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Acinetobacter baumannii is a notorious pathogen that commonly thrives in hospital environments and is responsible for numerous nosocomial infections in humans. The burgeoning multi-drug resistance leaves relatively minimal options for treating the bacterial infection, posing a significant problem and prompting the identification of new approaches for tackling the same. This motivated us to focus on non-canonical nucleic acid structures, mainly G-quadruplexes, as drug targets. G-quadruplexes have recently been gaining attention due to their involvement in multiple bacterial and viral pathogenesis. Herein, we sought to explore conserved putative G-quadruplex motifs in A. baumannii. In silico analysis revealed the presence of eight conserved motifs in genes involved in bacterial survival and pathogenesis. The biophysical and biomolecular analysis confirmed stable G-quadruplex formation by the motifs and showed a high binding affinity with the well-reported G-quadruplex binding ligand, BRACO-19. BRACO-19 exposure also decreased the growth of bacteria and downregulated the expression of G-quadruplex-harboring genes. The biofilm-forming ability of the bacteria was also affected by BRACO-19 addition. Taking all these observations into account, we have shown here for the first time the potential of G-quadruplex structures as a promising drug target in Acinetobacter baumannii, for addressing the challenges posed by this infamous pathogen., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Neisseria gonorrhoeae is an obligate human pathogen that causes gonorrhea and has shown a vast emergence of multidrug resistance in recent times. It is necessary to develop novel therapeutic strategies to combat this multidrug-resistant pathogen. The non-canonical stable secondary structures of nucleic acids, G-quadruplexes (GQs), are reported to regulate gene expressions in viruses, prokaryotes, and eukaryotes. Herein, we explored the whole genome of N. gonorrhoeae to mine evolutionary conserved GQ motifs. The Ng-GQs were highly enriched in the genes involved in various important biological and molecular processes of N. gonorrhoeae. Five of these GQ motifs were characterized using biophysical and biomolecular techniques. The GQ-specific ligand, BRACO-19, showed a high affinity towards these GQ motifs and stabilized them in both in vitro and in vivo conditions. The ligand showed potent anti-gonococcal activity and modulated the gene expression of the GQ-harboring genes. Strikingly, BRACO-19 also altered the biofilm formation in N. gonorrhoeae and its adhesion and invasion of the human cervical epithelial cells. In summary, the present study showed a significant role of GQ motifs in N. gonorrhoeae biology and put forward a step closer towards the search for therapeutic measures in combating the emerging antimicrobial resistance in the pathogen. KEY POINTS: •Neisseria gonorrhoeae genome is enriched in non-canonical nucleic acid structures-G-quadruplexes. •These G-quadruplexes might regulate bacterial growth, virulence, and pathogenesis. •G-quadruplex ligands inhibit biofilm formation, adhesion, and invasion of the gonococcus bacterium., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The genome of a micro-organism contains all the information required for its survival inside its host cells. The guanine rich regions of the genome can form stable G-quadruplex structures that act as the regulators of gene expression. Herein, the completely sequenced genomes of Helicobacter pylori were explored for the identification and characterization of the conserved G-quadruplex motifs in this gastrointestinal pathogen. Initial in silico analysis revealed the presence of ~8241 GQ motifs in the H. pylori genome. Metal binding proteins of H. pylori are significantly enriched in the GQ motifs. Our study emphasizes the identification and characterization of four highly conserved G-quadruplex forming motifs (HPGQs) in the nickel transporter genes (nixA, niuB1, niuB2, and niuD) of the H. pylori. Nickel is a virulence determinant in H. pylori and is required as a co-factor for the urease and [NiFe] hydrogenase enzymes that are crucial for its survival in the stomach lining of humans. The presence of GQ motifs in these nickel transporter genes can affect their expression and may alter the functioning of Urease and [NiFe] hydrogenase. Similar to human and virus G-quadruplexes, targeting these conserved PGQs with bioactive molecules may represent a novel therapeutic avenue for combating infection of H. pylori. The identified HPGQs were characterized in-vitro by using CD spectroscopy, electrophoresis technique, and NMR spectroscopy at both acidic (4.5) and neutral pH (7.0). ITC revealed the specific interaction of these HPGQs with high affinity to the known G-quadruplex binding ligand, TMPyP4. The mTFP based reporter assay showed decrease in the gene expression of mTFP in the TMPyP4 treated cells as compared to the untreated and further affirmed the formation of stable G-quadruplex structures in the HPGQ motifs in vivo. This is the first report for characterizing G-quadruplex motifs in nickel transport-associated genes in the H. pylori bacterium., (Copyright © 2022. Published by Elsevier B.V.)

The noncanonical structures, G-quadruplexes (GQs), formed in the guanine-rich region of nucleic acids regulate various biological and molecular functions in prokaryotes and eukaryotes. Neisseria meningitidis is a commensal residing in a human's upper respiratory tract but occasionally becomes virulent, causing life-threatening septicemia and meningitis. The factors causing these changes in phenotypes are not fully understood. At the molecular level, regulatory components help in a clearer understanding of the pathogen's virulence and pathogenesis. Herein, genome analysis followed by biophysical assays and cell-based experiments revealed the presence of conserved GQ motifs in N. meningitidis . These GQs are linked to the essential genes involved in cell adhesion, pathogenesis, virulence, transport, DNA repair, and recombination. Primer extension stop assay, reporter assays, and quantitative real-time polymerase chain reaction (qRT-PCR) further affirmed the formation of stable GQs in vitro and in vivo . These results support the existence of evolutionarily conserved GQ motifs in N. meningitidis and uphold the usage of GQ-specific ligands as novel antimeningococcal therapeutics.

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The G-quadruplex (GQ) motifs have recently been gaining prominence because of their role as gene cis-regulatory elements in a variety of organisms and as potential druggable targets for anti-cancer therapy and ageing. Several studies have demonstrated the existence of GQs in the genomes of emerging and re-emerging human pathogens, such as hepatitis virus, herpesviruses, Ebola virus, Zika virus and Nipah virus. Human Adenovirus (HAdV) exhibits a large number of clinical manifestations especially infecting the children and the immunocompromised patients. Moreover, the HAdV-based vectors have been widely used to deliver foreign DNAs to cells in gene therapy. However, the DNA secondary structural elements in AdV-based vectors could significantly determine the gene delivery efficacy of the vectors. In this study, using a combination of whole genome sequence analysis, biochemical, biophysical and interaction assays, we revealed fifteen putative GQs that are conserved across the different species of HAdV. We further showed that the GQs are embedded in the sequences of essential viral genes, namely E1B, E2B, and L3 genes (among others), which are involved in the early and late stages of the viral life cycle. Notably, Braco-19 (a well-known GQ binding ligand) interacted specifically with the HAdV GQs and increased their stability and further blocked the HAdV multiplication in human cells. Taken together, our data strongly supported the existence of G-quadruplex structures in the HAdV genome that affect the virus multiplication and posit that such structures may influence the efficacy of the gene-delivery vectors or even the HAdV virus life-cycle., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The G-quadruplex (GQ) motifs are considered as potential drug-target sites for several human pathogenic viruses such as Zika, Hepatitis, Ebola, and Human Herpesviruses. The recent outbreaks of Nipah virus (NiV) in India, the highly fatal emerging zoonotic virus is a potential threat to global health security as no anti-viral drug or vaccine in currently available. Therefore, here in the present study, we sought to assess the ability of the putative G-quadruplex forming sequences in the NiV genome to form G-quadruplex structures and act as targets for anti-viral compounds. Bioinformatics analysis underpinned by various biophysical and biochemical techniques (such as NMR, CD, EMSA, DMS footprinting assay) confirmed the presence of two highly conserved G-quadruplex forming sequences (HGQs) in the G and L genes of NiV. These genes encode the cell attachment glycoprotein and RNA-dependent RNA polymerase, respectively and are essential for the virus entry and replication within the host cell. It remains possible that stabilization of these HGQs by the known G-quadruplex binding ligands like TMPyP4 and Braco-19 represents a promising strategy to inhibit the expression of the HGQ harboring genes and thereby stop the viral entry and replication inside the host cell. Accordingly, we report for the first time, that HGQs in Nipah virus genome are targets for G-quadruplex specific ligands; therefore, could serve as potential targets for anti-viral therapy.

Several G-quadruplex forming motifs have been reported to be highly conserved in the regulatory regions of the genome of different organisms and influence various biological processes like DNA replication, recombination and gene expression. Here, we report the highly conserved and three potentially G-quadruplex forming motifs (SP-PGQs) in the essential genes (hsdS, recD, and pmrA) of the Streptococcus pneumoniae genome. These genes were previously observed to play a vital role in providing the virulence to the bacteria, by participating in the host-pathogen interaction, drug-efflux system and recombination- repair system. However, the presence and importance of highly conserved G-quadruplex motifs in these genes have not been previously recognized. We employed the CD spectroscopy, NMR spectroscopy, and electrophoretic mobility shift assay to confirm the adaptation of the G-quadruplex structure by the SP-PGQs. Further, ITC and CD melting analysis revealed the energetically favorable and thermodynamically stable interaction between a candidate G4 binding small molecule TMPyP4 and SP-PGQs. Next, TFP reporter based assay confirmed the regulatory role of SP-PGQs in the expression of PGQ harboring genes. All these experiments together characterized the SP-PGQs as a promising drug target site for combating the Streptococcus pneumoniae infection.