Journal: Nature Communications

Article Title: A unique inhibitor conformation selectively targets the DNA polymerase PolC of Gram-positive priority pathogens

doi: 10.1038/s41467-025-65324-8

Figure Lengend Snippet: a Structure of ibezapolstat (IBZ) with the guanine nucleobase moiety in light blue. b Structure of ACX-801 with the position of R1 ( N 2 -subtitution), R2 and R3 marked in coloured squares, and the blue arrow indicating displacement of the ring nitrogen from position 9 to 8 in the ACX scaffold compared to guanine and IBZ. c MIC distribution of 46 compounds from the ACX library for different bacterial species. Only MIC values below 16 mg/L for VRE were used. Values ≥ 64 mg/L are grouped together. Abbreviations: VRE (vancomycin-resistant E. faecium ), Efa ( E. faecalis) , MRSA (methicillin-resistant S. aureus ), Sau (susceptible S. aureus ), PRSP (penicillin-resistant S. pneumoniae ), and Eco (E. coli) . The size of the circles corresponds the number of compounds, from 1 (smallest) to 46 (largest). d Gel-based primer extension assay showing DNA polymerase activity of E. faecium wild-type (WT) and exonuclease-inactivated PolC (Exo null ; D431A + E433A), but not for polymerase-inactivated PolC (Pol null ; D972A + D974A). Schematic of non-extended primer:template DNA substrate is shown at the bottom right and fully extended primer:template above. A complementary exonuclease assay is shown in Supplementary Fig. . A representative gel of multiple runs with reproducible results is shown. e Real-time assay (Supplementary Fig. ) measuring inhibition of polymerase activity by IBZ and 4 representative compounds from the ACX library using exonuclease-inactivated E. faecium PolC, with derived IC 50 values. Individual data points for each replicate ( n = 3) are shown. A normalized dose-response (three parameter) fit was used to determine IC 50 values and, where an IC 50 could be determined ( < 100 μM), the standard error of the mean ( n = 3) is given. f Real-time assay measuring susceptibility of DnaE-type polymerases E. coli Pol IIIα and E. faecium DnaE to IBZ and ACX-801. Individual data points for each replicate ( n = 3) are shown. A normalized dose-response (three parameter) fit was used to determine IC 50 values and, where an IC 50 could be determined ( < 100 μM), the standard error of the mean ( n = 3) is given.

Article Snippet: A selection of 48 ACX candidates with MICs <16 mg/L against VRE, MRSA and/or PRSP was evaluated in an in vitro DNA polymerase assay , using purified PolC from E. faecium (ATCC 700221).

Techniques: Primer Extension Assay, Activity Assay, Inhibition, Derivative Assay

Journal: Nature Communications

Article Title: A unique inhibitor conformation selectively targets the DNA polymerase PolC of Gram-positive priority pathogens

doi: 10.1038/s41467-025-65324-8

Figure Lengend Snippet: a Apo structure (PDB-9QRN) of exonuclease-inactivated E. faecium PolC with a 3-nucleotide ssDNA bound to the exonuclease domain (Exo). Other domains are labelled OB (oligonucleotide/oligosaccharide-binding) and PHP (polymerase and histidinol phosphate) as in 2c. The flexible N-terminal domain (N-term), not resolved in the density map and structure, is indicated in grey. b Structure of exonuclease-inactivated E. faecium PolC (yellow) bound to DNA (grey) and ACX-801 (blue) (PDB-9QPC). c Schematic representations of the domains of E. faecium PolC. The PHP domain is interrupted by the Exo domain, and the palm domain is split by the thumb (T) subdomain. The position of the catalytic residues of the Exo domain (D431 and E433) are indicated with an asterisk. d Ligand interaction map of IBZ as derived from the structure (PDB-9QRL). An alternative map containing further details is provided in Supplementary Fig. . Conserved interacting residues are highlighted. e Ligand interaction map of ACX-801 as derived from the structure (PDB-9QPC). An alternative map is provided in Supplementary Fig. . Residues uniquely identified as interacting with ACX-801 are indicated with a stroke. Conserved interacting residues are highlighted. f Base-pairing (represented by dashed lines) between the dCMP (grey) and dGTP (yellow, from PDB-3F2C), IBZ (pink, PDB-9QRL) and ACX-801 (blue, PDB-9QPC). Stick representations are coloured by atom but with different backbone colours. g Close-up of the binding pocket in the IBZ-bound PolC structure (PDB-9QRL). PolC is shown in blue with specific residues in yellow and IBZ as sticks with a purple backbone. h Close-up of the binding pocket in the ACX-801-bound PolC structure (PDB-9QPC). PolC is shown in yellow with specific residues in pink and ACX-801 in blue. Water is represented as a blue spheres and the dashed lines indicate interactions with residue Y1274. i Displacement of residues in the ACX-801-bound structure (yellow; PDB-9QPC) compared to the ligand-free, apo structure (purple; PDB-9QRN). Polymerase catalytic residues (D972 and D974) are annotated. The arrows highlight the rotation and displacement of F1276, and minor displacement of Y1274 and Y1284 to accommodate the inhibitor.

Article Snippet: A selection of 48 ACX candidates with MICs <16 mg/L against VRE, MRSA and/or PRSP was evaluated in an in vitro DNA polymerase assay , using purified PolC from E. faecium (ATCC 700221).

Techniques: Binding Assay, Derivative Assay, Residue

Journal: Nature Communications

Article Title: A unique inhibitor conformation selectively targets the DNA polymerase PolC of Gram-positive priority pathogens

doi: 10.1038/s41467-025-65324-8

Figure Lengend Snippet: a Sequence conservation plotted on the surface of PolC (PDB-9QPC), showing the highest conservation in the DNA binding cleft and exonuclease active site. The inset on the left shows the conservation of PolC sequences plotted on an enlargement of the surface of the inhibitor binding pocket, with relevant amino acid residues indicated. b Structure-based sequence alignment of C-family sequences from Gram-positive and Gram-negative bacteria. Species names are coloured according to PolC-type (black) and DnaE-type (blue) polymerase sequences, with a + or – indicating Gram-positive or -negative bacteria, respectively. The secondary structures (coils representing helices numbered according to PDBs) are shown above for E. faecium PolC (PDB-9QPC) and below for E. coli PolIIIα (PDB-5M1S). The residues that are part of the inhibitor-binding pocket of E. faecium PolC are indicated, with residues that are displaced in the inhibitor-bound conformation in pink. The red asterisk (*) marks the truncated helix in DnaE-type polymerases. c Superposition of E. faecium PolC in ACX-801-bound form (in yellow) and three DnaE-type polymerases (in three tones of grey): E. coli PolIIIα (PDB-5M1S), M. tuberculosis DnaE1 (PDB-7PU7), and Thermus aquaticus Pol IIIα (PDB-3E0D). The two arrows indicate the movement of residues required to create a full inhibitor binding pocket. The red asterisk (*) marks the end of the helix in DnaE-type polymerases; this helix is extended by half a turn in PolC-type polymerases and positions the fourth aromatic residue of the PolC inhibitor binding pocket, which has no structural equivalent in DnaEs. Numbering of residues is based on E. faecium PolC.

Article Snippet: A selection of 48 ACX candidates with MICs <16 mg/L against VRE, MRSA and/or PRSP was evaluated in an in vitro DNA polymerase assay , using purified PolC from E. faecium (ATCC 700221).

Techniques: Sequencing, Binding Assay, Bacteria, Residue

Journal: Nature Communications

Article Title: A unique inhibitor conformation selectively targets the DNA polymerase PolC of Gram-positive priority pathogens

doi: 10.1038/s41467-025-65324-8

Figure Lengend Snippet: a Close-up of the inhibitor binding pocket in exonuclease-inactivated E. faecium PolC (PDB-9QPC) with two residues involved in resistance to PolC inhibitors (F1276 and A1281) highlighted in purple. ACX-801 is shown in blue and other residues that make up the binding pocket are shown in grey. b Polymerase activity inhibition of F1276 mutants compared to wild-type PolC protein by IBZ (left) and ACX-801 (right), with derived IC 50 values. All proteins were exonuclease-inactivated. Individual data points for each replicate ( n = 3) are shown with a normalized dose-response (three parameter) fit and, where an IC 50 could be determined, the standard error of the mean is given. c Susceptibility of C. difficile carrying different plasmid-based polC alleles to IBZ and ACX-801. Cells were spotted onto BHI agar with increasing amounts of IBZ (left) or ACX-801 (right). Plasmids carrying the wild-type polC gene ( polC ), or mutant alleles polC p .F1258L (F1258L), polC p .F1258I (F1258I), polC p .F1258S (F1258S) or polC p .A1263T (A1263T) are shown. d DNA polymerase activity of different PolC variants in the absence of inhibitor. The PolC A1281T (A1281T) mutant shows an ~10-fold reduction in activity compared to wild-type PolC and two different F1276 variants. The activity is shown as an average of three replicates; the standard error is omitted as it is obscured by the size of the symbols.

Article Snippet: A selection of 48 ACX candidates with MICs <16 mg/L against VRE, MRSA and/or PRSP was evaluated in an in vitro DNA polymerase assay , using purified PolC from E. faecium (ATCC 700221).

Techniques: Binding Assay, Activity Assay, Inhibition, Derivative Assay, Plasmid Preparation, Mutagenesis

Journal: Molecules

Article Title: Silver Nanoparticles Synthesized by Using the Endophytic Bacterium Pantoea ananatis are Promising Antimicrobial Agents against Multidrug Resistant Bacteria

doi: 10.3390/molecules23123220

Figure Lengend Snippet: ZOI (mm) and MIC (μg/mL) values of the synthesized AgNPs against pathogenic microbes and MDR bacteria.

Article Snippet: The synthesized AgNPs were found to exhibit a pronounced antibacterial activity against MDR strains of S. pneumonia (ATCC 700677), E. faecium (ATCC 700221), S. aureus subsp. aureus (ATCC 33592) and E. coli (NCTC 13351) with ZOIs of 10.20 ± 0.07 mm (MIC: 2.75 μg/mL), 12.16 ± 0.05 mm (MIC: 2.25 μg/mL) and 12.24 ± 0.05 mm (MIC: 3.5 μg/mL), respectively.

Techniques: Synthesized, Bacteria, Control

Journal: Molecules

Article Title: Silver Nanoparticles Synthesized by Using the Endophytic Bacterium Pantoea ananatis are Promising Antimicrobial Agents against Multidrug Resistant Bacteria

doi: 10.3390/molecules23123220

Figure Lengend Snippet: Antimicrobial activity of the synthesized AgNPs against ( A ) S. aureus subsp. aureus (ATCC 11632) ( B ) E. coli (ATCC 10536) ( C ) C. albicans (ATCC 10231) ( D ) MDR E. faecium (ATCC 700221).

Article Snippet: The synthesized AgNPs were found to exhibit a pronounced antibacterial activity against MDR strains of S. pneumonia (ATCC 700677), E. faecium (ATCC 700221), S. aureus subsp. aureus (ATCC 33592) and E. coli (NCTC 13351) with ZOIs of 10.20 ± 0.07 mm (MIC: 2.75 μg/mL), 12.16 ± 0.05 mm (MIC: 2.25 μg/mL) and 12.24 ± 0.05 mm (MIC: 3.5 μg/mL), respectively.

Techniques: Activity Assay, Synthesized

Journal: Frontiers in Microbiology

Article Title: Insights in the Antimicrobial Potential of the Natural Nisin Variant Nisin H

doi: 10.3389/fmicb.2020.573614

Figure Lengend Snippet: Determination of purity, yield and cleavage efficiency of the pre-nisin variants. (A) Purity of the purified nisin A and the variants nisin H and nisin H F 1 I (Marker: Precision Plus Protein Dual Xtra standards Bio-Rad). (B) Yields after purification of nisin A and their corresponding variants via cation-exchange chromatography. (C) Quantification of the cleavage efficiency of NisP. (D) Chromatogram of nisin H. (E) Chromatogram of the nisin H F 1 I variant. The pre-nisin variants before NisP cleavage were shown by blue dashed lines and after NisP cleavage by black lines. Error bars represent the standard deviation of at least three biological replicates.

Article Snippet: All bacteria used for minimum inhibitory concentration (MIC) determination of nisin variants [ Bacillus subtilis 168; S. aureus : MSSA strain ATCC 29213, MRSA/VISA strain ATCC 700699; E. faecium : ATCC 35667, ATCC 700221 (vancomycin resistant); E. faecalis : ATCC 29212, ATCC 51299 (vancomycin resistant)] were cultivated in Mueller-Hinton broth (MHB) at 37°C and shaking at 150 rpm.

Techniques: Purification, Marker, Chromatography, Variant Assay, Standard Deviation

Journal: Frontiers in Microbiology

Article Title: Insights in the Antimicrobial Potential of the Natural Nisin Variant Nisin H

doi: 10.3389/fmicb.2020.573614

Figure Lengend Snippet: Nisin mediated pore formation, visualized with the SYTOX green assay. The NZ9000Cm strain incubated with the SYTOX dye. After a stable baseline (∼200 s), one of the nisin variants (100 nM) was added (indicated with an arrow). The fluorescence signal was measured using a fluorolog (Horiba III). The rapid increase in fluorescence indicates pore formation. The black line represents the addition of nisin A, the red line nisin H, and the blue line nisin H F 1 I. As a control we added buffer shown as green line.

Article Snippet: All bacteria used for minimum inhibitory concentration (MIC) determination of nisin variants [ Bacillus subtilis 168; S. aureus : MSSA strain ATCC 29213, MRSA/VISA strain ATCC 700699; E. faecium : ATCC 35667, ATCC 700221 (vancomycin resistant); E. faecalis : ATCC 29212, ATCC 51299 (vancomycin resistant)] were cultivated in Mueller-Hinton broth (MHB) at 37°C and shaking at 150 rpm.

Techniques: Incubation, Fluorescence, Control

Journal: Frontiers in Microbiology

Article Title: Insights in the Antimicrobial Potential of the Natural Nisin Variant Nisin H

doi: 10.3389/fmicb.2020.573614

Figure Lengend Snippet: MIC values for nisin A, nisin H, and nisin H F 1 I against different pathogenic strains.

Article Snippet: All bacteria used for minimum inhibitory concentration (MIC) determination of nisin variants [ Bacillus subtilis 168; S. aureus : MSSA strain ATCC 29213, MRSA/VISA strain ATCC 700699; E. faecium : ATCC 35667, ATCC 700221 (vancomycin resistant); E. faecalis : ATCC 29212, ATCC 51299 (vancomycin resistant)] were cultivated in Mueller-Hinton broth (MHB) at 37°C and shaking at 150 rpm.

Techniques:

Journal: Marine Drugs

Article Title: Insights into the Variation in Bioactivities of Closely Related Streptomyces Strains from Marine Sediments of the Visayan Sea against ESKAPE and Ovarian Cancer

doi: 10.3390/md19080441

Figure Lengend Snippet: Antibiotic activity screening of the extracts of marine sediment-derived Streptomyces from Visayan Sea against ESKAPE pathogens.

Article Snippet: The ESKAPE ( E. faecium ATCC 700221, S. aureus ATCC BAA-44, K. pneumoniae ATCC BAA-1705, A. baumannii ATCC BAA-1605, P. aeruginosa ATCC BAA-1744 and E. cloacae ATCC BAA-2341) pathogens that were used in this study were identified as drug resistant by American Type Culture Collection (ATCC).

Techniques: Activity Assay