Characterization of Vibrio cholerae’s Extracellular Nuclease Xds

Abstract

The Gram-negative bacterium Vibrio cholerae encodes two nucleases, Dns and Xds, which play a major role during the human pathogen’s lifecycle. Dns and Xds control three-dimensional biofilm formation and bacterial detachment from biofilms via degradation of extracellular DNA and thus contribute to the environmental, inter-epidemic persistence of the pathogen. During intestinal colonization the enzymes help evade the innate immune response, and therefore promote survival by mediating escape from neutrophil extracellular traps. Xds has the additional function of degrading extracellular DNA down to nucleotides, which are an important nutrient source for V. cholerae. Thus, Xds is a key enzyme for survival fitness during distinct stages of the V. cholerae lifecycle and could be a potential therapeutic target. This study provides detailed information about the enzymatic properties of Xds using purified protein in combination with a real time nuclease activity assay. The data define an optimal buffer composition for Xds activity as 50 mM Tris/HCl pH 7, 100 mM NaCl, 10 mM MgCl2, and 20 mM CaCl2. Moreover, maximal activity was observed using substrate DNA with low GC content and ambient temperatures of 20–25°C. In silico analysis and homology modeling predicted an exonuclease domain in the C-terminal part of the protein. Biochemical analyses with truncated variants and point mutants of Xds confirm that the C-terminal region is sufficient for nuclease activity. We also find that residues D787 and H837 within the predicted exonuclease domain are key to formation of the catalytic center.

Escherichia coli Can Eat DNA as an Excellent Nitrogen Source to Grow Quickly

“…Extracellular DNA degradation activity of S. aureus and E. coli was examined using a previously described method (Jeffries et al, 1957;Pressler et al, 2019). Briefly, a 10.0 µl aliquot of an overnight culture (washed with PBS buffer (pH 7.2), then resuspended in PBS buffer) was spotted onto nutrient broth agar plates containing 2.0 g/L of DNA and incubated overnight at 37 • C. For visualization, plates were flooded with 3.0 ml of perchloric acid solution (50% v/v perchloric acid in distilled water) and kept for 5 min.…”

Fitness of Outer Membrane Vesicles From Komagataeibacter intermedius Is Altered Under the Impact of Simulated Mars-like Stressors Outside the International Space Station

“…Nucleases perform different roles to fit bacterial lifestyle ( Al-Wahaibi et al, 2019 ; Pressler et al, 2019 ). Extracellular nucleases are known as enzymes to accommodate invading bacteria in hosts via the formation of a set of diverse oligonucleotides with immunomodulatory properties ( Takahashi et al, 2006 ) or help evade the innate immune response ( Jhelum et al, 2018 ).…”

Pathogen-Derived Nucleases: An Effective Weapon for Escaping Extracellular Traps

“…Infection with a Dns/Xds deletion mutant of V. cholerae resulted in prolonged stability of the NET. The C-terminus of Xds has an exonuclease domain, and residues D787 and H837 within the exonuclease domain are the key sites of the catalytic center ( 13 ).…”