Rv0494 Mediates Tolerance to Antibiotics in Mycobacterium Tuberculosis

Background: Fatty acid metabolism plays an important role in the survival and pathogenesis of Mycobacterium tuberculosis. During dormancy, lipids are considered to be the main source of energy. The previous studies nd that Rv0494 is a starvation-inducible, lipid-responsive transcriptional regulator. However, the role of Rv0494 in bacterial persister survival has not been studied. Methods: We constructed Rv0494 deletion mutant and assessed the susceptibility of the mutant to various antibiotics conditions in persister assays. Results: We found that mutations in Rv0494 caused a defect in persister survival as shown by their increased susceptibility to Isoniazid. Conclusions: We conclude that Rv0494 is important for persister survival and may serve as a good target for developing new antibiotics that kill persister bacteria for improved treatment of persistent bacterial infections.


Background
Persisters are assumed to be non-replicating or slow-growing bacteria that are not killed by antibiotics, can return to a growth state upon removal of antibiotics, and are sensitive to the same antibiotics (1).
Persisters may be medically important and pose a major challenge to the treatment of many bacterial infections such as tuberculosis and may be responsible for latent and persistent infections, long-term treatment and relapse after treatment (2). The mechanisms of persister formation are so complex that not well understood.
Bacteria have evolved various survival mechanisms under stressful conditions. The success of Mycobacterium tuberculosis as a pathogen lies in its ability to survive asymptomatically within the host for long periods of time and to reactivate when host immunity is compromised. During infection, M. tuberculosis is exposed to harsh environmental conditions such as hypoxia, low pH and nutrient deprivation (3)(4)(5). Mycobacteria utilize their resources by e ciently coordinating gene expression according to prevailing conditions. The correct use of promoters in concert with transcriptional regulators plays an important role in mycobacterial physiology.
FadR acts as a sensor of the fatty acid level in bacteria. FadRs of Escherichia coli, Vibrio vulni cus and Corynebacterium glutamicum have been extensively studied and are reported to play important roles in cell physiology and virulence (6)(7)(8)(9)(10). A FadR homologue in M. tuberculosis, Rv0494, is a starvationinducible, auto-regulatory FadR-like regulator (11). Fatty acid metabolism plays an important role in the survival and pathogenesis of M. tuberculosis, lipids are assumed to be the major source of energy during persistence. Therefore, we assume that FadR plays a very important role in the persistence of M. tuberculosis.
In this study, we constructed the Rv0494 deletion mutant in M. tuberculosis H 37 Rv, and exposed the mutants to various antibiotics to exploring whether Rv0494 is important for persister survival. Our results demonstrate that Rv0494 is indeed involved in persister survival and tolerance to antibiotics.
All oligonucleotides and plasmids used in this study are listed in Tables 1 and 2.  The Rv0494 knockout mutants was constructed as described previously (12)(13)(14). Amplicons between 640 and 940 bases anking the gene were PCR generated with primer sets (LFP/LRP and RFP/RRP (Table 1)) to generate gene-speci c LHS and RHS. Plasmid p0004s was digested with Van91I, This fragment was ligated in one step to Van91I-digested LHS and RHS fragments corresponding to the gene Rv0494. The ligation mix was transformed in E.coli DH5α, and the clones were con rmed by sequencing. Thus the p0004s-AES plasmid was constructed.
The ligation mix was transformed in E.coli HB101, single colonies growing on hygromycin -resistant plates were picked into LB + Hyg 150 µg/ml broth. The plasmids were extracted and identi ed using PacI restriction endonuclease digestion. Thus the phAE159-AES phasmid was constructed.
The phasmid phAE159-AES was transformed into M. smegmatis mc 2 155 to obtain phages that could be transfected with M. tuberculosis H 37 Rv. Phages were transfected with M. tuberculosis H 37 Rv and then screened for positive clones using Middlebrook 7H10 + OADC + Hyg 75µg/ml . The primer sets (LYZFP/LYZRP and RYZFP/RYZRP (Table 1)) were then used to verify that the mutants were constructed.
Complementation of the Rv0494 knock-out mutants was performed utilizing the plasmid vector pMV361. A functional wild type copy of Rv0494 was ampli ed by primers Rv0494FP and Rv0494RP. PCR products were digested with restriction enzymes EcoRI and HindIII and cloned into pMV361. The recombinant pMV361 containing Rv0494 (pMV361-Rv0494) was veri ed by DNA sequencing. The resulting constructs were transformed along with the empty vector pMV361 into mutant for complementation.

Susceptibility to antibiotics in exposure assays
The susceptibilities of stationary phase Rv0494 mutants, complemented strains and the parent strain M.
tuberculosis H 37 Rv to various antibiotics, including isoniazid (4 µg mL − 1 ) and rifampin (8 µg mL − 1 ), were evaluated in drug exposure experiments on Middlebrook 7H10 supplemented with OADC. The stationary phase cultures (diluted 1:100 with Middlebrook 7H9) were exposed to different antibiotics, where undiluted cultures were used for incubation without shaking at 37℃ for various times, after which the cultures were plated for CFU determination on Middlebrook 7H10 + OADC plates.

Results And Discussion
Construction of phasmid The LHS and RHS products were recovered by Van91I digestion and ligated with Van91I digested plasmid p0004s and transformed into E. coli DH5α to screen positive clones and sequenced. The positive clones obtained by identi cation were further cleaved by PacI and recovered, and ligated with the plasmid phAE159, also PacI cleaved, packaged and transformed into E. coli HB101 cells to screen for phasmid, and the results of PacI cleavage validation were shown in Fig. 1.

Phage-based mutant construction
After transform the phasmid phAE159-AES into M. smegmatis mc 2 155, we obtain the phages that could be transfected with M. tuberculosis H 37 Rv. Finally, the ΔRv0494 strain was successfully completed using the phage we constructed. PCR was used to con rm the successful construction of the knockout strain ( Fig. 2, 3).

Reduced persister levels of the Rv0494 mutants in antibiotic exposure assays
To determine the persister levels of the Rv0494 mutant, the stationary phase cultures of the mutants and wild-type strain M. tuberculosis H 37 Rv were exposed to no antibiotics as control (Fig. 4a) and various antibiotics, including isoniazid (4 µg mL − 1 ) (Fig. 4b)and rifampin (8 µg mL − 1 ) ( Fig. 4c), and the survival of the bacteria was monitored at different time points. Overall, the results showed that Rv0494 mutant was more susceptible than the wild-type strain M. tuberculosis H 37 Rv to isoniazid and that complementation of the Rv0494 mutant restored the level of persisters to close to wild-type levels in the antibiotic exposure assay (Fig. 4). It is worth noting that, this result did not occur in the rifampicin exposure experiment.

Conclusions
Different phenotypes emerged after treatment of the mutants with different antibiotics. The rifampicintreated mutant showed no signi cant persisters reduction, whereas, the mutant showed a signi cant persisters reduction after isoniazid treatment, suggesting that Rv0494 may play an important role in maintaining the persister in response to isoniazid stress conditions. Suhail Yousuf's(11) research suggests that Rv0494 is a starvation-induced, auto-regulated fadR-like regulator, whereas this study was conducted after the strain entered the stationary phase with antibiotic stress applied, and it is expected that the absence of Rv0494 during this period resulted in the absence of its regulatory role, leading to the reduction of the persisters, and further studies are needed to address this hypothesis. Since FadR likely to be involved in persistence in other bacteria, our ndings support the idea that FadR could serve as a novel target for the development of new antibiotics that target persister bacteria for improved treatment of persistent bacterial infections. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Figure 1 Con rm the completion of phasmid construction after digestion of phasmid using PacI enzyme. Lane M: DNA Ladder, Lane 1-3: Results of phasmid digestion.