Pyrrole-based inhibitors of RND-type efflux pumps reverse antibiotic resistance and display anti-virulence potential.

Efflux pumps of the resistance-nodulation-cell division (RND) superfamily, particularly the AcrAB-TolC, and MexAB-OprM, besides mediating intrinsic and acquired resistance, also intervene in bacterial pathogenicity. Inhibitors of such pumps could restore the activities of antibiotics and curb bacterial virulence. Here, we identify pyrrole-based compounds that boost antibiotic activity in Escherichia coli and Pseudomonas aeruginosa by inhibiting their archetype RND transporters. Molecular docking and biophysical studies revealed that the EPIs bind to AcrB. The identified efflux pump inhibitors (EPIs) inhibit the efflux of fluorescent probes, attenuate persister formation, extend post-antibiotic effect, and diminish resistant mutant development. The bacterial membranes remained intact upon exposure to the EPIs. EPIs also possess an anti-pathogenic potential and attenuate P. aeruginosa virulence in vivo. The intracellular invasion of E. coli and P. aeruginosa inside the macrophages was hampered upon treatment with the lead EPI. The excellent efficacy of the EPI-antibiotic combination was evidenced in animal lung infection and sepsis protection models. These findings indicate that EPIs discovered herein with negligible toxicity are potential antibiotic adjuvants to address life-threatening Gram-negative bacterial infections.

Identification of diphenylurea derivatives as novel endocytosis inhibitors that demonstrate broad-spectrum activity against SARS-CoV-2 and influenza A virus both in vitro and in vivo.

Rapid evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A virus (IAV) poses enormous challenge in the development of broad-spectrum antivirals that are effective against the existing and emerging viral strains. Virus entry through endocytosis represents an attractive target for drug development, as inhibition of this early infection step should block downstream infection processes, and potentially inhibit viruses sharing the same entry route. In this study, we report the identification of 1,3-diphenylurea (DPU) derivatives (DPUDs) as a new class of endocytosis inhibitors, which broadly restricted entry and replication of several SARS-CoV-2 and IAV strains. Importantly, the DPUDs did not induce any significant cytotoxicity at concentrations effective against the viral infections. Examining the uptake of cargoes specific to different endocytic pathways, we found that DPUDs majorly affected clathrin-mediated endocytosis, which both SARS-CoV-2 and IAV utilize for cellular entry. In the DPUD-treated cells, although virus binding on the cell surface was unaffected, internalization of both the viruses was drastically reduced. Since compounds similar to the DPUDs were previously reported to transport anions including chloride (Cl-) across lipid membrane and since intracellular Cl- concentration plays a critical role in regulating vesicular trafficking, we hypothesized that the observed defect in endocytosis by the DPUDs could be due to altered Cl- gradient across the cell membrane. Using in vitro assays we demonstrated that the DPUDs transported Cl- into the cell and led to intracellular Cl- accumulation, which possibly affected the endocytic machinery by perturbing intracellular Cl- homeostasis. Finally, we tested the DPUDs in mice challenged with IAV and mouse-adapted SARS-CoV-2 (MA 10). Treatment of the infected mice with the DPUDs led to remarkable body weight recovery, improved survival and significantly reduced lung viral load, highlighting their potential for development as broad-spectrum antivirals.

Brevicillin, a novel lanthipeptide from the genus Brevibacillus with antimicrobial, antifungal, and antiviral activity.

AIM: This study was aimed to determine antimicrobial and antiviral activity of a novel lanthipeptide from a Brevibacillus sp. for disinfectant application. METHODS AND RESULTS: The antimicrobial peptide (AMP) was produced by a bacterial strain AF8 identified as a member of the genus Brevibacillus representing a novel species. Whole genome sequence analysis using BAGEL identified a putative complete biosynthetic gene cluster involved in lanthipeptide synthesis. The deduced amino acid sequence of lanthipeptide named as brevicillin, showed >30% similarity with epidermin. Mass determined by MALDI-MS and Q-TOF suggested posttranslational modifications like dehydration of all Ser and Thr amino acids to yield Dha and Dhb, respectively. Amino acid composition determined upon acid hydrolysis is in agreement with core peptide sequence deduced from the putative biosynthetic gene bvrAF8. Biochemical evidence along with stability features ascertained posttranslational modifications during formation of the core peptide. The peptide showed strong activity with 99% killing of pathogens at 12 µg ml-1 within 1 minute. Interestingly, it also showed potent anti-SARS-CoV-2 activity by inhibiting ∼99% virus growth at 10 µg ml-1 in cell culture-based assay. Brevicillin did not show dermal allergic reactions in BALB/c mice. CONCLUSION: This study provides detailed description of a novel lanthipeptide and demonstrates its effective antibacterial, antifungal and anti-SARS-CoV-2 activity.

Structural characterization of Myxococcus xanthus MglC, a component of the polarity control system, and its interactions with its paralog MglB.

The δ-proteobacteria Myxococcus xanthus displays social (S) and adventurous (A) motilities, which require pole-to-pole reversal of the motility regulator proteins. Mutual gliding motility protein C (MglC), a paralog of GTPase-activating protein Mutual gliding motility protein B (MglB), is a member of the polarity module involved in regulating motility. However, little is known about the structure and function of MglC. Here, we determined ∼1.85 Å resolution crystal structure of MglC using Selenomethionine Single-wavelength anomalous diffraction. The crystal structure revealed that, despite sharing <9% sequence identity, both MglB and MglC adopt a Regulatory Light Chain 7 family fold. However, MglC has a distinct ∼30° to 40° shift in the orientation of the functionally important α2 helix compared with other structural homologs. Using isothermal titration calorimetry and size-exclusion chromatography, we show that MglC binds MglB in 2:4 stoichiometry with submicromolar range dissociation constant. Using small-angle X-ray scattering and molecular docking studies, we show that the MglBC complex consists of a MglC homodimer sandwiched between two homodimers of MglB. A combination of size-exclusion chromatography and site-directed mutagenesis studies confirmed the MglBC interacting interface obtained by molecular docking studies. Finally, we show that the C-terminal region of MglB, crucial for binding its established partner MglA, is not required for binding MglC. These studies suggest that the MglB uses distinct interfaces to bind MglA and MglC. Based on these data, we propose a model suggesting a new role for MglC in polarity reversal in M. xanthus.

Disruption of MenT2 toxin impairs the growth of Mycobacterium tuberculosis in guinea pigs.

Toxin-antitoxin (TA) systems are abundantly present in the genomes of various bacterial pathogens. TA systems have been implicated in either plasmid maintenance or protection against phage infection, stress adaptation or disease pathogenesis. The genome of Mycobacterium tuberculosis encodes for more than 90 TA systems and 4 of these belong to the type IV subfamily (MenAT family). The toxins and antitoxins belonging to type IV TA systems share sequence homology with the AbiEii family of nucleotidyl transferases and the AbiEi family of putative transcriptional regulators, respectively. Here, we have performed experiments to understand the role of MenT2, a toxin from the type IV TA system, in mycobacterial physiology and disease pathogenesis. The ectopic expression of MenT2 using inducible vectors does not inhibit bacterial growth in liquid cultures. Bioinformatic and molecular modelling analysis suggested that the M. tuberculosis genome has an alternative start site upstream of the annotated menT2 gene. The overexpression of the reannotated MenT2 resulted in moderate growth inhibition of Mycobacterium smegmatis. We show that both menT2 and menA2 transcript levels are increased when M. tuberculosis is exposed to nitrosative stress, in vitro. When compared to the survival of the wild-type and the complemented strain, the ΔmenT2 mutant strain of M. tuberculosis was more resistant to being killed by nitrosative stress. However, the survival of both the ΔmenT2 mutant and the wild-type strain was similar in macrophages and when exposed to other stress conditions. Here, we show that MenT2 is required for the establishment of disease in guinea pigs. Gross pathology and histopathology analysis of lung tissues from guinea pigs infected with the ∆menT2 strain revealed significantly reduced tissue damage and inflammation. In summary, these results provide new insights into the role of MenT2 in mycobacterial pathogenesis.

Antifungal Azoles as Tetracycline Resistance Modifiers in Staphylococcus aureus.

Staphylococcus aureus has developed resistance to antimicrobials since their first use. The S. aureus major facilitator superfamily (MFS) efflux pump Tet(K) contributes to resistance to tetracyclines. The efflux pump diminishes antibiotic accumulation, and biofilm hampers the diffusion of antibiotics. None of the currently known compounds have been approved as efflux pump inhibitors (EPIs) for clinical use. In the current study, we screened clinically approved drugs for possible Tet(K) efflux pump inhibition. By performing in silico docking followed by in vitro checkerboard assays, we identified five azoles (the fungal ergosterol synthesis inhibitors) showing putative EPI-like potential with a fractional inhibitory concentration index of ≤0.5, indicating synergism. The functionality of the azoles was confirmed using ethidium bromide (EtBr) accumulation and efflux inhibition assays. In time-kill kinetics, the combination treatment with butoconazole engendered a marked increase in the bactericidal capacity of tetracycline. When assessing the off-target effects of the azoles, we observed no disruption of bacterial membrane permeability and polarization. Finally, the combination of azoles with tetracycline led to a significant eradication of preformed mature biofilms. This study demonstrates that azoles can be repurposed as putative Tet(K) EPIs and to reduce biofilm formation at clinically relevant concentrations. IMPORTANCE Staphylococcus aureus uses efflux pumps to transport antibiotics out of the cell and thus increases the dosage at which it endures antibiotics. Also, efflux pumps play a role in biofilm formation by the excretion of extracellular matrix molecules. One way to combat these pathogens may be to reduce the activity of efflux pumps and thereby increase pathogen sensitivity to existing antibiotics. We describe the in silico-based screen of clinically approved drugs that identified antifungal azoles inhibiting Tet(K), a pump that belongs to the major facilitator superfamily, and showed that these compounds bind to and block the activity of the Tet(K) pump. Azoles enhanced the susceptibility of tetracycline against S. aureus and its methicillin-resistant strains. The combination of azoles with tetracycline led to a significant reduction in preformed biofilms. Repurposing approved drugs may help solve the classical toxicity issues related to efflux pump inhibitors.

Floodplains landforms, clay deposition and irrigation return flow govern arsenic occurrence, prevalence and mobilization: A geochemical and isotopic study of the mid-Gangetic floodplains.

This article attempts to understand the evolution of groundwater chemistry in the mid Gangetic floodplain through the identification of hydrogeochemical processes including the impact of surface recharge and geological features. Isotopic investigations identified that irrigation return flow is partly responsible for arsenic (As) enrichment through preferential vertical recharge. Further, the floodplain geomorphological attributes and associated As hydrogeochemical behaviour traced through isotopes tracers highlighted that meandering and ox-bow like geomorphological features owing to clay deposition leads to the anoxic condition induced reductive microbial dissolution of As-bearing minerals causing the arsenic contamination in the investigated aquifer of the mid-Gangetic plain (MGP). To achieve the objectives, 146 water samples for water chemistry and 62 samples for the isotopic study were collected from Bhojpur district, Bihar (district bounded by the river Ganges in the north and Son in the east) located in MGP during the pre-monsoon season of 2018. The chemical results revealed high arsenic concentration (BDL to 206 µg.L-1, 32% samples are exceeding the 10 µg.L-1 limit) in the Holocene recent alluviums which are characterized by various geomorphological features such as meander scars and oxbow lake (northern part of the district). Arsenic is more concentrated in the depth range of 15-40 m below ground surface. All other trace metals viz. Ni, Pb, Zn, Cd and Al were found in low concentration except Fe and Mn. The geochemical analyses suggest that rock-water interaction is controlling the hydro-geochemistry while the chemical constituent of the groundwater is mainly controlled by carbonate weathering with limited contribution from silicate weathering. The isotopic signatures revealed that the Son river is recharging groundwater while the groundwater is contributing to the Ganges river. A clear pattern of fast vertical recharge in the arsenic contaminated area is observed in the proximity to the river Ganges with an elevated nitrate concentration resulted from the reduced As dissolution. The origin of groundwater is local precipitation with low to high evaporation enrichment effect which is further indicating the vertical mixing of groundwater from the irrigation return flow and/or recharge from domestic discharge causing enhanced As mobilization through microbial assisted reductive dissolution of As-bearing minerals.

Development of indices for surface and ground water quality assessment and characterization for Indian conditions.

Water quality indices are mathematical equations that transform water quality data into a particular number which describe the status of water. A number of water quality indices have been developed by various researchers for categorizing the water quality for different uses. These indices are developed based on classification criteria, sub-indices, and aggregation function. In the present study, a generalized Composite Water Quality Index (CWQI) is developed to classify the water into five categories, viz excellent, good, fair, poor, and polluted. For this purpose, the concentration ranges have been categorized on the basis of the Indian Standards (IS) and Central Pollution Control Board (CPCB) standards and considering International standards of World Health Organization (WHO) and European Commission (EC). Twenty-five water quality parameters are selected based on the social and environmental impacts, and weights are computed using the Saaty's Analytic Hierarchy Process (AHP) Multiple Criteria Decision Analysis (MCDA) tool. These parameters are selected such that the same indices can be used to assess the quality of both surface and ground water. The computed weights minimize the subjectivity in assigning the parameter weights. The proposed index improves understanding of water quality issues by integrating complex data and generates a score which describes the status of water quality. The index will be very useful for the water management authorities to maintain good health of surface water resources.

System-Wide Analysis Unravels the Differential Regulation and In Vivo Essentiality of Virulence-Associated Proteins B and C Toxin-Antitoxin Systems of Mycobacterium tuberculosis.

Toxin-antitoxin (TA) systems are bicistronic genetic modules that are ubiquitously present in bacterial genomes. The Mycobacterium tuberculosis genome encodes 90 putative TA systems, and these are considered to be associated with maintenance of bacterial genomic stability or bacterial survival under unfavorable environmental conditions. The majority of these in M. tuberculosis have been annotated as belonging to the virulence-associated protein B and C (VapBC) family. However, their precise role in bacterial physiology has not been elucidated. Here, we functionally characterized VapC toxins from M. tuberculosis and show that overexpression of some homologs inhibits growth of Mycobacterium bovis bacillus Calmette-Guérin in a bacteriostatic manner. Expression profiling of messenger RNA revealed that these VapC toxins were differentially induced upon exposure of M. tuberculosis to stress conditions. We also unraveled that transcriptional cross-activation exists between TA systems in M. tuberculosis. This study provides the first evidence for the essentiality of VapBC3 and VapBC4 systems in M. tuberculosis virulence.

Iron oxide labeling does not affect differentiation potential of human bone marrow mesenchymal stem cells exhibited by their differentiation into cardiac and neuronal cells.

Mesenchymal stem cells (MSCs) have shown promising outcomes in cardiac and neuronal diseases. Efficient and noninvasive tracking of MSCs is essential to harness their therapeutic potential. Iron oxide nanoparticles (IONPs) have emerged as effective means to label stem cells and visualize them using magnetic resonance imaging (MRI). It is known that IONPs do not affect viability and cell proliferation of stem cells. However, very few studies have demonstrated differentiation potential of iron oxide-labeled MSCs and their differentiation into specific lineages that can contribute to cellular therapies. The differentiation of IONP-labeled human bone marrow mesenchymal stem cells (hBM-MSCs) into cardiac and neuronal lineages has never been studied. In this study, we have shown that IONP-labeled hBM-MSCs retain their differentiation potential to cardiac and neuronal cell lineages. We also confirmed that labeling hBM-MSCs with IONP does not affect their characteristic properties such as viability, cellular proliferation rate, surface marker profiling, and trilineage differentiation capacity. This study shows that IONP can be efficiently tracked, and its labeling does not alter stemness and differentiation potential of hBM-MSCs. Thus, the labeled hBM-MSCs can be used in clinical therapies and regenerative medicine.

Index-based assessment of suitability of water quality for irrigation purpose under Indian conditions.

Agriculture is a major sector in India which contributes around 14% of country's gross domestic product (GDP). Being an agriculture-based country, good quality of water for irrigation has been a prime requisite. Highly growing population and accelerated industrial development are causing anthropogenic pollution to both surface and groundwater on one side and geogenic contamination like arsenic, fluoride, high dissolved solids, sodicity, and iron in groundwater on other side. As a result, ensuring safe water quality for the irrigation has become a major challenge to both the central and state governments. The present irrigation water quality standards being followed in India have been set by the Central Pollution Control Board (CPCB) and Central Ground Water Board (CGWB) in the year 2000. These standards are solely based on four parameters, namely electrical conductivity, sodium percentage, sodium absorption ratio, and residual sodium carbonate, which are quite subjective and many times are not capable to exactly decide the quality of irrigation water particularly when there are large variations in the source water quality. Therefore, in the present paper, an indices-based approach is presented for categorization of irrigation water quality. These indices are mathematical equations that transform water quality data into a numeric value, which describes the quality of irrigation water. The proposed irrigation water quality index (IWQI), which is based on 12 parameters, classifies the water into five categories, viz. excellent, good, medium, bad, and very bad in the same manner as given by the CPCB and CGWB. In order to give proper rating to various parameters of the index, weights are computed using Saaty's analytic hierarchy process (AHP)-based multiple criteria decision analysis (MCDA) approach. This approach minimizes the subjectivity in assessment of weights and improves understanding of water quality issues by generating an overall index to describe the status of water quality. The proposed index will be beneficial for the water management authorities in ensuring safe water to the stakeholders.

Crystal structure of the N-terminal domain of human SIRT7 reveals a three-helical domain architecture.

Human SIRT7 is an NAD(+) dependent deacetylase, which belongs to sirtuin family of proteins. SIRT7, like other sirtuins has conserved catalytic domain and is flanked by N- and C-terminal domains reported to play vital functional roles. Here, we report the crystal structure of the N-terminal domain of human SIRT7 (SIRT7(NTD) ) at 2.3 Å resolution as MBP-SIRT7(NTD) fusion protein. SIRT7(NTD) adopts three-helical domain architecture and comparative structural analyses suggest similarities to some DNA binding motifs and transcription regulators. We also report here the importance of N- and C-terminal domains in soluble expression of SIRT7. Proteins 2016; 84:1558-1563. © 2016 Wiley Periodicals, Inc.

Mycobacterium tuberculosis keto-mycolic acid and macrophage nuclear receptor TR4 modulate foamy biogenesis in granulomas: a case of a heterologous and noncanonical ligand-receptor pair.

The cell wall of Mycobacterium tuberculosis is configured of bioactive lipid classes that are essential for virulence and potentially involved in the formation of foamy macrophages (FMs) and granulomas. Our recent work established crosstalk between M. tuberculosis cell wall lipids and the host lipid-sensing nuclear receptor TR4. In this study, we have characterized, identified, and adopted a heterologous ligand keto-mycolic acid from among M. tuberculosis lipid repertoire for the host orphan NR TR4. Crosstalk between cell wall lipids and TR4 was analyzed by transactivation and promoter reporter assays. Mycolic acid (MA) was found to transactivate TR4 significantly compared with other cell wall lipids. Among the MA, the oxygenated form, keto-MA, was responsible for transactivation, and the identity was validated by TR4 binding assays followed by TLC and nuclear magnetic resonance. Isothermal titration calorimetry revealed that keto-MA binding to TR4 is energetically favorable. This keto-MA-TR4 axis seems to be essential to this oxygenated MA induction of FMs and granuloma formation as evaluated by in vitro and in vivo model of granuloma formation. TR4 binding with keto-MA features a unique association of host nuclear receptor with a bacterial lipid and adds to the presently known ligand repertoire beyond dietary lipids. Pharmacologic modulation of this heterologous axis may hold promise as an adjunct therapy to frontline tuberculosis drugs.

Crystal structure of Mycobacterium tuberculosis CarD, an essential RNA polymerase binding protein, reveals a quasidomain-swapped dimeric structural architecture.

Mycobacterium tuberculosis (Mtb) CarD is an essential transcriptional regulator that binds RNA polymerase and plays an important role in reprogramming transcription machinery under diverse stress conditions. Here, we report the crystal structure of CarD at 2.3 Å resolution, that represents the first structural description of CarD/CdnL-Like family of proteins. CarD adopts an overall bi-lobed structural architecture where N-terminal domain resembles 'tudor-like' domain and C-terminal domain adopts a novel five helical fold that lacks the predicted leucine zipper structural motif. The structure reveals dimeric state of CarD resulting from ß-strand swapping between the N-terminal domains of each individual subunits. The structure provides crucial insights into the possible mode(s) of CarD/RNAP interactions.

Structural basis for the redox sensitivity of the Mycobacterium tuberculosis SigK-RskA σ-anti-σ complex.

The host-pathogen interactions in Mycobacterium tuberculosis infection are significantly influenced by redox stimuli and alterations in the levels of secreted antigens. The extracytoplasmic function (ECF) σ factor σ(K) governs the transcription of the serodominant antigens MPT70 and MPT83. The cellular levels of σ(K) are regulated by the membrane-associated anti-σ(K) (RskA) that localizes σ(K) in an inactive complex. The crystal structure of M. tuberculosis σ(K) in complex with the cytosolic domain of RskA (RskAcyto) revealed a disulfide bridge in the -35 promoter-interaction region of σ(K). Biochemical experiments reveal that the redox potential of the disulfide-forming cysteines in σ(K) is consistent with its role as a sensor. The disulfide bond in σ(K) influences the stability of the σ(K)-RskAcyto complex but does not interfere with σ(K)-promoter DNA interactions. It is noted that these disulfide-forming cysteines are conserved across homologues, suggesting that this could be a general mechanism for redox-sensitive transcription regulation.

Biochemical and structural characterization reveals Rv3400 codes for ß-phosphoglucomutase in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) adapt to various host environments and utilize a variety of sugars and lipids as carbon sources. Among these sugars, maltose and trehalose, also play crucial role in bacterial physiology and virulence. However, some key enzymes involved in trehalose and maltose metabolism in Mtb are not yet known. Here we structurally and functionally characterized a conserved hypothetical gene Rv3400. We determined the crystal structure of Rv3400 at 1.7 Å resolution. The crystal structure revealed that Rv3400 adopts Rossmann fold and shares high structural similarity with haloacid dehalogenase family of proteins. Our comparative structural analysis suggested that Rv3400 could perform either phosphatase or pyrophosphatase or ß-phosphoglucomutase (ß-PGM) activity. Using biochemical studies, we further confirmed that Rv3400 performs ß-PGM activity and hence, Rv3400 encodes for ß-PGM in Mtb. Our data also confirm that Mtb ß-PGM is a metal dependent enzyme having broad specificity for divalent metal ions. ß-PGM converts ß-D-glucose-1-phosphate to ß-D-glucose-6-phosphate which is required for the generation of ATP and NADPH through glycolysis and pentose phosphate pathway, respectively. Using site directed mutagenesis followed by biochemical studies, we show that two Asp residues in the highly conserved DxD motif, D29 and D31, are crucial for enzyme activity. While D29A, D31A, D29E, D31E and D29N mutants lost complete activity, D31N mutant retained about 30% activity. This study further helps in understanding the role of ß-PGM in the physiology of Mtb.

Mycobacterium tuberculosis strain with deletions in menT3 and menT4 is attenuated and confers protection in mice and guinea pigs.

The genome of Mycobacterium tuberculosis encodes for a large repertoire of toxin-antitoxin systems. In the present study, MenT3 and MenT4 toxins belonging to MenAT subfamily of TA systems have been functionally characterized. We demonstrate that ectopic expression of these toxins inhibits bacterial growth and this is rescued upon co-expression of their cognate antitoxins. Here, we show that simultaneous deletion of menT3 and menT4 results in enhanced susceptibility of M. tuberculosis upon exposure to oxidative stress and attenuated growth in guinea pigs and mice. We observed reduced expression of transcripts encoding for proteins that are essential or required for intracellular growth in mid-log phase cultures of ΔmenT4ΔT3 compared to parental strain. Further, the transcript levels of proteins involved in efficient bacterial clearance were increased in lung tissues of ΔmenT4ΔT3 infected mice relative to parental strain infected mice. We show that immunization of mice and guinea pigs with ΔmenT4ΔT3 confers significant protection against M. tuberculosis infection. Remarkably, immunization of mice with ΔmenT4ΔT3 results in increased antigen-specific TH1 bias and activated memory T cell response. We conclude that MenT3 and MenT4 are important for M. tuberculosis pathogenicity and strains lacking menT3 and menT4 have the potential to be explored further as vaccine candidates.

Arsenic pollution and associated human health hazards in Rupnagar district, Punjab, India.

The hydrosphere although covering almost 70% of the Earth contributes only 3% of fresh water out of which groundwater covers almost 98%. The presence of some unwanted substance in this limited natural resource causes pollution when the substance causes serious harm to human beings and to the total ecosystem in a way. Arsenic is such a pollutant that is most naturally released in groundwater and long-term exposure to As-rich groundwater causes skin lesions and often leads to different types of cancers in humans. Rupnagar district in the Malwa region of Punjab is situated alongside the river Satluj which is one of the five important tributaries of Indus. The lowest reported concentration of As in this district is 10 µg/L and the highest is 91 µg/L. The higher values of As (> 50 µg/L) that are above the permissible limit of IS 10500, 2004 in drinking water, are dominantly found in the western and south-western parts of the district. The average hazard quotient (HQ) indicates high risk for the consumers of the As-polluted groundwater in the district. The present study deals with the major cause of high arsenic (As) concentration in groundwater and its correlation with intensive agriculture in the Rupnagar district. Owing to the large size of the district, GIS techniques like ArcGIS 10.4.1 and QGIS 3.22.8 software were used for analysis in this study. The study reveals that high As concentration (> 50 µg/L) is mostly found in agricultural lands and moderate concentration of As (10-50 µg/L) in groundwater is distributed all over the district and are mostly reported from the urbanised areas. Overall, the water table shows a declining trend but no such decline is observed in the western and south-western parts of the district. As pollution in groundwater can also be caused due to water level decline owing to intensive agriculture and rapid water abstraction though As is naturally sourced in groundwater. A detailed study using the geochemical analysis of groundwater in the district can be effective in clearing out the scenario in the study area.