Resultados de investigación

A—Volcano plot showing dRNA-seq data of global transcription of ΔcavA+cavA complemented strain (having high cAMP levels) compared to the control ΔcavA EV deleted mutant with empty vector (having low cAMP levels). The results indicated 234 differentially expressed genes in total, of which 143 genes were up-regulated (red dots) and 91 were down-regulated (blue dots). Highlighted are up-regulated genes linked to twitching motility (pil and com gene clusters), cAMP signalling (vfr) and c-di-GMP synthesis (ABUW_2135) and degradation (ABUW_1138) and down-regulated genes linked to biofilm formation and attachment (ABUW_2052–55 and csu operons), exopolysaccharides production (pga gene cluster), quorum sensing (abaI), c-di-GMP degradation (ABUW_2631). The crp homologue ABUW_2344 was amongst the not significantly differentially expressed genes (black dots). B & C—The impact of different cAMP levels in ΔcavA mutant and complemented ΔcavA+cavA strain compared to wild-type (WT) AB5075 on exopolysaccharide (EPS) production (B) and twitching motility (C). For the EPS assay colonies of each strain were grown in triplicates on Congo red (40 μg/ml) agar plates at 37°C for 5 days and the images shown are representatives of three independent repeats (B). The twitching zone diameter was measured in millimetres 48 h post-inoculation of soft agar plates. Data represents the mean ± SD of three biological repeats. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001—One-Way ANOVA with Tukey post-hoc test. Strains with empty miniTn7 were used as controls in both assessments (S3A and S4A Figs)., Peer reviewed

MICIU/AEI/ 10.13039/501100011033 and by “ESF Investing in your future” MICIU/AEI/10.13039/501100011033 MICIU/AEI/ 10.13039/501100011033 and by “FSE+, Peer reviewed

A—Biofilm formation of wild-type (WT) AB5075 compared to three ABUW_2208::T26 transposon mutants labelled with their corresponding number from the Manoil transposon mutant library. B—Proposed cell localisation and function of CavA adenylate cyclase based on domains present in the protein. The transmembrane domains anchor the protein to the inner membrane the cells. The catalytic domain forms a dimer and converts ATP molecules to cyclic AMP (cAMP). Figure was created using PyMOL and BioRender.com. C–Biofilm levels of ΔcavA clean deletion mutant and ΔcavA+cavA complemented strain. D–cAMP concentrations in ΔcavA mutant and complemented ΔcavA+cavA strain, as well as ΔcavB and ΔcavAΔcavB double mutant which was complemented with either cavA (ΔcavAΔcavB+cavA) or cavB (ΔcavAΔcavB+cavB). Cultures grown in LB broth were harvested and lysed via sonication. Bradford assay was used to quantify the total protein concentration and cAMP concentrations in each strain were determined using Cyclic Nucleotide XP Enzymatic Immunoassay kit. Data shown is the mean cAMP concentration per milligram protein from three independent repeats. E—Biofilm formation of ΔcavB deleted mutant compared to WT. Biofilm biomass was assessed after 24 h incubation at 37°C shaking and is presented as the optical density measured at 570 nm (OD570). All data represents the averages of three biological replicates ± standard deviations (SD). Bacterial growth was assessed at OD600 prior to staining the biofilms (S1 Fig). ns p>0.05, *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001—One-Way ANOVA with Dunnett (A) or Tukey (C, D) post-hoc tests and Unpaired t-test (D). Strains with empty miniTn7 were used as controls (S1 Fig)., Peer reviewed

Gene set enrichment analysis results of the significantly regulated genes in ΔcavA+cavA complemented vs ΔcavA EV strain in the dRNA-seq experiment., Peer reviewed

A—Cyclic di-GMP levels in WT AB5075, WT overexpressing constitutively active DGC gene pleD* (WT+pleD*) and WT with empty miniTn7 used for the strain construction. This demonstrates the activity of the modified CensYBL-Ab in detecting the elevated c-di-GMP levels in the WT+pleD* strain. The inactive CensYBL*-Ab biosensor was used to demonstrate that the increase in the signal was due to the changing c-di-GMP levels. B–Cyclic di-GMP levels in cavA related strains compared to the parental WT AB5075. The empty miniTn7 was used as control which demonstrates the empty vector did not have an effect on the c-di-GMP levels in the WT or the deleted ΔcavA mutant. ns p>0.05, ** p<0.01, **** p<0.0001—Two-Way ANOVA (A) and One-Way ANOVA (B) with Tukey post-hoc test., Peer reviewed

AHL production (indicated by the presence of a blue halo around the colonies) by the AV-T variants of the wild-type (WT) AB5075 and its cavA related derivatives. Dramatic difference was observed in the AHL synthesis, as the deleted ΔcavA mutant had increased AHL production compared to the WT and complemented ΔcavA+cavA strains where AHL secretion was abolished. AHL production was unaffected by the chromosomal insertion of the empty miniTn7 vector (EV) in the WT and ΔcavA backgrounds., Peer reviewed

Expression of pilA gene (A) and pga operon (B) is regulated by CavA and Vfr. Promoter regions of pilA and pga fused with gfpmut3 fluorescent reporter (PpilA::gfpmut3 and Ppga::gfpmut3 respectively) were used to assess the effect of CavA and Vfr on their expression. Bacterial cells were harvested from diluted bacterial cultures grown for 4.5 h, after which were resuspended in sterile PBS. Fluorescence (A.U.) at 470-15/515-20 nm excitation/emission was measured to determine the expression of Gfp and thus the expression of each promoter. Optical density at 600 nm (OD600) was measured to determine growth. Data represents the average of three independent repeats ± SD. **p<0.01, ****p<0.0001 –One-Way ANOVA with Tukey post-hoc test., Peer reviewed

Growth (A), biofilm formation (B), EPS production (C) and motility (D) of cavA and vfr related strains demonstrating that the observed phenotypes were not attributed to growth alternations of the strains. Moreover, the empty miniTn7 system (EV) with Tetracycline (Tc) or Tellurate (Tel) resistance markers, used for the complementations and genes expression in different backgrounds, did not have an effect on any of the tested phenotypes. Growth and biofilm formation were assessed after 24 h at 37°C shaking. EPS production was assessed on Congo agar after 5 days incubation of the strains at 37°C and motility was tested after 48 h on soft agar at 37°C. ns p>0.05, *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001—One-Way ANOVA with Tukey post-hoc test., Peer reviewed

A–Twitching motility of ΔcavA related strains showing that miniTn7 EV has no impact on A. baumannii motility. B–Natural transformation of ΔcavA mutant was bellow the detection limit (< d. l.) and was significantly decreased compared to the WT AB5075. ns p>0.05, ** p<0.01, ***p<0.001, **** p<0.0001—One-Way ANOVA with Tukey post-hoc test (A) and Unpaired t-test (B)., Peer reviewed

A–Representative images of Congo agar plates showing that the empty miniTn7 vector does not affect A. baumannii EPS production. B–Representative image of Congo red plates demonstrating the effect of pgaA disruption (pgaA::Tn) on Congo red dye binding to A. baumannii EPS and on the increased EPS production in the ΔcavA mutant. This shows that the effect of cavA on EPS production is mainly due to changes in the production of poly-β-1,6-N-acetylglucosamine (PNAG) and pgaABCD operon expression. C & D–Growth (C) and biofilm formation (D) of AB5075 WT and its derivative ΔabaI, csuC::Tn, pgaA::Tn and ΔcavA single and double mutants after being incubated at 37°C shaking for 24 h. Deletion of the autoinducer synthase gene abaI in ΔcavA did not affect the increased ΔcavA biofilm. In contrast, disruption of csuC or pgaA in the ΔcavA mutant significantly decreased the high biofilm levels caused by the deletion of cavA but did not completely reversed the ΔcavA phenotype. This data demonstrates that the regulation of A. baumannii biofilm formation by CavA is dependent on Csu pili and EPS production and is the additive effect of the global simultaneous regulation of multiple genes. ns p>0.05, **p<0.01, **** p<0.0001—One-Way ANOVA with Tukey post-hoc test., Peer reviewed