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. 2020 Feb 6;10(1):1991.
doi: 10.1038/s41598-020-58522-5.

A Cyclic-di-GMP signalling network regulates biofilm formation and surface associated motility of Acinetobacter baumannii 17978

Irfan Ahmad  1   2 , Evelina Nygren  3 , Fizza Khalid  4 , Si Lhyam Myint  3 , Bernt Eric Uhlin  5
Affiliations

A Cyclic-di-GMP signalling network regulates biofilm formation and surface associated motility of Acinetobacter baumannii 17978

Irfan Ahmad et al. Sci Rep. .

Abstract

Acinetobacter baumannii has emerged as an increasing multidrug-resistant threat in hospitals and a common opportunistic nosocomial pathogen worldwide. However, molecular details of the pathogenesis and physiology of this bacterium largely remain to be elucidated. Here we identify and characterize the c-di-GMP signalling network and assess its role in biofilm formation and surface associated motility. Bioinformatic analysis revealed eleven candidate genes for c-di-GMP metabolizing proteins (GGDEF/EAL domain proteins) in the genome of A. baumannii strain 17978. Enzymatic activity of the encoded proteins was assessed by molecular cloning and expression in the model organisms Salmonella typhimurium and Vibrio cholerae. Ten of the eleven GGDEF/EAL proteins altered the rdar morphotype of S. typhimurium and the rugose morphotype of V. cholerae. The over expression of three GGDEF proteins exerted a pronounced effect on colony formation of A. baumannii on Congo Red agar plates. Distinct panels of GGDEF/EAL proteins were found to alter biofilm formation and surface associated motility of A. baumannii upon over expression. The GGDEF protein A1S_3296 appeared as a major diguanylate cyclase regulating macro-colony formation, biofilm formation and the surface associated motility. AIS_3296 promotes Csu pili mediated biofilm formation. We conclude that a functional c-di-GMP signalling network in A. baumannii regulates biofilm formation and surface associated motility of this increasingly important opportunistic bacterial pathogen.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Predicted GGDEF/EAL proteins in A. baumannii 17978. Domain architecture of GGDEF/EAL proteins created by SMART protein analysis tool highlights the trans-membrane domains (Blue bars), PAS domains (Pink Box) and PAC domains (Pink arrowheads) in N terminal of GGDEF/EAL proteins.
Figure 2
Figure 2
Conserved domain signature alignment and classification of predicted GGDEF/EAL proteins in A. baumannii 17978. The amino acid sequence of the GGDEF domain signatures of STM 0385 (AdrA), a diguanylate cyclase from S. typhimurium and RocR a phosphodiesterase from P. aeruginosa were used as reference sequences to align GGDEF/EAL proteins of A. baumannii. The conserved residues of GGDEF/EAL signature that perfectly aligned with the reference sequence are shown in bold face.
Figure 3
Figure 3
Assessment of the enzymatic activity GGDEF/EAL domain proteins of A. baumannii 17978 by phenotypic testsing in model organisms. (A) Rdar morphotype formation of S. typhimurium SR11 after 72 hours. (B) Rugose morphotype formation of V. cholera C6706luxOcafter 48 hours. Derivatives of the two model strains were assessed upon the expression of individual GGDEF/EAL domain proteins of A. baumannii from the plasmid pMMB67EH on congo red agar plates.
Figure 4
Figure 4
Development of macro colony formation in A. baumannii17978. (A) Stereo macroscopic images of single colonies of A. baumannii upon the expression of individual GGDEF/EAL domain proteins from the plasmid pMMB67EH. Growth was done on congo red agar plates supplemented with 1 mM IPTG and 100 μg/ml carbenicillin. The diguanylate cyclases A1S_2506 and A1S_3296 mediated expression of reddish matrix component at 30 °C and 37 °C after 48 hours.
Figure 5
Figure 5
Biofilm formation assay of A. baumannii 17978 upon the expression of individual GGDEF/EAL domain proteins from the plasmid pMMB67EH. Biofilm formation assays were carried out at 37 °C (A) and 30 °C (B) as described in materials and methods with bacterial strains carrying the indicated plasmid clones. Error bars represent mean ± SD values of 6 replicates of three independent experiments. P values are shown on the top of columns with statistical significant alterations as compared to WT VC (Wild type vector control) were calculated by student paired t test using Graph Pad Prism software.
Figure 6
Figure 6
Analysis of biofilm formation by A. baumannii and of bacterial surface alterations upon deletion of the A1S_3296 coding sequence. (A) Biofilm formation assays were carried out at 37 °C and 30 °C as described in materials and methods with bacterial strains carrying the indicated plasmid clones. (B) Congo red binding monitored at the centre of colonies after growth at 37 °C and 28 °C. (C) Scanning electron microscopic images of colonies to reveal presence of extra cellular matrix component(s).
Figure 7
Figure 7
Surface associated motility of A. baumannii 17978. The assay with quantification of surface associated motility was performed as described in materials and methods. Error bars represent mean ± SD values of three experiments. (A) Motility upon the expression of individual GGDEF/EAL domain proteins from the plasmid pMMB67EH after 7 hours of incubation. (B) Motility upon deletion of coding sequences for A1S_2986 and A1S_3296, respectively, from the genome A. baumannii. *Represents p of value of less than 0.05 upon student paired t test as compared to WT VC (Wild type vector control).
Figure 8
Figure 8
A1S_3296 promoted biofilm formation through Csu pili mediated pathway. The effect of A1S_3296 in csuA and pilA mutants regarding congo red binding in macro colonies (A) and biofilm formation. (B) Immunoblot blot to illustrate alteration in CsuAB expression in 2396 mutant as compare to wild type and complemented strain. (C) The strain lacking the expression of csuABC operon (∆csuA) was used as negative control for CsuAB expression.
Figure 9
Figure 9
Summary of GGDEF/EAL proteins A, baumannii 17978 that regulate biofilm formation and surface associated motility. Arrows represent enhancement of the phenotype whereas line bars represent suppression of the phenotype.
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