Essential Gene(s) Targeted by Peptide Nucleic Acids Kills Mycobacterium smegmatis in Culture and in Infected Macrophages

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.


Introduction
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 [1]. Tuberculosis is one of the major causes of human death in the world. Around one-third of the human population is estimated to be infected with Mtb. Antibiotics commonly used to treat the Mtb in humans require prolonged therapy and they have many toxic side effects. The antibiotics used against Mtb cannot readily penetrate through the lipid-rich mycobacterial cell wall that represents a hurdle in the treatment of clinical disease [2] [3]. Moreover, emergence of multidrug-resistant (MDR) strains of Mtb is a great challenge to treat tuberculosis with a combination of antibiotics. It is very essential to develop an effective treatment for MDR Mtb without toxicity and that enables highly targeted delivery of antimicrobials to their intracellular niche.
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) [4]. It binds to form double stranded like DNA and complexes with complementary DNA or RNA. Short antisense oligonucleotides (10 -12 mers) PNAs can bind at start codon and upstream regions of essential genes. Addition of cell penetrating peptide (CPP) to the PNA transports it across cell membranes and bacterial cell envelopes, which is essential for its antimicrobial effects [5].
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 [6] [7] [8].
A previous study showed antisense PNA targeting only inhA gene inhibited the growth of M. smegmatis in pure culture [9]. As Mycobacterium spp. are intracellular pathogens, the growth inhibitory effects of antisense PNA must be studied inside the macrophage. To the best of our knowledge, no study has been performed so far to evaluate the growth inhibitory functions of anti-sense PNAs against Mycobacterium targeting one of its essential genes inside the macrophage. In this study, we have designed 6 antisense PNAs complementary to the translation start codon region on the mRNA of six essential genes of M. smegmatis; these are inhA, rpsL, gyrA, pncA, polA and rpoC. PNAs were conjugated with cell penetrating peptide (CPP = KFFKFFKFFK) to aid cell membrane passage [10]. The objectives of this study were to evaluate the inhibitory function of six anti-sense PNAs against M. smegmatis in pure culture and inside the macrophages and to identify the most suitable gene targets of Mycobacterium for antisense therapy.

Bacterial Growth in Pure Culture
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 OD 600 0.5 was reached.

Peptide Nucleic Acids
The list of PNAs shown in Table 1 was synthesized by Panagene Inc. (Daejeon, Republic of Korea). PNAs conjugated with cell penetrating peptide (KFF) 3 K, where K is lysine and F is phenylalanine, to facilitate PNAs uptake through bacterial cell envelope.

Growth of M. smegmatis in Pure Culture
An overnight culture of M. smegmatis was diluted in 7H9 broth to obtain 6 × 10 4 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

Inhibition of Growth in Pure Culture
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 Table   2.

Inhibition of Growth inside Murine Macrophage
Growth

Macrophage Viability and Toxicity Assay
The

Discussion
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 [11] [12]. Bacteria living within eukaryotic host tissue constitute a significant challenge for antisense treatment [13]. The PNAs linked with cell penetrating peptides are found effective in inhibiting growth of microbes both in vitro and in vivo [8]. The high prevalence of extreme drug resistant Mtb leaves no treatment option for patients that leads to increased morbidity and mortality rate worldwide. In this situation there is an urgent need to develop new drugs against Mtb that could be alternatives to traditional antibiotics. The present study evaluated six PNAs against M. smegmatis both in vitro and in vivo. In this study six PNAs were selected using the bioinformatics resources and tools. The PNAs were linked with cell penetrating peptide (CPP): CPP-O-PNA. These were significantly more effective in inhibiting expression of their targets as compared to non-CPP linked PNAs [8]. Cationic CPPs are known to bind with proteoglycans of eukaryotic cells and then enter into the cells by endocytosis [14] [15]. To develop a new antimicrobial, selection of essential genes as targets in bacteria for antisense PNAs is important [16]. The present study selected six essential genes (inhA, rpsL, gyrA, pncA, polA and rpoC) of M. smegmatis for development of effective antisense treatment targets. PNAs in this study were found to be effective in inhibiting the growth of M. smegmatis both in pure culture and murine macrophages. The antibacterial effect of PNA depends on bioavailability of PNA inside the bacterial cell while the lipid enriched cell wall is the major barrier of entrance to PNA inside Mycobacterium [2] [6]. Although gyrA PNA did not show growth inhibition of M. smegmatis in pure culture, it was found to have growth inhibitory effect against M. smegmatis inside murine macrophage. Growth inhibitory effects of gyrA PNA against Klebsiella pneumonia were observed both in pure culture and inside human epithelial cells at 20 µM concentration [17]. PNAs targeting rpsL and inhA were found to be most effective in killing M. smegmatis as compared to other essential gene targets. A dose dependent growth inhibition of M. smegmatis was observed for both rpsL and inhA PNAs. The susceptibility of M. smegmatis by inhA PNA to growth inhibition was originally shown by Kulyte et al., [9] and occurred at concentrations between 2 and 15 uM.
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 [17] [18]. Data from this study indirectly indicates that PNAs exerted antimicrobial activity by inhibiting the expression of essential gene(s) involved in different types of macromolecular synthesis.
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

Conclusion
M. smegmatis is susceptible in pure culture to PNA mediated growth inhibition by targeting genes involved in cell envelope synthesis, DNA and mRNA synthe-sis, 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.