Functional and structural establishment of a novel cyclic di-GMP signalling pathway in pseudomonas aeruginosa

Abstract

The pathogenicity of Pseudomonas aeruginosa can be attributed to not only virulence factors causing acute infection but also biofilm formation leading to antibiotic resistance and chronic infection. The global bacterial second messenger cyclic di-GMP, constituting signalling pathways with its effectors and their downstream targets, contributes to motility, biofilm formation, virulence and biofilm tolerance to antimicrobials. Many cyclic di-GMP effector proteins including enzymatic inactive GGDEF/EAL domain proteins, PilZ domain proteins and transcriptional regulators have been characterised functionally and structurally. YajQ family proteins are known to exhibit regulations on bacterial biofilm formation, virulence, antifungal activity and antibiotic resistance and are a newly identified class of cyclic di-GMP effectors. Transcriptional regulators are normally YajQ downstream targets to complete this cyclic di-GMP turnover. PA4395, a YajQ family protein exhibiting high binding affinity with cyclic di-GMP was proposed as a new cyclic di-GMP effector in P. aeruginosa previously. However, specific functional roles of PA4395 and how it structurally recruits cyclic di-GMP are still unclear. In addition, PA2692, a transcriptional regulator belonging to Rrf2 family proteins, displaying interacting affinity with PA4395 in previous works, was proposed as the downstream target of PA4395. Regulatory roles of PA2692 and how PA4395 interact with it are also unknown. In this study, I aimed to determine functions co-regulated by PA4395 and PA2692, to investigate binding mechanisms among cyclic di-GMP, PA4395 and PA2692, and to build up a new cyclic di-GMP signalling pathway. Firstly, functions of PA4395 and PA2692 were elucidated. PA4395 and PA2692 revealed significant essentiality for cell adhesion and aggregation under antimicrobial-replete environment, and have remarkably upregulation in downstream gene PA14_72360, a homologue of ivy for host cell colonization. PA2692 but not PA4395 also exhibited regulations in swarming motility, biofilm formation, and virulence. Secondly, the binding mechanism between cyclic di-GMP and PA4395 was investigated by solution NMR spectroscopy. Cyclic di-GMP having week binding affinity with PA4395 can be specifically recognized by this protein when it is at low concentration. R28, R29, F30, D31 of PA4395 were identified as key residues involved in the accommodation of cyclic di-GMP. Mutation of these residues undermined the cyclic di-GMP-binding ability of PA4395 and its promotion in PA14_72360 expression. In the last chapter of results, in vitro functional properties of PA2692 and strategy of PA4395 to contact PA2692 were studied. PA2692 was found forming stable and solid dimeric structure with conservative cysteine-coordinated iron cofactors, which is a prerequisite of its DNA-binding and protein-linking ability. It seems that six lysine residues K104, K111, K131, and K114, K116, K132 with opposite orientations were adopted by PA4395 to stabilize PA2692-interaction at two binding sites. In addition, in vitro assays displayed PA4395 facilitated the binding activity between PA2692 and DNA, which cannot be disturbed by the further addition of cyclic di-GMP, indicating the collaborated contribution of cyclic di-GMP, PA4395, and PA2692 to downstream gene expression from in vitro perspective. Taken together, a new cyclic di-GMP signalling pathway for the regulation of biofilm cell resistance to antimicrobials in P. aeruginosa was established by in vivo and in vitro assays. The formation of cyclic di-GMP-PA4395-PA2692 complex activate the transcription of PA14_72360, promoting cell colonization under antimicrobial-present conditions.

More information

Identifiers

ORCID for Jeremy Webb: orcid.org/0000-0003-2068-8589

ORCID for Joern Werner: orcid.org/0000-0002-4712-1833

Catalogue record

Export record