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.

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.

Structural insights into DarT toxin neutralization by cognate DarG antitoxin: ssDNA mimicry by DarG C-terminal domain keeps the DarT toxin inhibited.

In the DarTG toxin-antitoxin system, the DarT toxin ADP-ribosylates single-stranded DNA (ssDNA), which stalls DNA replication and plays a crucial role in controlling bacterial growth and bacteriophage infection. This toxic activity is reversed by the N-terminal macrodomain of the cognate antitoxin DarG. DarG also binds DarT, but the role of these interactions in DarT neutralization is unknown. Here, we report that the C-terminal domain of DarG (DarG toxin-binding domain [DarGTBD]) interacts with DarT to form a 1:1 stoichiometric heterodimeric complex. We determined the 2.2 Å resolution crystal structure of the Mycobacterium tuberculosis DarT-DarGTBD complex. The comparative structural analysis reveals that DarGTBD interacts with DarT at the DarT/ssDNA interaction interface, thus sterically occluding substrate ssDNA binding and consequently inactivating toxin by direct protein-protein interactions. Our data support a unique two-layered DarT toxin neutralization mechanism of DarG, which is important in keeping the toxin molecules in check under normal growth conditions.

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.

UVC-Based Air Disinfection Systems for Rapid Inactivation of SARS-CoV-2 Present in the Air.

The World Health Organization (WHO) declared in May 2021 that SARS-CoV-2 is transmitted not only by close contact with infectious respiratory fluids from infected people or contaminated materials but also indirectly through air. Airborne transmission has serious implications for the control measures we can deploy, given the emergence of more transmissible variants. This emphasizes the need to deploy a mechanism to reduce the viral load in the air, especially in closed and crowded places such as hospitals, public transport buses, etc. In this study, we explored ultraviolet C (UVC) radiation for its ability to inactivate the SARS-CoV-2 particles present in aerosols and designed an air disinfection system to eliminate infectious viruses. We studied the virus inactivation kinetics to identify the UVC dosage required to achieve maximum virus inactivation. Based on the experimental data, UVC-based devices were designed for the sanitization of air through HVAC systems in closed spaces. Further, a risk assessment model to estimate the risk reduction was applied which showed that the use of UVC radiation could result in the reduction of the risk of infection in occupied spaces by up to 90%.

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.

Amyloidogenic proteins in the SARS-CoV and SARS-CoV-2 proteomes.

The phenomenon of protein aggregation is associated with a wide range of human diseases. Our knowledge of the aggregation behaviour of viral proteins, however, is still rather limited. Here, we investigated this behaviour in the SARS-CoV and SARS-CoV-2 proteomes. An initial analysis using a panel of sequence-based predictors suggested the presence of multiple aggregation-prone regions (APRs) in these proteomes and revealed a strong aggregation propensity in some SARS-CoV-2 proteins. We then studied the in vitro aggregation of predicted aggregation-prone SARS-CoV and SARS-CoV-2 proteins and protein regions, including the signal sequence peptide and fusion peptides 1 and 2 of the spike protein, a peptide from the NSP6 protein, and the ORF10 and NSP11 proteins. Our results show that these peptides and proteins can form amyloid aggregates. We used circular dichroism spectroscopy to reveal the presence of ß-sheet rich cores in aggregates and X-ray diffraction and Raman spectroscopy to confirm the formation of amyloid structures. Furthermore, we demonstrated that SARS-CoV-2 NSP11 aggregates are toxic to mammalian cell cultures. These results motivate further studies about the possible role of aggregation of SARS proteins in protein misfolding diseases and other human conditions.

Molecular docking and dynamic simulation approach to decipher steroidal sapogenins (genus Trillium) derived agonists for glucocorticoid receptor.

Steroidal sapogenins (SS) are structural analogues of steroidal drugs, which are frequently used for the treatment of several diseases including reproductive, malignancies, neurological, and inflammation-related diseases. The glucocorticoid receptor (GR) is a nuclear receptor that regulates development, metabolism, and inflammation, in response to steroidal ligands. Therefore, GR is considered as a potential therapeutic target for steroidal agents to the treatment of inflammation-related diseases. We hypothesized that SS may act as an agonist for GR due to structural similarity with corticosteroids. In this study, we carried out in silico screening of various SS from the genus Trillium to check their potential as an agonist for GR. Our data suggest that out of 42 SS, only 7 molecules have interacted with GR. However, molecular mechanics with generalized Born and surface area (MM-GBSA) analysis revealed that only two SS (SS 38 and SS 39) molecules bind favorably to GR. Among these, SS 38 (docking score: -9.722 Kcal/mol and MM-GBSA ΔGbind: -50.192 Kcal/mol) and SS 39 (docking score: -11.20 Kcal/mol and MM-GBSA ΔGbind: -58.937 Kcal/mol) have best docking and MM-GBSA scores. Molecular dynamics (MD) simulation studies of SS 38, SS 39, and dexamethasone-GR complex revealed that both SS shows hydrogen bonding and hydrophobic interaction with GR over the 120 ns simulation with mild fluctuations. The current study suggests that SS 38 and SS 39 may be further explored as a potential agonist to treat several disease conditions mediated by GR.

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.

Identification of small molecules targeting homoserine acetyl transferase from Mycobacterium tuberculosis and Staphylococcus aureus.

There is an urgent need to validate new drug targets and identify small molecules that possess activity against both drug-resistant and drug-sensitive bacteria. The enzymes belonging to amino acid biosynthesis have been shown to be essential for growth in vitro, in vivo and have not been exploited much for the development of anti-tubercular agents. Here, we have identified small molecule inhibitors targeting homoserine acetyl transferase (HSAT, MetX, Rv3341) from M. tuberculosis. MetX catalyses the first committed step in L-methionine and S-adenosyl methionine biosynthesis resulting in the formation of O-acetyl-homoserine. Using CRISPRi approach, we demonstrate that conditional repression of metX resulted in inhibition of M. tuberculosis growth in vitro. We have determined steady state kinetic parameters for the acetylation of L-homoserine by Rv3341. We show that the recombinant enzyme followed Michaelis-Menten kinetics and utilizes both acetyl-CoA and propionyl-CoA as acyl-donors. High-throughput screening of a 2443 compound library resulted in identification of small molecule inhibitors against MetX enzyme from M. tuberculosis. The identified lead compounds inhibited Rv3341 enzymatic activity in a dose dependent manner and were also active against HSAT homolog from S. aureus. Molecular docking of the identified primary hits predicted residues that are essential for their binding in HSAT homologs from M. tuberculosis and S. aureus. ThermoFluor assay demonstrated direct binding of the identified primary hits with HSAT proteins. Few of the identified small molecules were able to inhibit growth of M. tuberculosis and S. aureus in liquid cultures. Taken together, our findings validated HSAT as an attractive target for development of new broad-spectrum anti-bacterial agents that should be effective against drug-resistant bacteria.

A novel plant lectin, NTL-125, interferes with SARS-CoV-2 interaction with hACE2.

COVID-19 caused by SARS-CoV-2 virus has had profound impact on the world in the past two years. Intense research is going on to find effective drugs to combat the disease. Over the past year several vaccines were approved for immunization. But SARS-CoV-2 being an RNA virus is continuously mutating to generate new variants, some of which develop features of immune escape. This raised serious doubts over the long-term efficacy of the vaccines. We have identified a unique mannose binding plant lectin from Narcissus tazetta bulb, NTL-125, which effectively inhibits SARS-CoV-2 replication in Vero-E6 cell line. In silico docking studies revealed that NTL-125 has strong affinity to viral Spike RBD protein, preventing it from attaching to hACE2 receptor, the gateway to cellular entry. Binding analyses revealed that all the mutant variants of Spike protein also have stronger affinity for NTL-125 than hACE2. The unique α-helical tail of NTL-125 plays most important role in binding to RBD of Spike. NTL-125 also interacts effectively with some glycan moieties of S-protein in addition to amino acid residues adding to the binding strength. Thus, NTL-125 is a highly potential antiviral compound of natural origin against SARS-CoV-2 and may serve as an important therapeutic for management of COVID-19.

A Microbe-Derived Efflux Pump Inhibitor of the Resistance-Nodulation-Cell Division Protein Restores Antibiotic Susceptibility in Escherichia coli and Pseudomonas aeruginosa.

The use of efflux pump inhibitors (EPIs) as potentiators along with the traditional antibiotics assists in the warfare against antibiotic-resistant superbugs. Efflux pumps of the resistance-nodulation-cell division (RND) family play crucial roles in multidrug resistance in Escherichia coli and Pseudomonas aeruginosa. Despite several efforts, clinically useful inhibitors are not available at present. This study describes ethyl 4-bromopyrrole-2-carboxylate (RP1) isolation, an inhibitor of RND transporters from the library of 4000 microbial exudates. RP1 acts synergistically with antibiotics by reducing their minimum inhibitory concentration in strains overexpressing archetype RND transporters (AcrAB-TolC and MexAB-OprM). It also improves the accumulation of Hoechst 33342 and inhibits its efflux (a hallmark of EPI functionality). The antibiotic-RP1 combinations prolong the postantibiotic effects and reduce the mutation prevention concentration of antibiotics. Additionally, from Biolayer Interferometry spectra, it appears that RP1 is bound to AcrB. RP1 displays low mammalian cytotoxicity, no Ca2+ channel inhibitory effects, and reduces the intracellular invasion of E. coli and P. aeruginosa in macrophages. Furthermore, the RP1-levofloxacin combination is nontoxic, well-tolerated, and notably effective in a murine lung infection model. In sum, RP1 is a potent EPI and worthy of further consideration as a potentiator to improve the effectiveness of existing antibiotics.

Potential Inhibitors of SARS-CoV-2 Main Protease (Mpro) Identified from the Library of FDA-Approved Drugs Using Molecular Docking Studies.

The Corona Virus Infectious Disease-2019 (COVID-19) outbreak originated at Wuhan, China, in December 2019. It has already spread rapidly and caused more than 6.5 million deaths worldwide. Its causal agent is a beta-coronavirus named SARS-CoV-2. Many efforts have already been made to develop new vaccines and drugs against these viruses, but over time, it has changed its molecular nature and evolved into more lethal variants, such as Delta and Omicron. These will lead us to target its more-conserved proteins. The sequences' BLAST and crystal structure of the main protease Mpro suggest a high sequence and structural conservation. Mpro is responsible for the proteolytic maturation of the polyprotein essential for the viral replication and transcription, which makes it an important drug target. Discovery of new drug molecules may take years before getting to the clinics. So, considering urgency, we performed molecular docking studies using FDA-approved drugs to identify molecules that could potentially bind to the substrate-binding site and inhibit SARS-CoV-2's main protease (Mpro). We used the Glide module in the Schrödinger software suite to perform molecular docking studies, followed by MM-GBSA-based energy calculations to score the hit molecules. Molecular docking and manual analysis suggest that several drugs may bind and potentially inhibit Mpro. We also performed molecular simulations studies for selected compounds to evaluate protein-drug interactions. Considering bioavailability, lesser toxicity, and route of administration, some of the top-ranked drugs, including lumefantrine (antimalarial), dipyridamole (coronary vasodilator), dihydroergotamine (used for treating migraine), hexoprenaline (anti asthmatic), riboflavin (vitamin B2), and pantethine (vitamin B5) may be taken forward for further in vitro and in vivo experiments to investigate their therapeutic potential.

Repurposing Approved Drugs as Fluoroquinolone Potentiators to Overcome Efflux Pump Resistance in Staphylococcus aureus.

Staphylococcus aureus is a versatile human commensal bacteria and pathogen that causes various community and hospital-acquired infections. The S. aureus efflux pump NorA which belongs to the major facilitator superfamily, confers resistance to a range of substrates. Many efflux pump inhibitors (EPIs) have been discovered, but none is clinically approved due to their undesirable toxicities. In this study, we have screened clinically approved drugs for possible NorA EPI-like activity. We identified six drugs that showed the best efflux pump inhibition in vitro, with a fractional inhibitory concentration index of ≤0.5, indicating synergism with hydrophilic fluoroquinolones. The mechanistic validation of efflux inhibitory potential was demonstrated in ethidium bromide-based accumulation and efflux inhibition assays. We further confirmed the functionality of EPIs by norfloxacin accumulation assay depicting more realistic proof of the conjecture. None of the EPIs disturbed membrane function or depleted the ATP synthesis levels in bacteria. Both raloxifene and pyrvinium displayed an increase in bactericidal activity of ciprofloxacin in time-kill kinetics, prolonged its post-antibiotic effect, and reduced the frequency of spontaneous resistant mutant development. The combination of EPIs with ciprofloxacin caused significant eradication of preformed biofilms. Moreover, in the murine thigh infection model, a single dose of pyrvinium combined with ciprofloxacin reduced the bacterial burden significantly compared to untreated control and ciprofloxacin alone, indicating the efficacy of the combination. Conclusively, this study represents approved drugs that can be repurposed and combined with antibiotics as NorA EPIs, having anti-biofilm properties to treat severe S. aureus infections at clinically relevant concentrations. IMPORTANCE Staphylococcus aureus is a frequent pathogen bacterium and the predominant cause of worsened nosocomial infections. Efflux pumps contribute to drug efflux and are reportedly associated with biofilm formation, thereby promoting difficult-to-treat biofilm-associated S. aureus infections. One strategy to combat these bacteria is to reduce active efflux and increase pathogen sensitivity to existing antibiotics. Repurposing approved drugs may solve the classical toxicity issues with previous efflux pump inhibitors and help reach sufficient plasma concentrations. We describe the in silico-based screening of FDA-approved drugs that identified six different molecules able to inhibit NorA pump (Major Facilitator Superfamily). Our study highlights that these compounds bind to and block the activity of the NorA pump and increase the sensitivity of S. aureus and methicillin-resistant S. aureus to fluoroquinolones. These drugs combined with fluoroquinolones significantly reduced the preformed biofilms and displayed significant efficacy in the murine thigh infection model when compared to untreated control and ciprofloxacin alone.

Corrigendum: Isolation and Taxonomic Characterization of Novel Haloarchaeal Isolates From Indian Solar Saltern: A Brief Review on Distribution of Bacteriorhodopsins and V-Type ATPases in Haloarchaea.

[This corrects the article DOI: 10.3389/fmicb.2020.554927.].

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.

Chlorhexidine: An effective anticovid mouth rinse.

CONTEXT: Dentists across the globe are witnessing a completely unforeseen and uncertain professional situation during these times of COVID-19 pandemic. There is conflicting evidence regarding the effectiveness of routinely used mouthwashes and especially Chlorhexidine, to reduce the viral load in oral cavity and the aerosols during oral procedures. AIMS: Comparative evaluation of the effectiveness of the current 'gold standard' chlorhexidine and povidone iodine as a control agent, through an in-vitro analysis. SETTINGS AND DESIGN: In-vitro laboratory analysis. METHODS AND MATERIAL: All the experiments for analysis of antiviral efficacy of chlorhexidine digluconate (2%)and povidone iodine(1%), against SARS-CoV-2 virus were performed in the BSL3 facility at the Council of Scientific and Industrial Research-Institute of Microbial Technology, using the VeroE6 cell lines. The analysis of the virus inactivation was based on quantification of viral RNA (Cycle threshold (Ct) profile) present in the culture supernatant using Real-Time Quantitative Reverse Transcription PCR (qRT-PCR). STATISTICAL ANALYSIS USED: Descriptive analysis (Statistical package for social sciences (SPSS Inc., Chicago, IL, version 15.0 for Windows). RESULTS: Chlorhexidine digluconate in 0.2% concentration inactivated more than 99.9% of SARS CoV 2 virus, in minimal contact time of 30 seconds, which was considered better efficacy than povidone-iodine utilized for 30 and 60 seconds. Subtle differences were observed in the activity of both the compounds in terms of percent inactivation of virus, though a greater relative change in Ct values was observed for chlorhexidine. CONCLUSIONS: Within the limitations of the present study, it can be concluded that Chlorhexidine digluconate in 0.2% concentration inactivated SARS CoV 2 in minimal contact time i.e 30 secs, however both compounds tested i.e Chlorhexidine and povidone-iodine were found to have antiviral activity against SARS CoV2 virus.

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.