Journal: bioRxiv

Article Title: Structural modeling reveals the mechanism of motor ATPase coordination during type IV pilus retraction

doi: 10.1101/2025.10.30.685630

Figure Lengend Snippet: AF3 models (left) of the indicated V. cholerae proteins with their respective PAE maps and confidence scores (right). ( A ) PilT(x6)-PilC(x3) and ( B ) PilT(x6)-PilU(x6) were used to generate the PilC-PilT-PilU hybrid complex shown in . ( C ) The PilU(x6)-PilC(x3) AF3 model had very low confidence metrics, suggesting that these proteins may not directly interact.

Article Snippet: Molecular dynamics simulation of the AF3 PilT-PilU model. A 100-ns span of the simulation is show for PilT K36 -PilU D366,E368 .

Techniques:

Journal: bioRxiv

Article Title: Structural modeling reveals the mechanism of motor ATPase coordination during type IV pilus retraction

doi: 10.1101/2025.10.30.685630

Figure Lengend Snippet: ( A ) AF3 model of PilT (green)-PilU (cyan) interactions. Inset highlights the interaction interfaces targeted for mutagenesis. The PilT residues that putatively participate in intermolecular salt bridges are shown in magenta. ( B ) NT assays of the indicated V. cholerae strains. Dark gray bars denote strains that report on PilT-dependent retraction ( i.e. , in a pilT + background where PilT ATPase activity is intact), while light grey bars denote strains that report on PilT-PilU-dependent retraction ( i.e. , in a pilT WA background where retraction is dependent on PilU). ( C ) Representative images of surface piliation (top panel) and cellular aggregation (bottom panel) for the indicated strains. Scale bars on micrographs, 3µm. Data are representative of two independent experiments. ( D ) Motor localization assay of cells expressing mCherry-pilU WA and the indicated pilT alleles. For each strain, the percentage of cells ( n = 120 cells analyzed) with one or more fluorescent mCherry-PilU WA foci is reported. ( E ) Motor localization assay of ΔpilU cells expressing an mCherry-pilT WA construct with the additional mutations indicated. For each strain, the percentage of cells ( n = 120 cells analyzed) with one or more fluorescent mCherry-PilT WA foci is reported. Data in B , D , and E are from 4 independent biological replicates and shown as the mean ± SD.

Article Snippet: Molecular dynamics simulation of the AF3 PilT-PilU model. A 100-ns span of the simulation is show for PilT K36 -PilU D366,E368 .

Techniques: Mutagenesis, Activity Assay, Expressing, Construct

Journal: bioRxiv

Article Title: Structural modeling reveals the mechanism of motor ATPase coordination during type IV pilus retraction

doi: 10.1101/2025.10.30.685630

Figure Lengend Snippet: AF3 model of the PilT (green)-PilU (cyan) complex. Insets show the residues involved in the three putative intermolecular salt bridges with PilT residues colored in magenta and PilU residues colored in blue.

Article Snippet: Molecular dynamics simulation of the AF3 PilT-PilU model. A 100-ns span of the simulation is show for PilT K36 -PilU D366,E368 .

Techniques:

Journal: bioRxiv

Article Title: Structural modeling reveals the mechanism of motor ATPase coordination during type IV pilus retraction

doi: 10.1101/2025.10.30.685630

Figure Lengend Snippet: ( A ) Multiple sequence alignment of PilT and PilU homologs from the following bacterial species: Vc = Vibrio cholerae , Ab = Acinetobacter baylyli , Pa = Pseudomonas aeruginosa , Lp = Legionella pneumophila , and Xf = Xylella fastidiosa . Red boxes denote conservation of the PilT Vc residues studied (D2, R35, K36, and E53), the PilT AIRNLIRE motif, and the extended C-terminus that is unique to PilU homologs. The red asterisk highlights conservation of the PilU Vc K348 residue among PilU homologs. ( B ) AF3 models of PilT-PilU complexes from the indicated species highlights the predicted conservation of the experimentally validated PilT Vc D2,E53 -PilU Vc K348 interfacial salt bridge.

Article Snippet: Molecular dynamics simulation of the AF3 PilT-PilU model. A 100-ns span of the simulation is show for PilT K36 -PilU D366,E368 .

Techniques: Sequencing, Residue

Journal: bioRxiv

Article Title: Structural modeling reveals the mechanism of motor ATPase coordination during type IV pilus retraction

doi: 10.1101/2025.10.30.685630

Figure Lengend Snippet: ( A ) AF3 model of PilT (green)-PilU (cyan) interactions. The inset highlights the residues that participate in the three putative salt bridges targeted for mutagenesis. PilT residues are shown in magenta with nitrogen atoms colored blue, and PilU residues are shown in dark blue with oxygen atoms colored red. ( B ) NT assay of the indicated V. cholerae strains. Data are from five independent biological replicates and shown as the mean ± SD. ( C ) Representative images of surface piliation (top panel) and cellular aggregation (bottom panel) for the indicated strains. Scale bar for micrographs, 3µm. Data are representative of two independent experiments. ( D ) Distance frequency histogram for the indicated residue pairs during the MD simulation of the AF3 PilT-PilU model. Distances between the side chains of the indicated residues were measured at 0.1-ns intervals over the 500-ns simulation. Percentage on graph denotes the amount of time that the indicated side chains are <3.5Å (dotted line). Data are compiled from two independent MD simulations.

Article Snippet: Molecular dynamics simulation of the AF3 PilT-PilU model. A 100-ns span of the simulation is show for PilT K36 -PilU D366,E368 .

Techniques: Mutagenesis, Residue

Journal: bioRxiv

Article Title: Structural modeling reveals the mechanism of motor ATPase coordination during type IV pilus retraction

doi: 10.1101/2025.10.30.685630

Figure Lengend Snippet: ( A ) Distance frequency histogram for the indicated residue pairs during the MD simulation of the AF3 PilT-PilU model. Distances between the side chains of the indicated residues were measured at 0.1-ns intervals over the 500 ns simulation. Percentage on graph denotes the amount of time that the indicated side chains are <3.5Å (dotted line). Data are compiled from two independent MD simulations. ( B ) Time-course scatter plots of distance measurements for the indicated residue pairs. Trajectories 1 and 2 represent two independent replicates of the MD simulations.

Article Snippet: Molecular dynamics simulation of the AF3 PilT-PilU model. A 100-ns span of the simulation is show for PilT K36 -PilU D366,E368 .

Techniques: Residue

Journal: bioRxiv

Article Title: Structural modeling reveals the mechanism of motor ATPase coordination during type IV pilus retraction

doi: 10.1101/2025.10.30.685630

Figure Lengend Snippet: ( A ) AF3 model of PilT Ab (orange)- PilU Ab (pink) interactions. Inset shows the conserved PilT Ab D2,D53 -PilU Ab K351 salt bridge. ( B ) NT assays of the indicated A. baylyi strains. Dark gray bars denote strains that report on PilT-dependent retraction ( i.e. , in a pilT + background where PilT ATPase activity is intact), while light grey bars denote strains that report on PilT-PilU-dependent retraction ( i.e. , in a pilT WA background where retraction is dependent on PilU). Data are from three independent biological replicates and shown as the mean ± SD.

Article Snippet: Molecular dynamics simulation of the AF3 PilT-PilU model. A 100-ns span of the simulation is show for PilT K36 -PilU D366,E368 .

Techniques: Activity Assay

Journal: bioRxiv

Article Title: Structural modeling reveals the mechanism of motor ATPase coordination during type IV pilus retraction

doi: 10.1101/2025.10.30.685630

Figure Lengend Snippet: AF3 model of the PilC(3x)-PilT(6x) complex. Residues of the AIRNLIRE motif (PilT A288-E295 ) are colored in magenta. The inset shows a slice of the PilT-PilC model for a better view of the AIRNLIRE motif at the PilC-PilT interface.

Article Snippet: Molecular dynamics simulation of the AF3 PilT-PilU model. A 100-ns span of the simulation is show for PilT K36 -PilU D366,E368 .

Techniques:

Journal: PLoS Pathogens

Article Title: Pseudomonas aeruginosa type IV minor pilins and PilY1 regulate virulence by modulating FimS-AlgR activity

doi: 10.1371/journal.ppat.1007074

Figure Lengend Snippet: (A) SK assays for PA14 pilA , fimU , pilV , pilW , pilX , pilY1 , and pilE mutants. Synchronized L4 worms were seeded onto SK plates and scored for death every 24 h, then plotted as “percent survival” over the course of the assay. “Day” represents the number of days after L4 on which the plates were scored. PA14 fimU and pilE mutants had similar virulence to WT, pilA and pilV mutants were slightly less virulent than WT, and pilW , pilX , and pilY1 mutants killed more slowly than all other strains tested. (B) SK assays for PAO1 pilA , fimU , pilV , pilW , pilX , pilY1 , and pilE mutants. The PAO1 pilE mutant had similar virulence to WT, the pilA mutant was slightly less virulent, and fimU , pilV , pilW , pilX , and pilY1 mutants showed significant delays in killing. In (A) and (B), asterisks indicate strains that were significantly different from a pilA mutant by Gehan-Breslow-Wilcoxon test at p = 0.05 (p = 0.00625 with a Bonferroni correction), n = 3 trials.

Article Snippet: Deletion constructs for PA14 fimU , pilV , pilW , pilX , pilY1 , and pilE were synthesized by Genscript in the pUC57Kan vector. pEX18Gm- sadC was created by amplifying the sadC deletion region from PA14 sadC roeA [ ], followed by digestion and ligation into pEX18Gm. pEX18Gm- fimS , pEX18Gm- algR D54A , and pEX18Gm- algR D54E were made by overlap extension PCR [ ].

Techniques: Mutagenesis

Journal: PLoS Pathogens

Article Title: Pseudomonas aeruginosa type IV minor pilins and PilY1 regulate virulence by modulating FimS-AlgR activity

doi: 10.1371/journal.ppat.1007074

Figure Lengend Snippet: (A) cdrA promoter activity in PA14 sadC and algR deletion and overexpression strains. pMS402-P cdrA , containing the lux genes under expression of the cdrA promoter, was introduced into strains of interest, along with pBADGr (vector-only control), pBADGr- sadC , or pBADGr- algR . Assays were set up in technical triplicate in SK media, with or without 0.05% L-arabinose to induce expression of the pBADGr promoter, and measurements were taken every 15 min over 5 h. Loss of sadC or algR led to a subtle decrease in cdrA promoter activity, while SadC overexpression led to a dramatic increase in cdrA promoter activity. Overexpression of AlgR also led to a subtle increase in cdrA promoter activity that was enhanced upon addition of L-arabinose. n = 3 trials. (B) cdrA promoter activity in PA14 pilA , fimU , pilV , pilW , pilX , pilY1 , and pilE mutants. Loss of pilW , pilX , or pilY1 led to a decrease in cdrA promoter activity. n = 3 trials.

Article Snippet: Deletion constructs for PA14 fimU , pilV , pilW , pilX , pilY1 , and pilE were synthesized by Genscript in the pUC57Kan vector. pEX18Gm- sadC was created by amplifying the sadC deletion region from PA14 sadC roeA [ ], followed by digestion and ligation into pEX18Gm. pEX18Gm- fimS , pEX18Gm- algR D54A , and pEX18Gm- algR D54E were made by overlap extension PCR [ ].

Techniques: Activity Assay, Over Expression, Expressing, Plasmid Preparation, Control

Journal: PLoS Pathogens

Article Title: Pseudomonas aeruginosa type IV minor pilins and PilY1 regulate virulence by modulating FimS-AlgR activity

doi: 10.1371/journal.ppat.1007074

Figure Lengend Snippet: (A) BACTH assays for FimS, AlgR, PilA, and MPs. Protein fusions with T18 and T25 fragments of the CyaA adenylate cyclase were screened for interactions on MacConkey and LB + X-gal plates. FimS interacted with itself, AlgR, PilA, FimU, PilV, PilW, PilX, and PilE. Positive (+) or negative (-) interactions are indicated below each image, n = 3. (B) fimU promoter activity in PA14 pilA , fimU , pilV , pilW , pilX , pilY1 , pilE , fimS , algR , pilY1 fimS , or pilY1 algR mutants. pMS402-P fimU , containing the fimU promoter upstream of the lux genes, was introduced into strains of interest. Loss of pilV , pilW , pilX , or pilY1 led to highly elevated fimU promoter activity. pilA and fimU mutants had moderately increased fimU promoter activity relative to WT. fimS and algR mutants had negligible luminescence, and loss of fimS or algR also reverted fimU promoter activity in the pilY1 mutant to baseline. n = 3 trials.

Article Snippet: Deletion constructs for PA14 fimU , pilV , pilW , pilX , pilY1 , and pilE were synthesized by Genscript in the pUC57Kan vector. pEX18Gm- sadC was created by amplifying the sadC deletion region from PA14 sadC roeA [ ], followed by digestion and ligation into pEX18Gm. pEX18Gm- fimS , pEX18Gm- algR D54A , and pEX18Gm- algR D54E were made by overlap extension PCR [ ].

Techniques: Activity Assay, Mutagenesis

Journal: PLoS Pathogens

Article Title: Pseudomonas aeruginosa type IV minor pilins and PilY1 regulate virulence by modulating FimS-AlgR activity

doi: 10.1371/journal.ppat.1007074

Figure Lengend Snippet: (A) Loss or inactivation of FimS-AlgR results in sustained WT (acute) virulence towards C . elegans . Under normal conditions, PilVWXY1 suppress FimS activation of AlgR, leading to reduced expression of the MPs and increased expression of acute virulence factors. These phenotypes are mimicked by genetic inactivation of AlgR (D54A) or deletion of fimS or algR . (B) Loss of PilVWXY1 frees FimS to activate AlgR, leading to increased expression of the MPs, reduced expression of acute virulence factors, and delayed nematode killing. Hyperactivating mutations in AlgR (D54E) phenocopy this mechanism. Abbreviations: fimU , U (magenta); pilV , V (orange); pilW , W (teal); pilX , X (pink); pilY1 , Y1 (purple); pilE , E (green). Yellow star indicates phosphorylation.

Article Snippet: Deletion constructs for PA14 fimU , pilV , pilW , pilX , pilY1 , and pilE were synthesized by Genscript in the pUC57Kan vector. pEX18Gm- sadC was created by amplifying the sadC deletion region from PA14 sadC roeA [ ], followed by digestion and ligation into pEX18Gm. pEX18Gm- fimS , pEX18Gm- algR D54A , and pEX18Gm- algR D54E were made by overlap extension PCR [ ].

Techniques: Activation Assay, Expressing, Phospho-proteomics