EXPRESSION OF Mycobacterium tuberculosis RpsA IN Mycobacterium smegmatis INCREASES SUSCEPTIBILITY TO PYRAZINAMIDE

Pyrazinamide (PZA) is one of the most important drugs used in combined antituberculous therapy. After the drug enters Mycobacterium tuberculosis it is hydrolyzed by pyrazinamidase (PZAse) to the bactericidal molecule pyrazinoic acid (POA). Ribosomal protein S1 (RpsA) was recently identified as a possible target of PZA based on its binding activity to POA and capacity to inhibit trans-translation. However, its role is not completely understood. It has been proposed that Mycobacterium smegmatis RpsA is not capable of binding POA, unlike M. tuberculosis RpsA. This may be due to the different amino acid sequence in the carboxy-terminal region of the two molecules: in M. smegmatis RpsA it is much closer to the sites that may interact with POA than in M. tuberculosis RpsA. These differences could be contributing, along with the presence of highly active POA efflux, to the natural resistance to PZA in M. smegmatis. To further understand the mechanisms of action of PZA and the role of RpsA in PZA susceptibility, we evaluated the effect of complementing M. tuberculosis RpsA expression in M. smegmatis using pNIT mycobacterial non-integrative expression vector and then performed a PZA susceptibility test determining the minimum inhibitory concentration (MIC) of PZA. It was expected that chimeric ribosomes comprising M. tuberculosis RpsA may be present and may affect PZA susceptibility. Our results showed a reduction in PZA MIC in M. smegmatis complemented with overexpressed M. tuberculosis RpsA compared to non-overexpressed M. smegmatis (468 µg/mL and >7500 µg/mL respectively).


INTRODUCTION
Pyrazinamide (PZA) is one of the most important drugs in the treatment of tuberculosis (TB), being used in both first and second line regimens. It is uniquely effective against slowly dividing sub-populations of bacteria in TB infection, and its inclusion in first-line treatment regimens makes it possible to shorten the duration of treatment from 9 to 6 months (1). PZA susceptibility has been associated with clinical outcomes and although testing for resistance is recommended, this is not universally done due to technical challenges (2).
PZA is a pro-drug that enters Mycobacterium tuberculosis by passive diffusion. In the cytoplasm, PZA is converted by the enzyme pyrazinamidase (PZAse) to its active form, pyrazinoic acid (POA). The POA is then expelled out of the mycobacterium by an efflux mechanism. If the pH of the extracellular environment is acidic, the POA is protonated to POAH, re-enters the cell, and the proton is released into the cytosol. This cycle repeats itself, causing an intra-cellular accumulation of POA and a reduction in the pH of the cytoplasm. This leads to a lethal alteration of membrane permeability; however, the exact mechanism by which this occurs is not yet known (3). It is important to note that other recent studies found that extracellular acid pH is not required in order for PZA/POA achieve its lethal effect (4,5,6). This confirms that the real mechanism of action of PZA is actually more complicated than previously thought, and further research is required.
Mechanisms of resistance to PZA in M. tuberculosis have been mostly associated with the loss of PZAse activity due to mutations in the pncA gene (pncA mutations in >80% of clinical isolates) (7,8,9). However, there are strains of M. tuberculosis that are resistant to PZA yet have active PZAse. The mechanism by which these strains are resistant to PZA is not fully known, although several studies suggest the involvement of other target proteins (10,11,12).
In contrast to M. tuberculosis, Mycobacterium smegmatis is naturally resistant to PZA.
While it actively produces POA through two PZAse enzymes (13), it was shown that in M. smegmatis POA is pumped into the extracellular environment 900 fold faster than in M. tuberculosis, preventing POA from accumulating intracellularly at a minimal required critical concentration, and consequentially from exerting any lethal effect on the mycobacterium (14).
Recent studies have identified the ribosomal protein S1 (RpsA) as a probable target of POA (15,16). RpsA is a protein involved in the ribosomal trans-translation process, which has been associated with bacterial survival in states of stress, bacterial virulence, and nutrient recovery in periods of deprivation. Blocking trans-translation prevents the recovery of stalled ribosomes and increases the accumulation of toxic or harmful proteins, causing cell death under conditions of metabolic stress (17

Bacterial strains and growth conditions
Escherichia coli Novablue was routinely grown in Luria Bertani (LB) medium for its use in DNA cloning procedures. Mycobacterium smegmatis mc 2 155 was grown at 37°C in Middlebrook (MB) 7H9 liquid medium or on MB 7H10 agar supplemented with 0.5% (v/v) glycerol and 0.05% (v/v) Tween 80. When required, kanamycin (20 μg/mL) was added. M. smegmatis cells were grown to approximately reach an optical density measured at a wavelength of 600 nm (OD 600 ) of 1, harvested, and washed 3 times in 10% gliceroltween 80, with a reduction in the volume of 10% glycerol-tween 80 used for each wash.
Finally, the washed pellet was resuspended in 1 mL of the original culture medium (20). RNA quality and integrity were verified by observing the staining intensity of the major ribosomal RNA (rRNA) bands and any degradation products in a 1% agarose gel electrophoresis using TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA).
Absorbance at 260 nm of the extracted RNA was measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific). The extracted RNA was then treated with DNAse I (RNAse free): 0.4 U of DNAse/g RNA was added to give a reaction volume of 100 µL. DNAse buffer 1X and DEPC water were then added and this was incubated at 37˚C for 30 minutes followed by DNAse inactivation at 75˚C for 5 min.
Reverse transcription of M. tuberculosis Rv1630 and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) housekeeping gene was performed using 200 ng RNA in a T100 Thermal Cycler (Bio-Rad). The reaction was carried out with Reverse Transcription Reagents (Applied Biosystems) in a 20 µL reaction volume, which contained 4 µg of total RNA, retro-transcription buffer 1X, 5.5 mM MgCl 2 , 2 mM dNTPs, 0.5 µM of reverse primer (primers used are listed in Table 1), 0.4 U/l RNAse inhibitor, 3 U/µL Multi Scribe Reverse Transcriptase, and DEPC treated water. The following thermal parameters were used: 1 cycle to 48˚C for 45 min, 1 cycle to 95˚C for 5 min, and the stable phase to 5˚C for 5 min.
To verify that there was no genomic DNA contamination, a reaction without reverse transcriptase was added. qPCR was performed independently using specific primers that hybridize a specific sequence of the M. tuberculosis Rv1630 (RpsA) gene but not the homologue gene coding for the RpsA protein of M. smegmatis. The primer sequences for qPCR for both the Rv1630 and housekeeping genes are found in Table I. Values obtained were normalized with respect to the housekeeping GAPDH gene using Livak's method (18). Each cDNA was amplified in a LightCycler Nano thermocycler (Roche)

Cloning of M. tuberculosis H37Rv Rv1630 into the pNIT vector
PCR of transforming colonies following the ligation process between the pNIT vector and the M. tuberculosis Rv1630 gene confirmed the presence of the gene (Figure 1).

Expression of Green Fluorescent Protein into the pNIT vector
Visualization of GFP fluorescence in the M. smegmatis pNIT-GFP confirmed that the expression vector is producing the recombinant protein appropriately ( Figure S1 supplementary material).  (Table III).

DISCUSSION
In contrast to M. tuberculosis, M. smegmatis is naturally resistant to PZA and is therefore a useful model in which to study PZA resistance mechanisms (20). This study evaluated the effect of introducing M. tuberculosis RpsA into M. smegmatis on PZA sensitivity. We Our previous studies showed that there is an association between PZA resistance and POA efflux rate, which correlates with extracellular POA concentration (11). In M.
smegmatis, resistance to PZA is associated to a POA efflux rate 900 times faster than that of M. tuberculosis, which prevents its intracellular accumulation and acidification of the cytosol and thus contributing to resistance to PZA. In a study performed by Boshoff      and with exposure to UV light (B).