA genes, ala (TUV): Alanine tRNA genes, arg (TUVW): Arginine tRNA genes, gly (TUV): Glycine tRNA genes, val (TV): Valine tRNA genes, met (TV): Methionine tRNA genes, thr (TUV): Threonine tRNA genes, lys (TU): Lysine tRNA genes, glu (TU): Glutamic acid tRNA genes, asp (t): Aspartic acid tRNA genes, phe (u): Phenylalanine tRNA genes, pro (TY): Proline tRNA genes, cys (u): Cysteine tRNA genes, his (t): Histidine tRNA genes, asn (t): Asparagine tRNA genes, rrs: 16S ribosomal RNA, rrf: 5S ribosomal RNA, bp: base pairs

In another viewpoint, Figure 2 expands on this by illustrating various genes positioned radially around a circular DNA strand. The fbiC gene is strategically located among several other genes, including Serine tRNA synthetase (serU), Valine tRNA synthetase (valU), and Leucine tRNA synthetase (leuT). This arrangement suggests potential functional relationships among these genes, indicating that they may participate in related metabolic or biosynthetic pathways [Diagram 1].

Diagram 1: The role of the F420 biosynthesis protein C gene in Mycobacterium tuberculosis. fbiC: F420 biosynthesis protein C

Annotations on the map provide insights into the roles of these genes, reflecting the complexity of the Mtb genome. In addition, specific regions, such as T-cell antigen 84 and other repeat sequences, are marked, pointing to areas that may contribute to genomic stability or variability.^[9] Understanding these aspects is essential for comprehending the bacterium’s adaptability to environmental pressures.

Figure 3 illustrates a circular map of a plasmid or genomic region measuring 6,461 base pairs in total length. This map highlights various genetic features, including the fbiC gene, which is prominently marked, alongside several other genes such as TB 8.4, 2,4-dienoyl-CoA reductase, and PE family protein 12. The map also indicates multiple restriction enzyme sites, including PshAI, EcoK, SnaB, BglII, and EcoRI, which are crucial for molecular cloning applications. The identification of these restriction sites is particularly valuable as they facilitate the construction of cloning vectors. By allowing the insertion of foreign DNA, researchers can perform gene expression studies or produce recombinant proteins. Cloning-specific genes like fbiC enable scientists to investigate their functions, interactions, and roles in metabolic pathways, especially within the context of Mtb. Furthermore, this circular map serves as a foundational tool for genetic manipulation, enabling precise modifications of the plasmid for various experimental purposes. Understanding the arrangement of genes and restriction sites aids in designing experiments aimed at assessing gene function, regulation, and potential applications in synthetic biology or therapeutics. Overall, this figure provides a comprehensive overview of the genetic elements and tools necessary for advancing research in molecular biology and biotechnology.

Figure 3: Restriction map and gene organization of the F420 biosynthesis protein C (fbiC) genomic region in Mycobacterium tuberculosis. The circular map spans coordinates 1,301,759–1,308,219 (6461 bp). Major features include the fbiC gene (bright pink), PE family protein 12 gene, 2,4-dienoyl-CoA reductase gene (purple), and tuberculosis protein 8.4 with associated repeat regions (grey). Restriction enzyme recognition sites are marked around the circle with their positions in parentheses. The map shows the relative positions of 22 restriction sites that could be useful for genetic manipulation of this region. fbiC: F420 biosynthesis protein C, fadH: 2,4-dienoyl-CoA reductase, PE12: PE family protein 12, TB8.4: Tuberculosis protein 8.4, bp: base pairs

Protein structure

From the perspective of protein structure, Figure 4 presents the three-dimensional conformation of the FbiC protein, depicted in a colorful ribbon format. This representation effectively illustrates various secondary structural elements, such as alpha-helices and beta-sheets, which are crucial for the protein’s stability and functionality. The distinct color gradient used in the visualization indicates different regions of the protein, providing a clear view of its complex folding pattern.

Figure 4: Three-dimensional structure of F420 biosynthesis protein C protein showing key functional domains. The protein consists of three major regions: an F0 synthase subunit 2 domain (green, residues 521–854), a Coenzyme F420 biosynthesis protein H (CofH)/Methylmenaquinol-futalosine cyclase (MqnC)-like C-terminal domain (purple, residues 735–830), and a 7,8-didemethyl-8-hydroxy-5-deazariboflavin synthase (cofG) domain (blue, residues 84–416). The structure reveals the spatial organization of these functional domains connected by linker regions (wheat color). The overlap between the F0 synthase subunit 2 and CofH/MqnC-like domain (residues 735–830) suggests potential functional interaction between these regions. FbiC: F420 biosynthesis protein C, F0: Precursor molecule of F420 coenzyme, CofH: Coenzyme F420 biosynthesis protein H, MqnC: Methylmenaquinol-futalosine cyclase, cofG: F420 biosynthesis gene G, F420: 8-hydroxy-5-deazaflavin derivative, a cofactor involved in redox reactions

Key features of the FbiC protein include several catalytic sites that are essential for its enzymatic activity.^[14] These sites facilitate the conversion of specific substrates into products, thereby enabling the protein to play a vital role in metabolic pathways critical for the growth and replication of Mtb.^[15] The substrate-binding region is particularly significant, as it determines the specificity of the FbiC protein for its target molecules, directly influencing its role in fatty acid metabolism.^[16] The overall architecture of the FbiC protein, highlighted by the intricate arrangement of its secondary structures, underscores the evolutionary adaptations that allow it to function effectively within the bacterium’s metabolic framework.

DISCUSSION

The genomic positioning and arrangement of the fbiC gene within Mtb offer critical insights into the bacterium’s metabolic capabilities and adaptive strategies. As shown in Figures 1 and 2, the fbiC gene (Rv1781) is located among several key genes, including notable members of the PPE family, which are known to play significant roles in modulating immune responses.^[17] This strategic placement suggests that fbiC may be functionally linked to these immunomodulatory genes, potentially enhancing Mtb’s ability to evade host defenses. The significance of the fbiC gene lies in its encoding of an enzyme essential for the biosynthesis of coenzyme F420, which is integral to various biochemical pathways, particularly those involved in fatty acid metabolism and oxidative stress resistance.^[18] The ability of Mtb to synthesize F420 enables its survival in hostile environments, such as the hypoxic conditions found within granulomas.^[19] The interaction between fbiC and neighboring genes, including tRNA synthetases, indicates a coordinated metabolic network that may bolster the bacterium’s adaptability to environmental pressures. Figure 3 emphasizes the value of understanding the genetic landscape surrounding fbiC. The identification of multiple restriction enzyme sites not only aids in molecular cloning but also provides a framework for exploring gene function and interactions. Furthermore, the three-dimensional structure of the FbiC protein, depicted in Figure 4, reveals its complex folding and functional sites critical for enzymatic activity. The presence of specific substrate-binding regions indicates that the FbiC protein is finely tuned to interact with its targets, influencing metabolic processes essential for bacterial growth and replication. Overall, the findings underscore the intricate genomic and structural characteristics of the fbiC gene and its product, highlighting their roles in the survival and pathogenicity of Mtb. This review stresses that while the fbiC gene is crucial for the biosynthesis of coenzyme F420 – an important cofactor for metabolic processes that enhance survival during dormancy, mutations or dysfunctions in this gene do not entirely abolish the bacterium’s ability to endure challenging environments.

One significant factor is the metabolic flexibility of Mtb. The bacterium has evolved to utilize various nutrient sources, allowing it to adapt even when its primary biosynthetic pathways are compromised.^[20] Consequently, if the fbiC gene is nonfunctional, Mtb can switch to alternative metabolic pathways to generate energy and sustain itself, relying on other substrates such as fatty acids or carbohydrates. In addition, Mtb can enter a dormant state characterized by significantly reduced metabolic activity.^[21] During dormancy, the bacterium minimizes its energy requirements, enabling it to survive in nutrient-poor and hypoxic conditions for extended periods. While the fbiC gene contributes to this dormancy process, the bacterium can still transition into dormancy through other genetic and metabolic pathways independent of fbiC. Another essential aspect is the presence of alternative pathways for maintaining redox balance. Although coenzyme F420 plays a role in protecting the bacterium from oxidative stress, Mtb possesses other antioxidant mechanisms that can mitigate oxidative damage.^[22] For instance, the bacterium can utilize molecules like glutathione to counteract oxidative stress, allowing survival even in the absence of functional F420.^[23] Mtb also exhibits genetic compensation, wherein other genes can upregulate or compensate for the loss of function in the fbiC gene. This adaptability means that if fbiC is mutated, other metabolic processes can adjust to sustain the bacterium’s viability under stress. Moreover, the bacterium is skilled at adapting to various environmental stressors, including fluctuations in oxygen levels and nutrient availability. Even without a functional fbiC gene, Mtb can modify its gene expression in response to environmental cues, activating alternative pathways that facilitate survival. In certain instances, Mtb can form biofilms, which provide a protective environment that enhances its survival under stress.^[24] Biofilms can shield bacteria from immune responses and antibiotics, allowing them to persist even when certain metabolic functions are compromised.^[25] The fbiC gene (Rv1173) in Mtb encodes an enzyme vital for coenzyme F420 biosynthesis, influencing fatty acid metabolism and oxidative stress resistance. Its strategic genomic positioning suggests interactions with immunomodulatory genes, enhancing bacterial adaptability in hostile environments, particularly during hypoxia within granulomas [Diagram 1]. The survival of Mtb with a nonfunctional fbiC gene highlights the challenges of effectively treating TB. Understanding the diverse mechanisms that enable the bacterium to endure adverse conditions is crucial for developing new therapeutic strategies. Targeting the pathways that provide alternative survival mechanisms, alongside conventional treatments, may improve the efficacy of TB therapies, particularly in cases involving drug-resistant strains. Furthermore, recognizing that Mtb can adapt and survive despite genetic mutations emphasizes the need for comprehensive approaches in TB management that consider both active and latent infections. This understanding can guide the development of new diagnostic tools and preventive measures to better control TB transmission and reactivation, ultimately improving public health outcomes.

CONCLUSIONS

Mtb exhibits a remarkable capacity for dormancy, enabling it to persist within the host despite various environmental challenges. The fbiC gene plays a critical role in this process by facilitating the production of coenzyme F420, which is essential for managing oxidative stress and maintaining redox balance. Mutations or impairments in fbiC can compromise the bacterium’s ability to survive under hostile conditions; however, Mtb has evolved robust metabolic adaptability that allows it to compensate for these deficits. Understanding the mechanisms behind dormancy and the role of fbiC in metabolic regulation is crucial for developing targeted therapeutic strategies against TB. By unraveling these complex interactions, we can advance our efforts to combat both active and latent forms of this persistent infection, ultimately improving public health outcomes in the fight against TB.

Outcome of the study

The study offers a detailed understanding of the fbiC gene’s genomic context and its functional importance within Mtb. In addition, it highlights the potential survival mechanisms that the bacterium may employ when the fbiC gene is dysfunctional.

Rationale of the study

The rationale for this study is to understand the genomic context and both the functional and dysfunctional aspects of the fbiC gene. This understanding is essential as it allows us to explore how Mtb can survive in adverse conditions, particularly within granulomas in the human host. In addition, examining the functionality of the fbiC gene and the potential compensatory mechanisms that arise when it is dysfunctional can provide valuable insights into the bacterium’s metabolic adaptations.

Limitations of the study

While there have been reports on the fbiC gene, its genetic context, and its significant role in Mtb dormancy, information regarding the dysfunction of the fbiC gene in relation to dormancy is currently unavailable.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.