Moraxella catarrhalis NucM is an entry nuclease involved in extracellular DNA and RNA degradation, cell competence and biofilm scaffolding

Abstract

Moraxella catarrhalis is a host-adapted bacterial pathogen that causes otitis media and exacerbations of chronic obstructive pulmonary disease. This study characterises the conserved M. catarrhalis extracellular nuclease, a member of the ββα metal finger family of nucleases, that we have named NucM. NucM shares conserved sequence motifs from the ββα nuclease family, including the DRGH catalytic core and Mg²⁺ co-ordination site, but otherwise shares little primary sequence identity with other family members, such as the Serratia Nuc and pneumococcal EndA nucleases. NucM is secreted from the cell and digests linear and circular nucleic acid. However, it appears that a proportion of NucM is also associated with the cell membrane and acts as an entry nuclease, facilitating transformation of M. catarrhalis cells. This is the first example of a ββα nuclease in a Gram negative bacteria that acts as an entry nuclease. In addition to its role in competence, NucM affects cell aggregation and biofilm formation by M. catarrhalis, with ΔnucM mutants having increased biofilm biomass. NucM is likely to increase the ability of cells to survive and persist in vivo, increasing the virulence of M. catarrhalis and potentially affecting the behaviour of other pathogens that co-colonise the otorhinolaryngological niche.

Figure 1

Extracellular nuclease activity of M. catarrhalis. (A) Nuclease activity in whole cell and secreted fractions of M. catarrhalis strain 25238 and 25239 cells grown in brain heart infusion media (BHI; top) and chemically defined media (CDM; bottom), co-incubated with plasmid DNA overnight. Fractions are whole cell (WC), cell free filtrate (F), supernatant from ultracentrifugation of OMVs (S) and outer membrane vesicles (OMV). Sizes are given for the marker (M; 1 kb ladder, NEB) in the bottom panel. (B) Nuclease activity in BHI (top panel) and CDM (bottom panel) at 2 hour intervals (representing early-, mid-, late-log and stationary phases during aerobic growth) for M. catarrhalis strains 25238 (8) and 25239 (9), showing shift in plasmid forms from closed circular to nicked and linear forms in CDM isolated filtrate. (C) Sequential degradation of plasmid DNA over time. Nuclease activity from cell-free filtrate from overnight aerobic growth of M. catarrhalis 25238 (8) and 25239 (9) strains in BHI, co-incubated with plasmid DNA for timed intervals of 5, 15, 30, 60, 120, 240 or 480 minutes, or overnight (O/N). Plasmid forms are indicated to the right of the gel, and sizes are given for the marker (1 kb ladder, NEB) to the left. The full-length gels from panels A–C are presented in Supplementary Fig. S3.

Figure 2

Conserved DRGH nuclease motif. Partial protein sequence alignment of the M. catarrhalis NucM consensus to the Serratia nuclease and pneumococcal End A. Alignment of the central region of the NucM protein consensus from M. catarrhalis (with non-conserved residues shown in lowercase) to the Serratia marcescens nuclease, S. pneumoniae EndA and consensus sequence from multispecies alignments as seen in. Matches to the consensus sequence are shown in red, with additional residues important for the mechanism of EndA shown in blue.

Figure 3

Growth and extracellular nuclease activity of M. catarrhalis wild type and isogenic ΔnucM mutants. (A) Growth curve of M. catarrhalis 25238 and 25239 strains, grown overnight in BHI at 37 °C with aeration, using 0.5 mL aliquots in a 48-well plate format. Assays were carried out in triplicate and OD₆₀₀ values were measured in a Tecan Infinite 200 Pro plate reader, with mean and standard deviation shown. (B) Degradation assays of genomic DNA (gDNA), plasmid DNA or RNA incubated with filtered broth without cells (media only, -ve), or grown with wild type (WT) or isogenic ΔnucM knock out 25238 and 25239 strains. The full-length gels from panel B are presented in Supplementary Fig. S4.

Figure 4

Cell-cell aggregation of M. catarrhalis wild type and isogenic ΔnucM mutants. (A) Growth curve of M. catarrhalis strains, grown overnight in CDM at 37 °C with aeration, using 0.5 mL aliquots in a 48-well plate format. Assays were carried out in triplicate and OD₆₀₀ values were measured in a Tecan Infinite 200 Pro plate reader, with mean and standard deviation shown. Significance of mean differences was tested by t-test for unpaired samples not assuming equal standard deviations in PRISM; ***p < 0.0005; **p < 0.005, *p < 0.05. (B) Example wells from plate assay, showing aggregates in wells with ΔnucM strains. The level of aggregation seen for each strain is indicated: ‘−’ for no aggregation, ‘+’ for minor aggregation, ‘++’ for aggregation. (C) Settling curves shown for M. catarrhalis 25238 (top) and 25239 (bottom). Assays were carried out in triplicate with OD₆₀₀ readings taken at the time points indicated. Average and standard deviations are shown. Significance was tested by t-test for unpaired samples not assuming equal standard deviations in PRISM; **p < 0.005, *p < 0.05.

Figure 5

Static plate biofilm assay of M. catarrhalis wild type and isogenic ΔnucM mutants. Assays were carried out in 48-well plate format over 20 hours, and biofilm biomass quantified by crystal violet staining and absorbance at 570 nm. The data presented represent the average of six replicates, and the standard deviation is shown. Significance was tested by t-test for unpaired samples not assuming equal standard deviations in PRISM; ****p ≤ 0.0001, ***p ≤ 0.0005; **p ≤ 0.005.

Figure 6

Scanning electron microscopy of static plate biofilm of wild type and isogenic ΔnucM mutants. Assays were carried out in 48-well plate format with coverslips over 20 hours, with biofilms washed, fixed and dehydrated in plates. Coverslips were removed, mounted and gold-sputtered for visualisation on SEM. Representative images of M. catarrhalis 25239 wild type and ΔnucM mutants are shown, taken at 50x, 400x and 2000x magnification (top, middle and bottom rows, respectively) and scale bars are indicated on each panel.

Figure 7

MBEC^TM biofilm assays of M. catarrhalis wild type and isogenic ΔnucM mutants. Assays were carried out in 96-well plates for 48 hours, at 37 °C with aeration. Viable colony forming units of biofilms attached to MBEC^TM pegs were assessed by sonication of pegs and enumeration on BHI agar for 25238 (A) and 25239 (B). Significance was tested by two-sided t-test for unpaired samples not assuming equal standard deviations in PRISM; ****p ≤ 0.0001, ***p ≤ 0.0005; **p ≤ 0.005. (C) Fluorescence live/dead staining with SYTO9 (green) and propidium iodide (red) of 25238 (top) and 25239 biofilms (bottom) on MBEC^TM pegs.