Precision control of bacterial biofilm formation signal via cell division factor

This study is led by Dr. Liang Yang (School of Medicine, Southern University of Science and Technology), Dr. Yingdan Zhang (School of Medicine, Southern University of Science and Technology) and Dr. Lianhui Zhang (Guangdong Province Key Laboratory of Microbial Signals and Disease Control, Integrative Microbiology Research Center, South China Agricultural University). Pseudomonas aeruginosa is one of the leading nosocomial pathogens that causes both severe acute and chronic infections. The strong capacity of P. aeruginosa to form biofilms can dramatically increase its antibiotic resistance and lead to treatment failure. The biofilm resident bacterial cells display distinct gene expression profiles and phenotypes compared to their free-living counterparts. Thus, elucidating the genetic determinants of biofilm formation is crucial for the development of antibiofilm drugs.

In this study, they employed a high-throughput transposon-insertion site sequencing (Tn-seq) approach to identify novel P. aeruginosa biofilm genetic determinants. Their Tn-seq results reasonably reflected the previous biofilm mechanistic studies, as the genes related to exopolysaccharide synthesis and motility were suggested to be biofilm determinants. What’s more, they also discovered that genes with unknown functions might play important roles in P. aeruginosa biofilm formation. They selected five hypothetical genes (PA0222, PA1112, PA2345, PA3797, and PA4438) for experimental validation. And their results suggest that each of the five genes, particularly PA4438, plays an important role in P. aeruginosa biofilm formation and is novel biofilm genetic determinant.

PA4438 is predicted to encode an AAA+ AFG1 ATPase and its predicted amino acid sequence has regions of high conservation to the cell division protein ZapE of E. coli (EcZapE). A previous study reported that the loss of EcZapE would result in cell elongation under oxygen-limited and high-temperature conditions. Their results suggest that zapE (PA4438) is also important for proper cell division under anaerobiosis and high-temperature conditions, hereafter, they name PA4438 as zapE.

To further investigate the regulatory mechanism of zapE in P. aeruginosa biofilm formation, they used RNA-seq-based transcriptomic analysis to elucidate the global effect of zapE on P. aeruginosa gene expression. Interestingly, almost all pqs quorum sensing (QS)-regulated genes were expressed at a lower level in ΔzapE compared to PAO1 wild-type strain, suggesting that the cell division factor ZapE regulates the P. aeruginosa pqs QS system. To elucidate the link between ZapE and pqs QS, the team applied immunoprecipitation and mass spectrometry (IP-MS) and surface plasmon resonance (SPR) assay to identify potential ZapE-interacting proteins. Their results suggested that ZapE could directly interact with PqsH, an NADH-dependent monooxygenase which converts 2-heptyl-4-quinolone (HHQ) to heptyl-3-hydroxy-4-(1H)-quinolone (PQS). They further proved that the ATPase enzyme activity of ZapE is required for the synthesis of PQS and the pqs QS.

The amino acid sequence of ZapE appeared to be highly conserved not only in P. aeruginosa strains (PAO1, PA14, and PAK), but also in other members of the Pseudomonas group, such as Pseudomonas stutzeri, Pseudomonas putida, Pseudomonas chloritidismutans, and Pseudomonas fluorescens. Their results suggested that ZapE plays a key role in the biofilm formation and cell division in other P. aeruginosa strains.

In summary, their work showed that ZapE, a cell division factor and an AAA+ protein, is a novel biofilm regulator through modulating the pqs QS. As the ZapE amino acid sequence is highly conserved among the Pseudomonas group, this study could also provide a novel target for the development of anti-Pseudomonas compounds.

See the article:

Cell division factor ZapE regulates Pseudomonas aeruginosa biofilm formation by impacting the pqs quorum sensing system

https://doi.org/10.1002/mlf2.12059