A Glutamine Insertion at Codon 432 of RpoB Confers Rifampicin Resistance in Mycobacterium tuberculosis

Tuberculosis (TB) is an infectious respiratory disease caused by Mycobacterium tuberculosis and one of the top 10 causes of death worldwide. Treating TB is challenging; successful treatment requires a long course of multiple antibiotics. Rifampicin (RIF) is a first-line drug for treating TB, and the development of RIF-resistant M. tuberculosis makes treatment even more difficult. To determine the mechanism of RIF resistance in these strains, we searched for novel mutations by sequencing. Four isolates, CDC-1, CDC-2, CDC-3, and CDC-4, had high-level RIF resistance and unique mutations encoding RpoB G158R, RpoB V168A, RpoB S188P, and RpoB Q432insQ, respectively. To evaluate their correlation with RIF resistance, plasmids carrying rpoB genes encoding these mutant proteins were transfected into the H37Rv reference strain. The plasmid complementation of RpoB indicated that G158R, V168A, and S188P did not affect the MIC of RIF. However, the MIC of RIF was increased in H37Rv carrying RpoB Q432insQ. To confirm the correlation between RIF resistance and Q432insQ, we cloned an rpoB fragment carrying the insertion (encoding RpoB Q432insQ) into H37Rv by homologous recombination using a suicide vector. All replacement mutants expressing RpoB Q432insQ were resistant to RIF (MIC > 1 mg/L). These results indicate that RpoB Q432insQ causes RIF resistance in M. tuberculosis.


INTRODUCTION
Mycobacterium tuberculosis is an important human pathogen that causes tuberculosis (TB). It was first isolated by Robert Koch in 1882, and numerous TB drugs have been developed since the 1930s, although the first effective anti-TB drug was not discovered until 1944 (Vilcheze and Jacobs, 2014;Islam et al., 2017;Long et al., 2019;Mabhula and Singh, 2019). TB is a common, chronic infectious respiratory disease that affects nearly one-third of the world's population (Long et al., 2019;Yong et al., 2019) and is transmitted via aerosol (e.g., cough) from infected persons. According to the World Health Organization, TB is one of the top 10 causes of death worldwide. In 2018, there were approximately 10 million people with TB and 1.4 million deaths due to TB worldwide (World Health Organization [WHO], 2019). Approximately 500,000 new cases were found to be resistant to the most effective first-line drug, rifampicin (RIF), of which 78% had multidrug-resistant TB (resistant to at least isoniazid and RIF) (World Health Organization [WHO], 2019). According to the Taiwan Centers for Disease Control, there were 9,179 new cases of TB in Taiwan in 2018, and 294 of these were RIF-resistant. Over the period of long-term anti-TB therapy, M. tuberculosis is exposed to the appropriate drug concentration, which might lead to the development of drugresistant TB and increase the risk of transmission (Yong et al., 2019). It takes at least 6 months to successfully treat TB, and the development of drug resistance makes therapy even more difficult and is a threat to public health. The treatment of drugresistant TB requires the administration of more than five drugs for more than 9 months (Vekemans et al., 2020). In addition, RIF-resistant TB is frequently not adequately treated because of a delay in the diagnosis of drug resistance. Such delayed treatment not only has a poor therapeutic effect on the infected patient but also increases the risk of transmission (Boyd et al., 2017;Yong et al., 2019). First-line RIF acts by binding the β-subunit of RNA polymerase, blocking RNA synthesis, and inducing hydroxyl radical formation, which likely contributes to its killing effect (Piccaro et al., 2014;Lohrasbi et al., 2018). It has been reported that drug resistance develops when mutations in rpoB block RIF binding to the β-subunit. Most point mutations causing RIF resistance occur in an 81-nucleotide region (codons 426-452 in M. tuberculosis or codons 507-533 in the Escherichia coli codon numbering system) of rpoB that is called the RIF resistancedetermining region (RRDR) (Pang et al., 2013;Chikaonda et al., 2017). According to previous studies, >90% of RIF-resistant TB strains have mutations in the RRDR within codon 435, 445, and 450 (codon 516, 526, and 531 in E. coli) (Pang et al., 2013;Swain et al., 2020). Drug resistance is a major challenge for TB control. Due to the slow growth rate of M. tuberculosis, conventional drug susceptibility testing takes several weeks. Therefore, sequencing drug resistance-related mutations can be used to quickly detect drug-resistant TB, significantly reducing the duration of therapy, and avoiding treatment delays. Thus, we examined the effects of novel mutations in known target gene regions and mutations outside of the target region on RIF resistance in M. tuberculosis with the aim to expand upon known RIF resistance-causing mutations for use in clinical molecular diagnostics.

Bacteria Strains
Clinical isolates were obtained from the Reference Laboratory of Mycobacteriology of the Taiwan Centers for Disease Control. M. tuberculosis H 37 Rv was used as the reference strain. M. tuberculosis H 37 Rv and clinical isolates were cultured in Difco TM Middlebrook 7H9 medium (BD REF: 271310; MD, NJ, United States) supplemented with 10% oleic acid/albumin/dextrose/catalase (OADC), 0.5% glycerol, and 0.05% tween-80 at 37 • C (Tan et al., 2006). E. coli DH10B was grown in Luria broth (LBL405.1; BioShop, Burlington, Canada). All experiments involving M. tuberculosis strains were conducted at the Biosafety level 3 lab in the National Taiwan University College of Medicine and followed institutional biosafety procedures.

Isolation of Clinical Strains and Drug Susceptibility Testing
Clinical isolates of M. tuberculosis were tested in the Reference Laboratory of Mycobacteriology of the Taiwan Centers for Disease Control. Each isolate was initially obtained from a patient and was inoculated in solid and liquid culture. The minimum inhibitory concentration (MIC) of RIF was determined using a Sensititre TM Mycobacterium tuberculosis MIC Plate (MYCOTB; Thermo Fisher Scientific, Cleveland, OH, United States) according to the manufacturer's instructions. The bacterial culture was adjusted to a 0.5 McFarland standard and then added to the Sensititre TM plate, which was covered with an adhesive plastic seal. After incubation at 37 • C, the results were recorded with a Sensititre TM Vizion TM Digital MIC Viewing System. The critical concentration of RIF was 1 mg/L.

Microplate Alamar Blue Assays (MABAs)
Microplate alamar blue assays were performed as previously described with minor modifications (Burke et al., 2017). Briefly, M. tuberculosis strains were cultured in Middlebrook 7H9 medium, and then the OD 600nm was adjusted to 0.1. A 200-µL aliquot of each prepared bacterial suspension was placed in a 96-well sterile plate (LabServ, Singapore) and incubated at 37 • C for 14 days with and without RIF (0.03-512 mg/L). After 14 days of incubation, 50 µL of a freshly prepared 1:1 mixture of Alamar blue (alamarBlue R ; BioRad, Hercules, CA, United States) and 10% tween 80 was added to each well. Then, the plate was incubated at 37 • C for 48 h, and the color was recorded. Blue indicates no growth, and pink indicates growth. During incubation and staining, the plate was sealed with an adhesive plate seal.

RpoB Expression Plasmids
The plasmid pMN437 was used to express RpoB in M. tuberculosis (Steinhauer et al., 2010). Derivative plasmids

Site-Directed Mutagenesis
The putative resistance-associated rpoB mutations were amplified by PCR from the genomic DNA of the resistant strains as a template and then cloned into the ScaI site of the suicide plasmid pGOAL19 (Addgene; MA, United States) (Parish and Stoker, 2000). The primers used are shown in Supplementary Table 1.
The resulting plasmids were transformed into M. tuberculosis H 37 Rv, and point mutants were selected after two rounds of homologous recombination, as previously described (Parish and Stoker, 2000;Larsen et al., 2007).

Identification of Novel rpoB Mutations in RIF-Resistant Clinical Isolates
Four clinical isolates, CDC-1, CDC-2, CDC-3, and CDC-4, had high-level RIF resistance and different novel mutations within the rpoB gene. The RIF resistance levels of clinical strains were determined using a Sensititre TM Mycobacterium tuberculosis MIC Plate. The MIC of RIF for CDC-1 was 8 mg/L, and the MICs of RIF for CDC-2, CDC-3, and CDC-4 were >16 mg/L ( Table 1). Sequencing showed that each of these four clinical isolates harbored genes encoding two amino acid changes in RpoB. When compared to the sequence of the reference strain H 37 Rv, in CDC-1, the G at codon 158 was replaced with R (G 158 R) and the V at codon 170 was replaced with F (V 170 F). In CDC-2, the V at codon 168 was replaced with A (V 168 A) and the V at codon 170 was replaced with F (V 170 F). In CDC-3, the V at codon 170 of RpoB was substituted with F (V 170 F), and the S at codon 188 was replaced with P (S 188 P). In CDC-4, the S at codon 431  Table 3).
was replaced with G (S 431 G) and a Q was inserted at codon 432 (Q 432 insQ). However, G 158 R, V 168 A, S 188 P, and Q 432 insQ had not previously been confirmed to be related to drug resistance.

An RpoB Q 432 insQ Expression Plasmid Increases the MIC of RIF in H 37 Rv
Clinical isolates CDC-1, CDC-2, CDC-3, and CDC-4 had highlevel RIF resistance and harbored unique RpoB amino acid changes (G 158 R, V 168 A, S 188 P, and Q 432 insQ, respectively). To evaluate the association between these unique amino acid changes and the RIF resistance of the clinical isolates, plasmids (pMN437-derived) expressing these RpoB proteins were transformed into H 37 Rv ( subcloned from pMN437-RpoB Q 432 insQ into the ScaI site of the pGOAL19 plasmid, and the resulting construct, pGOAL19-Rv RpoB Q 432 insQ ( Table 2) was checked by sequencing. Then, the plasmid was transformed into M. tuberculosis H 37 Rv, and the unmarked replacement mutant (RpoB Q 432 insQ) was selected after two rounds of homologous recombination. DNA sequencing confirmed that seven strains (#19, #112, #132, #133, #135, #136, and #137) had the CAA insertion in codon 432 of rpoB, which leads to a Q insertion (Figure 2). All tested strains were resistant to RIF, with an MIC >1 mg/L.

DISCUSSION
Clinical strains CDC-1, CDC-2, and CDC-3 have high-level RIF resistance and unique rpoB gene mutations. All of them also encoded V 170 F, which is a well-known RIF-resistance associated mutation, as it has been reported that M. tuberculosis isolates encoding the V 170 F mutation have high-level resistance to RIF (8-32 mg/L) (Heep et al., 2000). According to our data, G 158 R, V 168 A, and S 188 P mutations likely did not contribute to RIF resistance in these strains. However, strain CDC-2 had a RIF MIC of 256-512 mg/L, which was 32-64 fold higher than the MIC previously reported for a strain encoding RpoB V 170 F (Heep et al., 2000), suggesting that strain CDC-2 might have another drug-resistance mechanism not involving RpoB. Because we only sequenced the rpoB genes of RIFresistant strains, resistance mechanisms outside rpoB could not be detected. Mutations causing other antibiotic resistance could be missed too. Chromosome mutations comprise the major mechanism causing drug resistance in M. tuberculosis (Miotto et al., 2018). Drug resistance in strains could be caused by other resistance mechanisms such as antibiotic modifications or neutralization, augmented efflux pumps, porin alterations, and the downregulation of cell-wall permeability (Mnyambwa et al., 2017;Sharma et al., 2018).
Codons 431 and 432 of rpoB are located in the RRDR (Friehs, 2004), and mutations in this region are related to RIF resistance (Sandgren et al., 2009). Mutations at codon 431 (S 431 T, S 431 I, S 431 R, and S 431 G) were previously reported to be associated with RIF resistance (Kapur et al., 1994;Sajduda et al., 2004;Chan et al., 2007). Multiple mutations including codon 431 or 432 of RpoB resulted in higherlevel RIF resistance (MIC > 100 mg/L) (Bahrmand et al., 2009). We also demonstrated that plasmid complementation or chromosomal mutagenesis of Q 432 insQ alone could cause RIF resistance, whereas S 431 G plus Q 432 insQ complementation resulted in a two-fold higher MIC of RIF (Tables 2, 3,  Figure 1).
RIF interacts with the β-subunit of RpoB, and most RIF resistance in M. tuberculosis is caused by mutations in the RRDR of RpoB (Tupin et al., 2010). The domain structure of RpoB was previously deduced by analyzing the crystal structure, which showed that RpoB directly interacts with RIF via 12 hydrogen bonds (Nusrath Unissa et al., 2016). Molecular docking experiments showed stronger RIF binding by wild-type RpoB than by mutant RpoB proteins (Nusrath Unissa et al., 2016). Q432 is an energetically favorable binding site and is considered part of the active site that is involved in ligand binding (Campbell et al., 2001;Nusrath Unissa et al., 2016). RpoB Q 432 insQ might influence the binding enthalpy to weaken the molecular interaction between RpoB and RIF, which could result in RIF resistance.
We observed that CDC-4 grew slower compared to the H 37 Rv strain. Therefore, mutants survived better only in the presence of RIF. Multiple mutations in rpoB have been reported, shown to result in increased drug resistance (Bahrmand et al., 2009). The occurrence of multiple mutations could be accumulated due to continuous RIF usage after a first mutation or they could occur simultaneously in high RIF environments.
Rifampicin is an effective drug used to treat most cases of drug-susceptible TB (Long et al., 2019). However, cases of RIF resistance have been reported since the early 1990s, leading to problems in TB control (Zaw et al., 2018). In M. tuberculosis, drug resistance is mostly caused by genetic changes rather than gene transfer from other bacteria (Tupin et al., 2010;Zaw et al., 2018). Thus, sequencing wellknown mutation sites is important to detect drug-resistant M. tuberculosis. Several molecular diagnostic methods have been developed for the rapid detection of drug resistance in M. tuberculosis (Yong et al., 2019). Nevertheless, more comprehensive information on drug resistance-associated mutations must be established to improve the diagnosis and treatment of TB.

CONCLUSION
In summary, we studied four isolates with high-level RIF resistance and unique mutations encoding RpoB G 158 R, RpoB V 168 A, RpoB S 188 P, and RpoB Q 432 insQ. Results of plasmid complementation of RpoB indicated that G 158 R, V 168 A, and S 188 P of RpoB do not affect the MIC of RIF. However, the transfer of pMN437-RpoB Q 432 insQ plasmids to M. tuberculosis H 37 Rv or chromosomal mutagenesis generating RpoB Q 432 insQ turned sensitive strains into RIF-resistant strains. Therefore, RpoB Q 432 insQ confers RIF resistance in M. tuberculosis.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

AUTHOR CONTRIBUTIONS
J-TW designed the study. L-YL, P-FH, T-LL, and J-TW discussed the results and revised the manuscript. W-TL, H-YT, W-HL, and RJ provided and analyzed the clinical strains. L-YL, S-HW, S-EC, L-YH, and H-EK prepared materials and performed experiments. L-YL and P-FH analyzed the data. L-YL wrote the manuscript. All authors reviewed and approved the final version of the manuscript.