Functional determination of site-mutations in rdxA involved in metronidazole resistance of Helicobacter pylori

Background Metronidazole (MTZ) is among the first-line drugs against the human gastric pathogen Helicobacter pylori (H. pylori). MTZ is used as a prodrug that is activated by an oxygen-insensitive enzyme NADPH nitroreductase (RdxA). Loss-of-function mutations in rdxA make H. pylori MTZ resistant; however, experimental proof is lacking. Methods We collected 139 gastric biopsy samples from patients suspected of H. pylori infection in Shanghai, and amplified Hp-specific rdxA gene from 134 samples. All these rdxA genes were sequenced and phylogenetically compared. The effect of mutations on RdxA function was measured by expressing them in Escherichia coli DH5α by using the MTZ sensitivity test. Results In total, 134 gastric biopsy samples were identified as H. pylori positive. Of the 134 samples, 74 and 6 had point mutations at the various sites or promoter region of rdxA, generating truncated and extended fused proteins, respectively. The remaining 54 were full-length with single nucleotide variation (SNV) compared with the wild-type RdxA from H. pylori, with 49 clustering with hpEastAsia, 3 with hpEurope, and 2 with hpNEAfrica. All 134 rdxA were expressed in E. coli DH5α; 22 and 112 resultant strains showed MTZ-sensitive and MTZ-resistant phenotypes, respectively. Comparative analysis of single nucleotide polymorphisms (SNPs) in the functional and inactivated RdxA revealed 14 novel mutations in RdxA, 5 of which conferred MTZ resistance: S18F, D59S, L62I, S79N, and A187V. Conclusion The occurrence of MTZ resistance induced by site-mutation of RdxA in patients with H. pylori infection was 83.6% (112/134) in the Shanghai region. The major form of loss-of-function mutation was truncation of RdxA translation at a rate of 58/112 (51.8%). Molecular detection reliably determined the resistance of H. pylori to MTZ. Thus, the functional mutants involved in MTZ resistance facilitate clinical diagnosis and medication based on sequence analysis.


Introduction
Helicobacter pylori (H.pylori) is a gram-negative microaerophilic bacterium that was first isolated from the gastric antrum and cultivated in vitro (Marshall and Warren, 1984).In 1994, the World Health Organization classified H. pylori as a type-1 carcinogen (IARC, 1994).H. pylori infection is the major causative factor of peptic ulcer and chronic gastritis, affecting >50% of the world's population.The average infection rate of H. pylori in different regions of China is ~50% (Hooi et al., 2017).Because of the pathogenic and harmful nature of H. pylori, almost all patients with H. pylori infection require eradication therapy.
Treating H. pylori infection in a clinical setting is a huge challenge for physicians, and choosing the right antibiotic is critical for recovery from H. pylori infection (Savoldi et al., 2018).Traditional triple therapy, consisting of a proton pump inhibitor and two antibiotics, or quadruple therapy, consisting of a proton pump inhibitor, bismuth, and two antibiotics, is the most commonly used regimen (Kate et al., 2013).
Metronidazole (MTZ), a synthetic 5-nitroimidazole derivative of azomycin (Dingsdag and Hunter, 2018), is one of the constituents of standard triple therapy recommended by the National Institute of Health Consensus, 1994, for H. pylori eradication (NIH, 1994).In H. pylori, MTZ is activated by RdxA (Tomb et al., 1997) through the transfer of four electrons, which may produce intermediates, such as superoxide radicals, nitroso derivatives, and hydroxylamine, leading to DNA damage-mediated killing of H. pylori (Lindmark and Müller, 1976;Hu et al., 2017).This process is particularly active in anaerobic bacteria through the redox of pyruvate-ferredoxin oxidoreductase (PFOR) in concert with ferredoxin or effectors with a sufficiently negative midpoint redox potential (<−415 mV) (Lockerby et al., 1984).MTZ is used as an alternative in patients allergic or resistant to clarithromycin or amoxicillin (Gisbert and Pajares, 2005).However, MTZ, as a common antibiotic for anaerobic bacteria, is widely used in parasitic infections, gingivitis and vaginosis (Löfmark et al., 2010).Due to its mutation-inducing properties, its resistance is higher than that of clarithromycin and amoxicillin in developing countries, posing a great challenge for subsequent eradication of H. pylori as triple therapy (Savoldi et al., 2018).Therefore, it is necessary to perform a sensitivity test before usage.
Mutations in rdxA, which encodes an oxygen-insensitive NADPH nitroreductase, are the main causes of H. pylori resistance to MTZ (Goodwin et al., 1998).RdxA is a homodimer, which exhibits potent NADPH oxidase activity under aerobic conditions and MTZ reductase activity under anaerobic conditions with flavin mononucleotide (FMN) as cofactor (Olekhnovich et al., 2009).RdxA contains six cysteine residues with a pI of ca. 8, whereas the nitrogen regulators of enteric bacteria have 1-2 cysteine residues and more acidic pI values (pI 5.4-5.6).The C159 mutation leads to a significant decrease in MTZ reductase activity, indicating that the point mutations affect RdxA activity (Goodwin et al., 1998;Martínez-Júlvez et al., 2012).
Molecular testing methods, including restriction fragment length polymorphism (Suzuki et al., 2013), fluorescence in situ hybridization (Hansomburana et al., 2012), real-time polymerase chain reaction (RT-PCR) (Monno et al., 2012), and allele-specific-PCR (Nishizawa et al., 2007), and dual-priming oligonucleotidebased multiplex PCR (Woo et al., 2009), have been used to determine H. pylori resistance.The most commonly used target genes for the molecular detection of H. pylori include 23S rRNA and gyrA, because >90% of clarithromycin resistance is caused by mutations in A2142 and A2143 of 23S rRNA, whereas quinolone resistance is mainly caused by mutations in the quinolone resistance determination region of gyrA (Gong and Yuan, 2018).By comparison, mutations in rdxA are variable and random, and some are developmental signals rather than resistance related (Zhang et al., 2020).This greatly increases the limitations of direct determination of MTZ resistance by molecular testing.However, it is difficult to perform MTZ susceptibility test in most clinical laboratories because the cultivation of H. pylori is challenging and time-consuming, with a low success rate.
In this study, we employed heterologous expression of rdxA in E. coli DH5α deficient in DNA repair to establish the relationship between site mutations of rdxA and MTZ resistance.The sequence map for MTZ resistance-related mutations from Shanghai is presented.In combination with multiple sequence analysis, we determined five new resistance-associated mutation sites in rdxA, facilitating clinical diagnosis and medication, based only on sequence analysis.

Gastric biopsy sample
Participants (age >18 years) with positive 13 C urea breath test from Ruijin Hospital, Shanghai Jiao Tong University were invited to participate.Exclusion criteria included (i) use of proton pump inhibitors, H 2 -receptor antagonists, or antibiotics within the previous 30 d; (ii) prior gastrectomy; (iii) history of severe heart, liver, and renal disorders; and (iv) pregnancy or breastfeeding.The study protocol was approved by the Ethics Committee of Ruijin Hospital (Ethics Approval Number: (2022)321), and written informed consent was obtained from all participants.Gastric Rapid preparation of total DNA from H. pylori DNA was extracted from biopsies using carboxy magnetic beads (Tiangen Biotech, China).Briefly, the samples were incubated with 600 μL of lysis buffer (2% SDS) for 5 min at 95 °C; mixed with 600 μL of isopropyl alcohol and vigorously vortexed for 5 s; cooled to room temperature; and mixed and incubated with 10 μL of magnetic beads (10 mg/mL) for 5 min at room temperature.The supernatant was discarded, and the magnetic beads with absorbed nucleic acid were collected using a Magnetic Separation Device (Sangon Biotech, China).The magnetic beads were washed twice with 80% ethanol and incubated for 5 min with 200 μL of RNase-free water to elute nucleic acid.The supernatant was transferred to a new tube and stored at −40 °C.A simple flowchart for DNA extraction is shown in Supplementary Figure S1A.

PCR detection of H. pylori-specific genes
The sequences of 23S rRNA, oorD, rdxA, frxA, and 16S rRNA available for H. pylori strains were downloaded from the NCBI database to obtain conserved regions.The conserved fragments were PCR amplified using total DNA from the samples (all primers listed in Supplementary Table S1).The concentration of each primer was 200 nM.Primer specificity was verified by Primer-BLAST.PrimeSTAR Max DNA Polymerase was used for singleplex PCR and Ex Taq DNA Polymerase for multiplex PCR (TaKaRa).DNA quality was acceptable for PCR amplification of H. pylori 23S rRNA (Supplementary Figure S1B) and multiplex amplification of H. pylori 23S rRNA, oorD, rdxA, frxA, and 16S rRNA (Supplementary Figure S1C).
Construction of the rdxA expression vector and site-directed mutagenesis rdxA obtained from total DNA was amplified by PCR using the primers rdxA-F1 and rdxA-R1.The resulting amplicons were 860 bp in length.The product was then amplified using the primers rdxA-F2 and rdxA-R2, with restriction sites EcoRI and BamHI, respectively.The amplified product and pBluescript were digested with the same restriction enzymes and ligated using DNA Ligation Kit Ver.2.1 (Takara).RdxA mutant variants were constructed using whole-plasmid PCR and DpnI digestion.Primers used are listed in Supplementary Table S1.The sequence of rdxA was confirmed (Majorbio Co., Ltd., Shanghai).Confirmed sequences were transformed into E. coli DH5α (Invitrogen) for sensitivity testing.

Construction of the phylogenetic tree
The phylogenetic tree was constructed using MEGA-X.The reference protein sequences compared were obtained from a broad range of geographic locations and contained seven population types (Supplementary Table S2).All aligned with full-length RdxA from samples using the Clustal W algorithm.The neighbor-joining method was employed to construct a phylogenetic tree with default values in MEGA-X.The phylogenetic tree was visualized on iTOL (iTOL: Interactive Tree Of Life (embl.de)).

High performance liquid chromatography (HPLC) detection
The E. coli DH5α containing pBluescript rdxA-26695 or pBlueScript were cultured aerobically till an optical density of 0.6 was achieved at 600 nm in LB broth; mixed with MTZ to a final concentration of 16 μg/mL; and cultured overnight under anaerobic conditions.The supernatant was collected after centrifugation for HPLC detection using a YMC ODS-AQ chromatographic column.Solution A contained 0.5% (v/v) trifluoroacetic acid in water, whereas solution B was 100% acetonitrile.The ratios of solution A to B (v/v) in mobile phases A and B were (5:95) and (90:10), respectively.The flow rate, wavelength for determination, and temperature of the column were 1 mL/min, 320 nm, and 25 °C, respectively.

Sensitivity test
E. coli DH5α expressing rdxA from samples and H. pylori 26695 were cultured aerobically to OD 600 0.5 in LB broth and spotted (5 μL of each strain) onto LB agar plates supplemented with 0-16 μg of MTZ (Sigma-Aldrich) per mL.The plates were incubated under aerobic conditions at 37 °C and then scored for growth at 16-24 h.

Data analysis
Correlation analysis was performed by R. The effect of mutations was investigated based on the crystal structure of H. pylori RdxA strain 26695 [Protein Data Bank (PDB): 3QDL].Residue exchange was modeled and analyzed with PyMol.The energetic effect of the mutation was quantified using DUETweb.

Baseline characteristics and good consistency between clinical and molecular diagnosis
A total of 139 clinical samples were obtained from patients with positive 13 C urea breath tests at Shanghai Ruijin Hospital.
Frontiers in Cell and Developmental Biology frontiersin.org Overall, H. pylori positive results were concordant between pathological examination and PCR detection in 134 patients, and one sample was negative when assessed using either method.In one case, H. pylori was identified in pathological examination but not in PCR detection, and in three cases H. pylori was detected only by PCR.Clinical data showed that of the 134 patients with H. pylori infection and investigated by gastric endoscopy, 98 (73.13%) had gastritis, 10 (7.46%) had gastric ulcer, and 26 (19.41%) had duodenal ulcer (Table 1).

Mutation analysis of rdxA of H. pylori from clinical samples
H. pylori RdxA is responsible for susceptibility to the redox active prodrug MTZ (Goodwin et al., 1998).Therefore, rdxA from 134 clinical samples positive for H. pylori were cloned and sequenced to identify mutations associated with resistance phenotypes.Compared to the reference RdxA from H. pylori 26695, 74 and 6 had point mutations at the various sites or promoter region of rdxA, generating truncated and extended fused protein respectively, whereas 54 were full-length with missense point mutations.Of the 74 truncated variants, 27 were pretermination, 33 were deletions and 14 were insertions.The 6 fusion variants were promoter deletion, in which the start codon M was mutated to I (Table 2).

Identification of new mutation sites in MTZresistant strains
Because the cultivation of H. pylori in vitro is challenging and time-consuming and has a low success rate, we examined the activity of RdxA variants by heterologous expression of their coding genes in E. coli DH5α.There are two reasons to choose this host: 1) it's a DNA recombination-deficient strain that is susceptible to DNA damage (Supplementary Figure S2); and 2) it lacks a rdxA homolog, and is resistant to MTZ (Jackson et al., 1984).We individually cloned 134 rdxA genes with 102-bp upstream DNA sequences, into pBluescript and transformed these into E. coli DH5α for MTZ sensitivity testing.Compared with E. coli DH5α harboring rdxA from H. pylori 26695 (MIC ≤ 8 μg/mL), expression strains with 112 rdxA showed MTZ-resistant phenotypes (MIC > 8 μg/mL).Thus, in patients with H. pylori infection in Shanghai, MTZ resistance was 83.58%.For the 112 MTZ-resistant RdxA, 64 were truncations or fusions, and 48 were missense mutations with fulllength coding sequences.Comparative analysis of RdxA from MTZ- "R" means the mutation has been reported to occur in metronidazole resistance H. pylori strains."S" means the mutation has been reported to occur in metronidazole susceptible H. pylori strains.
Two new mutations occurred only in susceptible strains: S29T and S43L, and 1 new mutation P180S occurred in both susceptible and resistant strains (Table 4).The importance of these 14 sites identified from strains with resistant, susceptible and resistant/susceptible phenotype to MTZ were further functionally explored.

Effect of point mutations on the structure and function of RdxA
We obtained the crystal structure of RdxA from PDB to investigate the SNPs important for RdxA activity.RdxA is a homodimer that exhibits domain swapping and contains two molecules of FMN bound at the dimer interface (Martínez-Júlvez et al., 2012).The positions of S18, D59, L62, S79, and A187 are mapped onto the RdxA structure (Figure 3).The hydrogen bonds formed by FMN ribi tyl and hydroxyl of Ser18 of the same monomer decreased when S18 was changed to F18 with the bond of 3.0 Å disappeared which may explain the decrease in RdxA activity (Figures 3A, B).The other four mutations were >8 Å from FMN and could not interact with the latter.Among them, N and O of A187 form hydrogen bonds with O and N of C159, which is important for MTZ reductase activity.A187V mutation slightly increased the lengths of two bonds with C159, which finally influenced the positioning of FMN (Figures 3C, D).Previous studies have shown that two functional homodimers are present in the asymmetric unit of the tetragonal crystals in the RdxA structure.Interestingly, the remaining three mutation sites, D59S, L62I and S79N, located on α helixs, which were away from the dimer interface and did not interact with cofactor FMN (Figure 3E) (Martínez-Júlvez et al., 2012).Considering the importance of α helixes in stability of protein conformation, mutations of them may destruct the primary structure of RdxA and influence its activity.

Discussion
With the global increase in the prevalence of antibiotic resistance and poor availability of culture-based susceptibility testing, the empirical use of most popular triple therapies for H. pylori has resulted in poor cure rates (Graham and El-Serag, 2021;Nyssen et al., 2021).Studies have confirmed that quadruple therapy, including clarithromycin, levofloxacin, or MTZ, should only be used after confirmation of culture susceptibility (Graham and Moss, 2022).In China, antimicrobial resistance in H. pylori is especially prevalent, and molecular-based susceptibility testing is recommended to predict H. pylori resistance to antibiotics.Consistently, resistance to antibiotics, such as clarithromycin and levofloxacin, in H. pylori is well understood, and molecular-based tests could provide regional H. pylori susceptibility data to enhance cure rates.However, the molecular underpinnings of MTZ are controversial.
Gene sequencing alone cannot confirm whether H. pylori is resistant or susceptible to MTZ in a given patient because the exact site mutations that lead to the functional failure of RdxA remain unknown.Many spontaneous mutations that are not essential to RdxA function further complicate the determination of resistancerelated sites simply based on gene sequence.RdxA can reduce MTZ in E. coli and cause DNA lesions.In most cases, these lesions can be repaired using a DNA recombination system, such as RecA.However, DNA lesions introduced by MTZ in RdxA are lethal to E. coli DH5α, which is deficient in DNA repair.This feature offered a good opportunity to correlate between MTZ resistance with site mutations in RdxA.
Failures in the eradication of H. pylori may be due to preexisting drug-resistant H. pylori strains or the development of uniquely resistant strains due to the antimicrobial burden, and point mutations are a major mechanism of antibiotic resistance (Binh et al., 2015).MTZ resistance is predominantly caused by mutations in RdxA (Goodwin et al., 1998).RdxA sequences are highly heterogeneous, with the homology of sensitive strains in clinical samples ranging 95.73%-96.21%.Our study included 134 patients with H. pylori infection, and mutations in full-length RdxA identified in 54 patients could be classified into three types: (i) evolutionary traces rather than drug resistance mutations, such as T31E, H53R, D59N, L62V, S88P, G98S, R131K, and V172I, which are conservatively present in some MTZ-sensitive strains belonging to a few phylogenetic clades (Supplementary Table S2); (ii) low-frequency random mutations not associated with drug resistance, accounting for 40.4% of the total mutation sites; and (iii) mutations occurring only in drug-resistant strains.For these sites, we analyzed the effect of mutations on the function of RdxA combined with its protein structure.R16H, S18F, and G163V were expected to reduce the affinity of the apo protein for FMN cofactors.D59S, L62I, and S79N were located in the α helix.These mutations may affect the structural stability of RdxA.Moreover, molecular docking study predicted 24 mutation sites related to MTZ resistance (Chu et al., 2020).M21A, M21I, and Y47C revealed in our study are among them.
In addition to full-length RdxA, the majority (44.78%) of RdxA sequences from clinical samples were truncated due to frameshift or nonsense mutations, most of which showed reduced activity.Interestingly, some strains expressing truncated RdxA exhibited an MTZ-sensitive phenotype.We found by sequence alignment that these proteins lacked the Q50-V55 or A68-E75 fragment, indicating that these fragments may not be critical for the nitroreducing function of RdxA.Therefore, mutations in this region can be excluded from the MTZ resistance correlation analysis.Furthermore, 27/60 truncated sequences were due to a premature termination codon.Aminoglycoside antibiotics can restore fulllength expression of truncated proteins through premature termination codon readthrough (Zingman et al., 2007).Gentamicin, netilmicin, and tobramycin have bactericidal effects on H. pylori (Lee et al., 2019).Therefore, the combination of MTZ and aminoglycoside antibiotics may have unexpected effects during H. pylori treatment.Interestingly, four MTZ-resistant frameshift mutations occurred upstream of rdxA, suggesting that the regulation of RdxA expression could result in MTZ resistance, consistent with previous reports (Han et al., 2007).
In conclusion, our study suggests that molecular detection can replace culture-based susceptibility testing for H. pylori to MTZ.The findings provide new resistance-associated mutation sites in rdxA to increase predictive accuracy, particularly for MTZ resistance.We believe that sequence analysis will reliably predict outcomes of H. pylori infection treatment in the future.relationships that could be construed as a potential conflict of interest.

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FIGURE 1
FIGURE 1 Cluster analysis of full-length RdxA sequence from Shanghai samples.Comparison of the sequence homology of RdxA from samples from Shanghai with 57 typical sequences belonging to the seven major population clusters.Populations containing sample sequences are highlighted with shading.Most sample RdxA sequences are clustered with the East Asian group.

FIGURE 2
FIGURE 2Association of RdxA mutations with MTZ resistance (A) Correlation coefficient between 45 mutations present in full-length RdxA and MTZ resistance was calculated as Spearman's correlation (**P < 0.01; *P < 0.05).(B) The 16 mutation sites were randomly selected for verification, and their drug-resistant phenotypes were observed on LB plates containing 0, 8, 12, and 16 μg/mL MTZ.

FIGURE 3
FIGURE 3 Crystal structure of dimeric H. pylori RdxA with the cofactor FMN Structure of RdxA is shown in gray.Mutation sites, including S18, D59, L62, S79, and A187, and the cofactor FMN are highlighted using different colors.(A,B) Position S18 was mutated to F18, resulting in a decrease of three hydrogen bond interactions between the amino acid and FMN to one.(C,D) Position A187 was mutated to V187, resulting in the loss of hydrogen bonding with C159, which interacts with FMN.(E) The left three mutation sites D59, L62, and S79 are located at α helix.Hydrogen bonds are indicated by yellow dashed lines.

TABLE 1
Demographic characteristics and diagnosis of the recruited patients with positive H. pylori infection (n = 134).

TABLE 2
Statistics of mutations on RdxA.

TABLE 3
List of amino acid substitution sites detected in full-length RdxA.

TABLE 4
Comparison of SNPs detected in the 54 full-length RdxA proteins.

TABLE 4 (
Continued) Comparison of SNPs detected in the 54 full-length RdxA proteins.