key: cord-0974621-744n10r9
authors: Jain, Neha; Mishra, Subodh Kumar; Shankar, Uma; Jaiswal, Ankit; Sharma, Tarun Kumar; Kodgire, Prashant; Kumar, Amit
title: G-quadruplex stabilization in the ions and maltose transporters gene inhibit Salmonella enterica growth and virulence
date: 2020-09-06
journal: Genomics
DOI: 10.1016/j.ygeno.2020.09.010
sha: 31b780dd76474b2deb92dd5818fa3574592d2549
doc_id: 974621
cord_uid: 744n10r9

The G-quadruplex structure is a highly conserved drug target for preventing infection of several human pathogens. We tried to explore G-quadruplex forming motifs as promising drug targets in the genome of Salmonella enterica that causes enteric fever in humans. Herein, we report three highly conserved G-quadruplex motifs (SE-PGQ-1, 2, and 3) in the genome of Salmonella enterica. Bioinformatics analysis inferred the presence of SE-PGQ-1 in the regulatory region of mgtA, SE-PGQ-2 in ORF of entA, and SE-PGQ-3 in the promoter region of malE and malK genes. The G-quadruplex forming sequences were confirmed by biophysical and biomolecular techniques. Cellular studies affirm the inhibitory effect of G-quadruplex specific ligands on Salmonella enterica growth. Further, PCR inhibition, reporter based assay, and RT-qPCR assays emphasize the biological relevance of G-quadruplexes in these genes. Thus, this study confirmed the presence of G-quadruplex motifs in Salmonella enterica and characterized them as a promising drug target.

Salmonella enterica belongs to the Enterobacteriaceae family and is known to cause typhoid fever and food poisoning. Salmonella enterica contains six subspecies among those, S. enterica is known to infect humans and further divided into two subclasses ( Figure 1a ) [1, 2] . Typhoidal class of S. enterica included S. enterica subsp. enterica ser. Typhi (S. ser. Typhi) and S. ser. Paratyphi and are known to causes typhoid fever, whereas nontyphoidal class includes S. ser. Enteritidis, and S. ser. Typhimurium causes food poisoning in humans [3] . As per the Centers for Disease Control and Prevention (CDC), typhoid fever causes ~22 million new cases and ~200,000

deaths every year across the world [4] . The emergence of anti-microbial resistance for chloramphenicol, cotrimoxazole, ampicillin, ciprofloxacin, ofloxacin, azithromycin, and cephalosporin leads to increased death rate due to clinical treatment failure and make the situation more dangerous [5] [6] [7] . Due to its high prevalence and rapid emergence of drug resistance, Salmonella rings a global alarm for the development of novel and promising therapeutic approaches.

S. enterica is an intracellular pathogen that grows in phagocytes and macrophages. During the growth phase, the host innate immune system generates various nitro-oxidative and oxidative stresses to eradicate this pathogen. However, S. enterica possesses a magnesium homeostasis mechanism that is mainly controlled by mgtA gene and helps bacteria to survive in nitro-oxidative stressed conditions [8] . The mgtA encodes for an Mg 2+ transport ATPase that neutralizes the reactive nitrogen stress (RNS, nitro-oxidative) and plays a vital role in the bacterial survival inside macrophages (Supplementary data: Figure S1a ) [9] . Hence, targeting mgtA gene expression may serve as a promising therapeutic approach against S. enterica pathogenesis.

Similar to Mg 2+ , Iron(Fe) is also an essential nutrient element for the S. enterica. The S. enterica contains an entABCDEF operon that encodes for two low molecular weight siderophores (enterobactin and salmochelin) that have a high affinity for iron than the host iron-binding proteins and import iron inside the bacteria cell radioresistance [32] . Similarly, G4 present at the upstream of nasT in Paracoccus denitrificans is involved in nitrite assimilation [33] .

All these reports demonstrate the pivotal role of G-quadruplex in human pathogens, and their conservedness suggests them as a promising drug target for both drug-susceptible and drug-resistant strains of pathogens.

Therefore, a comprehensive study that discovers highly conserved G-quadruplex in the essential genes of S. enterica may provide a more suitable therapeutic approach for fighting against infection and overcome the emergence of the drug-resistant problem in this deadly pathogen.

In the present study, we sought to explore the highly conserved potential G-quadruplex forming sequences (Figure 2a) . In order to confirm the formation of G-quadruplex structure by SE-PGQs, CD, NMR, and EMSA were employed and to validate these SE-PGQs as a potential drug target, CD melting, and polymerase stop assays were performed. Disc diffusion assay and MTT assay confirmed the growth inhibition of S. enterica cells by G4 binders BRACO-19 (9-[4-(N,N-dimethylamino)phenylamino]-3,6-bis(3pyrrolodinopropionamido) acridine) and 9-Aminoacridine. Further, Real-time PCR (RT-PCR) revealed the reduced expression of genes that harbor these SE-PGQs in either their coding region or regulatory region upon the treatment with both BRACO-19 and 9-Aminoacridine. mTFP reporter based assay further strengthens the role of SE-PGQs in the gene expression regulation. This change in expression of PGQ harboring genes in the presence of G4 binding ligand suggested a G4 mediated regulatory mechanism in the expression of these genes. Therefore, G-quadruplex motifs found in these genes can be utilized as a potential drug target to develop a promising anti-microbial therapeutics. Moreover, the high conserved-ness of these SE-PGQs, even in the drug-resistant strain would be able to overcome the problem of the emergence of drug-resistance in S. enterica

J o u r n a l P r e -p r o o f Journal Pre-proof

Since the last several decades, the scientific community has been witnessed a rapid increase in the number of such human pathogenic bacterial species that acquired resistance to multiple anti-bacterial agents. Currently, the emergence of multidrug-resistant strains remains a significant public health concern for clinical investigators that rings a global alarm to search for novel and highly conserved drug targets. Recently, conserved G-quadruplexes and their binding with small molecules are being extensively investigated as a promising therapeutic approach for combating the various type of human pathogenic infection [27, 28] . Considering the suitability of G-quadruplex structure as a promising drug target in both drug-susceptible and drug-resistant strains of pathogens, here we sought to search for G-quadruplex motifs in S. enterica strains.

Comprehensive mining of potential G-quadruplex forming motifs (SE-PGQs) was performed on 412 completely sequenced strains of S. enterica (Supplementary data: Table S1 ). The bioinformatics analysis observed a total of 109400 PGQs in 412 strains of S. enterica (Document S2). Given that, a similar sequence may correspond to the same structure and evolutionarily conserved function, all the predicted PGQs were further clustered by Unweighted Pair Group Method with Arithmetic Mean clustering method using Clustal Omega tool. The conservedness is an essential parameter that makes these PGQ motifs suitable to work as promising drug targets. Therefore, next, we examined the conservation of each PGQ clustered using the following equation:

Where p is the frequency of occurrence, n = number of strains with specific G4 sequence, and N represents the total number of strains of S. enterica. These conservation analysis revealed 187 PGQ clusters that were observed to possess conversed-ness in more than 90% strains of S. enterica (Supplementary data: Table S2 ). G-quadruplex with loop length 1-7 and G tract of ≥3 forms a more stable G-quadruplex [34] . Therefore, for further study, we selected only those PGQs that satisfied the aforesaid criteria of G-quadruplex formation and were listed in Supplementary data: Table S3 .

These predictions were further crosschecked with QGRS Mapper and PQSFinder (Supplementary data: Guanine residues of SE-PGQ motifs during the evolutionary process ( Figure 2b ).

NMR spectroscopy is considered as the most reliable technique for confirming the formation of G-quadruplex structure formation by the nucleic acid sequences. Therefore 1D 1 H NMR spectroscopy was performed to confirm the formation of G-quadruplex conformation by SE-PGQs. The presence of a chemical shift in the range of 10-12 ppm in 1D 1 H NMR spectra depicts the presence of hoogsteen base pairing in characteristic G-tetrads of Gquadruplex structure whereas, canonical G-C Watson Crick base pairing can be characterized by a chemical shift in the range of 12-14 ppm. All the three SE-PGQs showed an imino proton resonance between 10-12 ppm and clearly affirmed the formation of G-quadruplex structure ( Figure 3 and Supplementary data: Figure S2 ). Whereas, mutant sequence NMR spectra analysis depicted an absence of imino proton peaks between 10-12 ppm. (Supplementary data: Figure S3) 

Circular dichroism is one of the widely used techniques for analyzing the topology of the G-quadruplex structure. G-quadruplex, depending upon its sequence, loop length, and bound cation, can form either a parallel, anti-parallel or hybrid conformation. A positive peak at ~260 nm and a negative peak at ~240 nm signifies for parallel G-quadruplex topology. However, a positive peak at ~290 nm and a negative peak at ~260 nm signifies for anti-parallel G-quadruplex topology. Two positive peaks at 260 nm and 290 nm with a negative peak at 240 nm depicts the mix or hybrid topology. Additionally, all the G-quadruplex conformers forms a positive peak at 210 nm [35] . Different cation affects the stability of the G-quadruplex structure to a different extent. The stabilizing ability of some well studied cations is ranked as follows : K + > Na + > Mg 2+ > Li + [20] . Therefore, we performed the CD spectroscopy of SE-PGQs in these four different cations (K + , Na + , Li +, and Mg 2+ ) containing buffers ( Figure 4 and Supplementary data: Figure S4 ).

CD spectra analysis revealed the predominant parallel G-quadruplex topology exhibited by SE-PGQ-1 and SE-PGQ-3 in the presence of the K + ion, whereas SE-PGQ-2 showed hybrid G-quadruplex topology ( Figure 4a ). As expected, CD spectral scanning performed in the increasing concentration of K + ion showed the maximum molar ellipticity in the highest K + ion concentration (Supplementary data: Figure S5 ). Figure To evaluate the significance of G-tracts for their G-quadruplex forming ability, the central Guanine was mutated to Adenine, and CD spectra analysis was performed in 50 mM K + ion (Supplementary data: Table S6 ). Mutants (mut-PGQ-1, mut-PGQ-2, and mut-PGQ-3) failed to show the characteristic CD signal of G-quadruplex i.e., a positive band at 210/260/290 nm and a negative band at 240 nm suggesting the mutation in G tract disrupted the G-quadruplex formation ( Figure 4a ).

Next, Electrophoretic Mobility Shift assay (EMSA) was performed to check the molecularity (inter or intra molecular G-quadruplex) of SE-PGQ in the solution. An intramolecular G-quadruplexes possess a compact topology and migrate faster than their linear counterpart, whereas intermolecular G-quadruplex contains a comparatively wider topology and exhibited slow migration than their linear counterpart [36] . All three SE-PGQs and positive control (Tel22 DNA G-quadruplex) showed faster mobility than their respective linear counterpart. They, therefore, suggested the formation of intramolecular G-quadruplex by SE-PGQs (Supplementary data: Figure S6 ).

Similar to the G-quadruplex motif present in the genome of another human pathogen, a G-quadruplex motif present in mgtA, malK, malE, and entA genes of S. enterica strains may also serve as a potential target for developing anti-bacterial therapy. These G-rich targets can also overcome the problem of the drug resistance due to their high conserved-ness in both drug-susceptible and drug-resistant bacterial strains. Recently, conserved Gquadruplexes and their binding with small molecules are being extensively investigated as a promising therapeutic approach for combating the various type of human pathogen infection [27, 28] . For example, HIV-1 promoter region possessed a G-quadruplex motif in the long terminal repeat (LTR) region of their genome and observed to be critical for its proliferation. BRACO-19, a tri-substituted Acridine derivative, has shown anti-HIV-1 activity by stabilizing the G-quadruplex motif present in the LTR region [27] . BRACO-19 is also reported to inhibit the viral multiplication in Adenoviruses and Herpes Simplex virus by stabilizing G-quadruplex motifs in their genome [30, 37] .

Similarly, stabilization of G-quadruplex structure present in the core gene of HCV genome by PDP, halts its replication, translation and, therefore, can be used as potential anti-hepatitis therapeutics [38] . Pyridostatin is also J o u r n a l P r e -p r o o f Journal Pre-proof observed to stabilize the G-quadruplex structure formed in the mRNA of the nuclear antigen 1 protein of EBV, leading to its translation suppression [39] . Recently, BRACO-19 has shown an inhibitory effect on Mycobacterium tuberculosis, and Klebsiella pneumoniae, while Quarfloxin inhibited Plasmodium falciparum, by stabilizing Gquadruplexes present in various regions of their genome [25, 40, 41] . Previously, 9-Aminoacridine and its derivatives have been observed for their anti-proliferative properties in cancer cells [42] by binding to the telomeric region [43] , the c-Myc gene [44] and c-Kit promoter [45] . Therefore, here we analyzed the effect of G-quadruplex specific ligands, BRACO-19, and 9-Aminoacridine on the S. enterica growth and performed MTT assay. An increase in color intensity with the decrease in the concentration of G4 specific ligands was observed giving maximum in control well (untreated) that suggested the inhibitory effect of both G4 ligands, BRACO-19 and 9-Aminoacridine on the S. enterica growth with an IC 50 value of 15.877 μM and 10.5 μM respectively (Supplementary data: Figure S7 and S8).

Isothermal Table S8 ). For 9-Aminoacridine, the association constant (K a 1) was found to J o u r n a l P r e -p r o o f Journal Pre-proof be 61.99, 52.02, and 62.98 folds higher for SE-PGQ-1, SE-PGQ-2, and SE-PGQ-3 respectively than the association constant for the negative control duplex DNA (Supplementary data: Figure S10 and Table S8 ). On comparative analysis, it was observed that the BRACO-19 showed the highest affinity for SE-PGQ-2 (2.73 x 10 6 M -1 ) while 9-Aminoacridine showed the highest binding against SE-PGQ-2 (2.81 x 10 6 M -1 ). In summary, ITC analysis depicted the higher affinity and specificity of BRACO-19 and 9-Aminoacridine with SE-PGQs as compared to that of the duplex DNA [46, 47] .

To (Supplementary data: Figure S12 ). of a planer hetero-aromatic chromophore that helps in π-π interaction with the G4 motif. A short alkyl chain substituents in this heterocyclic ring, usually terminated by an amino group, enhances the G4 specificity to a large extend [51] . 9-Aminoacridine satisfies this condition of the hetero-aromatic chromophore and can be substituted with the desired functional groups. This has been widely exploited for the synthesis of numerous di-substituted and tri-substituted Acridine Derivatives that have shown very high specificity towards G-quadruplexes as compared to the duplex DNA [42, 43, 45, [52] [53] [54] [55] [56] [57] [58] . Thus, this Acridine scaffold can be used as a starting point for the synthesis of novel G4 specific ligands with minimum cytotoxicity to the host cells. Herein, two Acridine derivatives, BRACO-19 and 9-Aminoacridine were observed to bind and reduced the expression of these G-quadruplex structures possessing genes and thereby proposed as novel G4 mediated therapeutic approach for combating the infection of Salmonella enterica in humans.

Completely sequenced strains of S. enterica (Supplementary data: Table S1 ) were downloaded from National Center for Biotechnology Information (NCBI). These strains were then extensively mined for the potential J o u r n a l P r e -p r o o f G-quadruplex motifs in both sense and antisense strand using our previously developed G-quadruplex predictor tool [59] . This prediction tool used the following regular expression.

Where T1 represents consecutive tracts of Guanine that can be any number from 2-7, X is any nucleotide (A, T, G, C), L1, L2, L3 represents the variable loop region and can be any number from 1-7. For our prediction, we used G-tracts -3 or 4 and loop length 1-20 nucleotide [59] . The results were further cross-verified by using QGRS Mapper [60] and PGSFinder [61] tools.

To find the conserved PGQ's that are available in all the strains, multiple sequence alignment (MSA) was performed by using Clustal Omega tool, and clustering was done using UPGMA method. Consensus sequences representing the whole G4 sequence with -5 and +5 flanking regions were constructed using the Glam2 tool of MEME Suite [62] .

The resultant PGQ clusters were then mapped for their gene location in the genome of the individual S. enterica strains using the coordinates extracted from our G4 prediction tool by using Graphics mode of GenBank Database (https://www.ncbi.nlm.nih.gov/nuccore/).

In order to check the conservation of predicted PGQs at the Salmonella genus level, NCBI nucleotide BLAST was performed by taking each consensus PGQ as a query sequence and Salmonella bongori genome sequences as a target(NCBI taxid: 590). The threshold e-value was set as 1e-3 to remove any results by chance.

Predicted G4 oligonucleotides sequences were procured from Sigma Aldrich Chemicals Ltd. 

Native PAGE was run using 20% polyacrylamide gel in 1X TBE buffer. Each sample was dissolved in Tris buffer(pH=7, 10mM) containing four different cations K + , Na + , Mg 2+ , Li + (50mM each) separately. For each PGQ, an oligonucleotide of similar length (G mutated with T nucleotide) was taken as a negative control, and standard Gquadruplex (Tel22 DNA) was taken as a positive control. 20 µl of each oligonucleotide sample were loaded, and electrophoresis was performed at 4 0 C, 90 Voltage in a vertical gel unit system. The gels were visualized by staining with ethidium bromide and analyzed on ImageQuant LAS 4000 gel doc (GE Healthcare Biosciences Ltd., Sweden).).

AVANCE 500 MHz BioSpin International AG, Switzerland equipped with a 5 mm broadband inverse probe was used to perform NMR spectroscopic analysis. The SE-PGQs were dissolved in 50 mM potassium phosphate buffer.

All the NMR experiments were performed using H 2 O/D 2 O solvent at 9:1 ratio. Temperature of 298K with 20 ppm spectral width and 3 -(Trimethylsilyl) propionic-2, 2, 3, 3-D4 acid sodium salt (TSP) as an internal reference were used. NMR data processing, integration, and analysis were done by using Topspin 1.3 software. 

The S. ser. Typhimurium strain ATCC 14028 was procured from HiMedia and streaked on Nutrient Agar (HiMedia). A single colony was inoculated in the in Nutrient Broth (HiMedia) and kept overnight at 37˚C and 220 rpm in incubator shaker. [63] .

MTT assay was performed for cytotoxic analysis of 9-Aminoacridine and BRACO-19 on S. enterica. 50 µl of the overnight grown culture of S. enterica, was inoculated in 5 ml Nutrient Broth (NB) at 220 rpm, 37˚C and allowed to grow till the O.D 600 =0.5. After that, 50 µl was transferred in a fresh 5 ml NB tube, and 100 µl was transferred in each well of 96 well plates. Dilution was prepared from the stock solution (200 µM) of 9-Aminoacridine/BRACO-19 of the following concentrations 100 µM -0.09 µM and added to the respective wells, last well served as blank (without 9-Aminoacridine). The plates were kept at 37˚C, 220 rpm for 3 hr. Afterward, 10 µl of MTT (5 mg/mL) was added to each well and incubated for 3 hr. Finally, 20 µl of DMSO was added in each well to dissolve formazan crystal, and the plate was examined using a microplate reader (BioTek) at 590 nm [64] .

Templates 

Reporter based assay was performed by utilizing a revised protocol described elsewhere [65] . were added to MIC flask. For 9-Amino acridine treatment assay, control culture DMSO was added as 9-Aminoacridine was dissolved in it. All the flasks were kept at 37˚C, 220 rpm for 45 min. Subsequently, samples were centrifuged at 12000 rpm and immediately preceded for RNA isolation. S. enterica culture prior to RNA isolation was treated with RNA protect reagent (Qiagen, USA) to stabilize RNA and prevent it from degradation. Table S10 and S11, respectively. Briefly, the thermo-cycle used in qRT-PCR was 94˚C for 2 min, subsequently 40 cycles of 94˚C for 15 sec and 57˚C for 1 min.

Data conceptualization and methodology were performed by A.K. 

 Highly conserved potential G-quadruplex forming sequences (SE-PGQs) are reported from completely sequenced strains of S. enterica which could serves as potential therapeutic targets.

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The authors are thankful to SIC Facility at IIT Indore for NMR and CD experiments. N.J. and A.J. acknowledges the 

The authors declare no conflict of interest.