Identification of genes essential for pellicle formation in Acinetobacter baumannii

Abstract

BackgroundAcinetobacter baumannii is an opportunistic pathogen, which has the ability to persist in the clinical environment, causing acute and chronic infections. A possible mechanism contributing to survival of A. baumannii is its ability to form a biofilm-like structure at the air/liquid interface, known as a pellicle. This study aimed to identify and characterise the molecular mechanisms required for pellicle formation in A. baumannii and to assess a broad range of clinical A. baumannii strains for their ability to form these multicellular structures.ResultsRandom transposon mutagenesis was undertaken on a previously identified hyper-motile variant of A. baumannii ATCC 17978 designated 17978hm. In total three genes critical for pellicle formation were identified; cpdA, a phosphodiesterase required for degradation of cyclic adenosine monophosphate (cAMP), and A1S_0112 and A1S_0115 which are involved in the production of a secondary metabolite. While motility of the A1S_0112::Tn and A1S_0115::Tn mutant strains was abolished, the cpdA::Tn mutant strain displayed a minor alteration in its motility pattern. Determination of cAMP levels in the cpdA::Tn strain revealed a ~24-fold increase in cellular cAMP, confirming the role CpdA plays in catabolising this secondary messenger molecule. Interestingly, transcriptional analysis of the cpdA::Tn strain showed significant down-regulation of the operon harboring the A1S_0112 and A1S_0115 genes, revealing a link between these three genes and pellicle formation. Examination of our collection of 54 clinical A. baumannii strains revealed that eight formed a measurable pellicle; all of these strains were motile.ConclusionsThis study shows that pellicle formation is a rare trait in A. baumannii and that a limited number of genes are essential for the expression of this phenotype. Additionally, an association between pellicle formation and motility was identified. The level of the signalling molecule cAMP was found to be controlled, in part, by the cpdA gene product, in addition to playing a critical role in pellicle formation, cellular hydrophobicity and motility. Furthermore, cAMP was identified as a novel regulator of the operon A1S_0112-0118.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0440-6) contains supplementary material, which is available to authorized users.

Global Dynamic Proteome Study of a Pellicle-forming Acinetobacter baumannii Strain

“…S5) (81,82), was accumulated in 1-day pellicles. This operon could be involved in the synthesis of a biosurfactant lipopeptide, or an N-Acyl homoserine lactone (AHL) (25,81).…”

“…This operon could be involved in the synthesis of a biosurfactant lipopeptide, or an N-Acyl homoserine lactone (AHL) (25,81). It is independently regulated by three different factors: (1) by the AHL, N-(3-hydroxydodecanoyl)-homoserine lactone (3-OH-C 12 -HSL), itself in a abaI-dependent manner (AbaI, A1S_0109, being the AHL synthase (81,83), (2) by the cAMP via the CpdA regulator (82), and (3) via an iron limitation (58). A1S_0112 and A1S_0115 proteins are an AcylCoA synthetase and an AMP-dependent synthetase, respectively.…”

“…A1S_0112 and A1S_0115 proteins are an AcylCoA synthetase and an AMP-dependent synthetase, respectively. Loss of these proteins in mutant strains induces an alteration of the pellicle formation, motility and hydrophobicity (82). In S-L biofilm, the A1S_0112-0118 genes have been shown overexpressed after 24 h of growth and an A1S_0114 deletion mutant (the corresponding protein being an acyl carrier protein ACP), also exhibited a reduced S-L biofilm formation compared with the wild-type (25).…”

Resistance to pentamidine is mediated by AdeAB, regulated by AdeRS, and influenced by growth conditions inAcinetobacter baumanniiATCC 17978

“…One 10 mL sample was treated with 7.8 mg/L of pentamidine (0.5 × MIC for ΔadeRS), whilst the other remained untreated. Cultures were grown for an additional 30 min before total RNA was extracted as outlined previously [34].…”

“…Plasmids were introduced into appropriate A. baumannii cells as previously described [33]. Primers used to generate complementation vectors are listed in S2 Table. Cell treatments and RNA isolation RNA was isolated from WT and ΔadeRS cells and Hiseq RNA transcriptome analysis performed following methodologies as outlined previously [34].…”