IolR, a negative regulator of the myo-inositol metabolic pathway, inhibits cell autoaggregation and biofilm formation by downregulating RpmA in Aeromonas hydrophila

Abstract

Aeromonas hydrophila is the causative agent of motile Aeromonad septicemia in fish. Previous studies have shown that the myoinositol metabolism is essential for the virulence of this bacterium. IolR is a transcription inhibitor that negatively regulates myoinositol metabolic activity. While in the process of studying the inositol catabolism in A. hydrophila Chinese epidemic strain NJ-35, we incidentally found that ΔiolR mutant exhibited obvious autoaggregation and increased biofilm formation compared to the wild type. The role of surface proteins in A. hydrophila autoaggregation was confirmed by different degradation treatments. Furthermore, calcium promotes the formation of aggregates, which disappear in the presence of the calcium chelator EGTA. Transcriptome analysis, followed by targeted gene deletion, demonstrated that biofilm formation and autoaggregation caused by the inactivation of iolR was due to the increased transcription of a RTX-family adhesion gene, rmpA. Further, IolR was determined to directly regulate the transcription of rmpA. These results indicated that iolR is negatively involved in autoaggregation and biofilm formation in A. hydrophila, and this involvement was associated with its inhibition on the expression of rmpA.

Bacteria autoaggregation: how and why bacteria stick together

“…Autoaggregation can be the indirect consequence of a secreted factor mediating aggregation indirectly by connecting other surface factors [28]. Proteinase K treatment more commonly alters or completely obliterates autoaggregation compared with sodium periodate ( polysaccharide removal) and DNase1 (extracellular DNA removal) treatment [29][30][31]. However, polysaccharides as well extracellular DNA, released during autolysis can mediate autoaggregation.…”

Essential role of calcium in extending RTX adhesins to their target

“…It was found that an overproduction of Ah Lap (named against convention within the study as RmpA) was responsible for this new phenotype, adding Ah Lap to the growing list of RTX adhesins vital to biofilm formation ( Guo et al, 2017 , Hinsa et al, 2003 , Martínez-Gil et al, 2010 , Syed et al, 2009 ). While deletions of Ah Lap removed the hyper biofilm-forming phenotype as expected, so too did the removal of Ca 2+ from the solution via chelating agents ( Dong et al, 2020 ). This result mirrors other such experiments on RTX adhesin-mediated behaviour, like studies on the RTX adhesin SiiE in Salmonella enterica , where the removal of calcium greatly reduced invasion of the bacteria into polarized epithelial cells ( Peters et al, 2017 ).…”

“…Several such cases have been reported ( D’Auria et al, 2008 , Guo et al, 2017 , Wrobel et al, 2018 ), and likely many more have yet to be caught. For instance, only a single repeat is reported in the Ah Lap sequence from the NJ-35 strain ( Dong et al, 2020 ), which could be an underestimate.

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“…Recently, Dong et al published a paper detailing hyper biofilm-forming mutants in A. hydrophila strains that infect fish ( Dong et al, 2020 ). It was found that an overproduction of Ah Lap (named against convention within the study as RmpA) was responsible for this new phenotype, adding Ah Lap to the growing list of RTX adhesins vital to biofilm formation ( Guo et al, 2017 , Hinsa et al, 2003 , Martínez-Gil et al, 2010 , Syed et al, 2009 ).…”

“…These regions are found in both multi-cellular organisms (ex. integrin and cadherin, which maintain contacts between cells and extracellular matrix components ( Campbell and Humphries, 2011 , Takeichi, 1990 )), and in single-celled organisms, where adhesion proteins allow bacteria to stick to surfaces ( Guo et al, 2012 , Ivanov et al, 2012 , Leo et al, 2010 , Schilling et al, 2001 , Wagner et al, 2014 ) and cluster together to make bacterial communities known as biofilms ( Borlee et al, 2010 , Dong et al, 2020 , Kikuchi et al, 2005 , Martínez-Gil et al, 2010 ).…”