Background: Antisense peptide nucleic acids (PNAs) exhibit growth inhibitory effects on bacteria by inhibiting the expression of essential genes and could be promising therapeutic agents for treating bacterial infections. A study was carried out to determine the efficacy of several antisense PNAs in inhibiting extracellular and intracellular growth of Mycobacterium smegmatis. Methods: Six PNAs obtained from a commercial supplier were tested to evaluate the inhibitory effect on bacterial growth by inhibiting the expression of the following essential genes: inhA (a fatty acid elongase), rpsL (ribosomal S12 protein), gyrA (DNA gyrase), pncA (pyrazinamidase), polA (DNA polymerase I) and rpoC (RNA polymerase β subunit) of M. smegmatis. Each PNA was tested at 20 μM, 10 μM, 5 μM and 2.5 μM concentrations to determine whether they caused a dose dependent killing of M. smegmatis cultured in Middlebrook 7H9 broth or in a J774A.1 murine macrophage cell line. Results: In Middlebrook broth, the strong growth inhibitory effect against M. smegmatis was observed by PNAs targeting the inhA and rpsL genes at all four concentrations. The PNAs targeting the pncA, polA and rpoC genes were found to exhibit strong growth inhibition against M. smegmatis but only at 20 μM concentration. No growth inhibition of M. smegmatis was seen in pure culture when treated with PNAs targeting gyrA and a mismatch PNA targeting dnaG (DNA primase). All six PNAs showed killing of M. smegmatis in J774A.1 macrophage cell line that were statistically significant (p < 0.05). Conclusion: It may be concluded from this study that PNAs could be potential therapeutics for mycobacterial infections.
Mycobacterium smegmatis is an avirulent bacterial genus present in the soil and smegma that shows many features similar to Mycobacterium tuberculosis (Mtb), responsible for causing tuberculosis in humans [
Antisense/antigene therapy using peptide nucleic acids (PNAs) has the potential to help control MDR bacterial infections. PNA is DNA mimic with a peptide backbone instead of sugar (deoxyribose) [
Recently PNA therapy has shown promising results in inhibiting the growth of Salmonella typhimurium,Staphylococcus aureus and Escherichia coli,by down regulating the functions of the essential genes of these bacteria [
M.smegmatis was obtained from the laboratory repository of the Center for Molecular Medicine and Infectious Disease (CMMID) now called Center for One Health Research (COHR), Virginia-Maryland College of Veterinary Medicine at Virginia Tech.
A frozen stock of M.smegmatis was diluted in Middlebrook 7H9 broth (Sigma) enriched with 10% ADC incubated at 37˚C aerobically for 48 h on a shaker incubator until the OD600 0.5 was reached.
The list of PNAs shown in
An overnight culture of M.smegmatis was diluted in 7H9 broth to obtain 6 × 104 colony forming units (CFUs)/ml and incubated with either 20 µM, 10 µM, 5 µM or 2.5 µM concentration PNAs (volume = 100 µl) in triplicate using a 96 well, low adhesion microtiter plate (Corning Inc. Cat. # 3474). The plate was sealed with an adhesive lid (Microseal B, Bio-Rad) and incubated at 37˚C for 48 h on a SPECTRA max 340PC with shaker incubator; growth was monitored by measuring optical density of the culture spectrophotometrically (600 nm). All PNA
| Gene target | Gene accession no. | CPP-PNA sequence sequencea | Proposed functions/Drug |
|---|---|---|---|
| inhA | MSMEG_3151 | H-KFFKFFKFFK-O-gtcatttggt-NH2 | Isoniazid Enoyl-ACP reductase (acyl carrier protein) of fatty acid synthase II needed to elongate long chain fatty acids for the synthesis of mycolic acid/Isoniazid (INH) |
| rpsL | MSMEG_1398 | H-KFFKFFKFFK-O-ttggcatgta-NH2 | Ribosomal protein S12/Streptomycin |
| gyrA | MSMEG_0456 | H-KFFKFFKFFK-O-catatcgtcgga-NH2 | DNA gyrase subunit A/Fluoroquinolones |
| pncA | MSMEG_6506 | H-KFFKFFKFFK-O-catgcctcga-NH2 | PZA inhibits the activity of FAS I (palmitic acid) in PZA-susceptible M. smegmatis strains/Pyrazinamide (PZA) |
| polA | MSMEG_3839 | H-KFFKFFKFFK-O-ttcatgcctgt-NH2 | DNA polymerase I |
| rpoC | MSMEG_1368 | H-KFFKFFKFFK-O-catcgtaactc-NH2 | RNA polymerase subunit β |
| dnaG (negative control PNA) | BR_1480 | H-KFFKFFKFFK-O-cattacagatt-NH2 | Mismatch PNA for DNA primase |
aThe PNAs and CPP were synthesized (Panagene Inc., Daejeon, South Korea) and joined by an ethylene glycol linker designated as “O”; a glycol linker of nine atoms used to distance the hybridization portion of the molecule from the CPP. The bolded “cat” indicates the PNA sequence complementary to ATG start codon contained in the specified gene.
treated cultures of M.smegmatis were removed after 48 h and were serially diluted and plated on 7H10 Middlebrook agar. The plates were incubated at 37˚C for 2 to 4 days to determine the number of viable cells (CFU/ml).
Intracellular activity of PNAs was evaluated in J774A.1 murine macrophage cell line (ATCC, Manassas, VA). J774A.1 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) (Sigma) supplemented with heat inactivated 10% fetal bovine serum. The cells were grown at 37˚C in an atmosphere containing 5% CO2. J774A.1 cells were plated in 200 µl of DMEM at a concentration of 6.5 × 104 cells per well in 96-well tissue culture white plates with clear bottoms (Corning Inc. Cat. #3596) and allowed to adhere overnight. For the infection, mid-log phase M.smegmatis (OD600 = 0.5) were washed twice with PBS, centrifuged at 2800 × G for 15 min and then resuspended in DMEM and sonicated for 2 × 30 seconds in a bath sonicator to disperse clumps. The bacteria in the supernatant were then diluted in DMEM to a concentration of 1.6 × 106 CFUs; 100 µl of bacterial suspension was added to each well of the J774A.1 cells. After 1h of infection at 37˚C in 5% CO2, J774A.1 cells were washed twice with gentamicin (10 µg/ml) to eliminate any extracellular bacteria. Cells were treated with PNAs designed to target inhA,rpsL,pncA, rpoC,polA, gyrA and dnaG. The concentrations of each PNA were 0.2 µM, 2 µM and 20 µM; rifampin concentration was 16 µg/ml. 100 µl of each PNA concentration or rifampin was added to infected J774A.1 cells in triplicate and incubated at 37˚C under 5% CO2 for 24 h. The infected macrophages were lysed with 100 µl of 0.1% TritonX-100 (Sigma) and serially diluted in PBS–Tween and 25 µl lysed cells were inoculated onto Middlebrook7H10 agar (Difco) plates for viable count determination. The CFU was determined just after the infection (day 0, i.e., post gentamicin treatment) and at the end of the experiment.
Cell viability assay using tetrazolium compound [CellTiter 96® Aqueous Non-Radioactive Cell Proliferation Assay (MTS); Promega, Fitchburg, WI] was conducted to measure the growth inhibitory effects of PNAs linked with CPP on J774A.1 cells. The macrophages were treated with 20 µM PNA linked with CPP and incubated at 37˚C under 5% CO2 for 24 h. The viable cell numbers were determined at 490 nm absorbance due to the formazone product. The trypan blue exclusion assay was used to measure cell viability after 24 h of PNA treatment. The number of viable macrophage cells was assessed using a hemocytometer. The percentage of living cells was determined by counting at least 100 cells.
CFU data of PNAs treated and untreated infected cells were analyzed for statistical significance using student’s t test (Microsoft Excel version 2010, USA). A P value of ≤0.05 was considered to be statistically significant.
The results of growth inhibitory assays of six PNA showed that only two PNAs (inhA and rpsL)out of six essential genes were found to exhibit strong growth inhibition at all concentrations. Three PNAs (pncA, polA and rpoC) showed growth inhibitory effect only at the highest concentration used i.e., 20 µM concentration. One PNA (gyrA)and a mismatch PNA did not show any growth inhibitory effect. The growth inhibition effects of PNAs are summarized in
Growth inhibitory effects of PNAs were measured at 24 h post-treatment. The results show that all six PNAs significantly decreased the intracellular growth of M.smegmatis (p > 0.05). PNAs targeting inhA and rpsL showed concentration dependent growth inhibition as compared to remaining four PNAs. Complete growth inhibition of intracellular growth was observed for rpsL PNA at 20 µm concentration. The ability of PNAs to inhibit growth of M.smegmatis inside murine macrophages is shown in
The viability and reproductive capacity of J774A.1 macrophage cell line was tested by treating the cell line with PNAs linked or unlinked to CPP or by CPP only at 24 h post treatment. No visible toxic effects were seen both in trypan blue exclusion assay and MTT assay (data not shown). None of the tested concentrations of PNAs affected either morphology or doubling time of J774.A1 cells (data not shown).
| Gene target | PNA concentration | |||
|---|---|---|---|---|
| 20 µM | 10 µM | 5 µM | 2.5 µM | |
| dnaG* | - | - | - | - |
| inhA | ++++ | ++++ | ++++ | ++++ |
| rpsL | ++++ | ++++ | ++++ | ++++ |
| gyrA | - | - | - | - |
| pncA | ++++ | - | - | - |
| polA | ++++ | - | - | - |
| rpoC | ++++ | - | - | - |
Abbreviations: ++++, Strong inhibition of growth; -, No growth inhibition, *, mismatch control PNA.
The identification of essential genes of microbes through microbial genomics helps selecting potential targets for antisense inhibition. Antisense therapy using PNAs has become an attractive tool for controlling bacterial growth. PNAs can produce strong complexes with complementary strands of DNA or RNA and inhibit expression of microbial genes required for their growth in dose dependent manner at low micromolar concentration [
There was no detectable toxic effect on macrophages noticed either in a trypan blue exclusion assay or in the cell viability MTS assay even at the highest concentration (20 µM) of PNA used suggesting that the PNA is specific for M.smegmatis.
Antisense oligonucleotides are designed to bind the target mRNA to prevent translation or bind DNA to prevent gene transcription respectively. They can bind to the specific sequence of RNA and DNA by Watson-Crick base pairing which leads to a variety of post-binding events [
Sequence homology is necessary for PNA binding in order to selectively hybridize to complementary nucleic acids of either chromosomal or messenger RNA of M.smegmatis.6 This study demonstrated sequence specific binding of PNAs with the gene targets since mismatch a PNA lacking sequence homology did not show growth inhibition against M. smegmatis both in pure culture and in M.smegmatis infected J774A.1 cells. The antimicrobial effect observed against M.smegmatis by antisense peptide PNAs in murine macrophage cell line shows the potential for the development of PNA based antibiotic against mycobacterial infection in humans.
M.smegmatis is susceptible in pure culture to PNA mediated growth inhibition by targeting genes involved in cell envelope synthesis, DNA and mRNA synthesis, and protein synthesis. M.smegmatis growing in a macrophage cell line is also susceptible to PNA targeting genes involved in cell envelope and nucleic acid synthesis of the bacterium. Data from this study suggest that PNAs targeting inhA and rpsL could be useful as antisense therapy for Mycobacterium infections. Further studies to examine the effect of PNAs administered as nanoparticles targeted to macrophages in mice infected with M.smegmatis will need to be carried out.
We thank Rebecca Wattam (Virginia Bioinformatics Institute) for help with PNA design.
This research was funded through grants from NIH to SMB (5R03AI083735-02) and from The Islamic Development Bank to MAI.
Not required.
A poster with interim findings was presented at the Euro-Scicon conference 2013 held on 21 March 2013 at the Royal College of Pathologist, London, UK. The poster’s abstract was published in https://www.euroscicon.com/documents/21March2013TB.pdf.
The authors declare no conflicts of interest regarding the publication of this paper.
Islam, M.A., Khatun, M.M.,Sriranganathan, N. and Boyle, S.M. (2021) Essential Gene(s) Targeted by Peptide Nucleic Acids Kills Mycobacterium smegmatis in Culture and in Infected Macrophages. Advances in Infectious Diseases, 11, 156-164. https://doi.org/10.4236/aid.2021.112015