Extracellular TatD from Listeria monocytogenes displays DNase activity and contributes to biofilm dispersion

Abstract

TatD is evolutionarily conserved in a variety of organisms and has been implicated in DNA repair, apoptosis, and the disruption of extracellular traps. The aim of our study was to investigate the effects of TatD on L. monocytogenes biofilms. In our previous study, the deletion of the TatD gene from L. monocytogenes (named LmTatD) increased biofilm formation. However, the underlying mechanism remains unclear. In this study, we present a detailed analysis of the structural characteristics of TatD. Bioinformatic analysis revealed that the amino acid residues DPGEGDQHEDP are fully conserved. LmTatD belongs to the Class II TatD family (TATDN3) and contains a signal peptide. Recombinant LmTatD exhibited DNase activity regardless of the DNA substrate. Mutagenesis experiments confirmed the importance of glutamic acid, histidine, and aspartic acid residues in enzymatic activity. Biofilm formation was evaluated via a crystal violet assay, confocal laser scanning microscopy, and scanning electron microscopy. rLmTatD impaired biofilm formation and reduced eDNA levels without disrupting the integrity of the bacteria within biofilms. Moreover, deficiency of LmTatD led to a significant decrease in the DNase activity of the extracellular proteins from L. monocytogenes, whereas there was an increase in biofilm formation and eDNA production during the dispersion stage. However, no significant change in the total number of biofilm or planktonic bacteria was observed at any of the time points. Additionally, the mRNA level of LmTatD in the biofilm formed by the wild-type strain at the dispersion stage was greater than that at the attachment and maturation stages. The number of planktonic bacteria for the wild-type strain at the dispersion stage was significantly greater than that for the ΔLmTatD mutant. Collectively, these data suggest that LmTatD exhibits extracellular DNase activity and regulates L. monocytogenes biofilm dispersion.