Prey killing without invasion by Bdellovibrio bacteriovorus defective for a MIDAS-family adhesin

Abstract

The bacterium Bdellovibrio bacteriovorus is a predator of other Gram-negative bacteria. The predator invades the prey's periplasm and modifies the prey's cell wall, forming a rounded killed prey, or bdelloplast, containing a live B. bacteriovorus. Redundancy in adhesive processes makes invasive mutants rare. Here, we identify a MIDAS adhesin family protein, Bd0875, that is expressed at the predator-prey invasive junction and is important for successful invasion of prey. A mutant strain lacking bd0875 is still able to form round, dead bdelloplasts; however, 10% of the bdelloplasts do not contain B. bacteriovorus, indicative of an invasion defect. Bd0875 activity requires the conserved MIDAS motif, which is linked to catch-and-release activity of MIDAS proteins in other organisms. A proteomic analysis shows that the uninvaded bdelloplasts contain B. bacteriovorus proteins, which are likely secreted into the prey by the Δbd0875 predator during an abortive invasion period. Thus, secretion of proteins into the prey seems to be sufficient for prey killing, even in the absence of a live predator inside the prey periplasm.

Fig. 1. Microscopy showing empty bdelloplasts of Δbd0875 predator and Bd0875 protein location.

a Microscopically imaged typical progress through predation, from attack phase (AP) outside prey to invasion, replication and exit; for cytoplasmically labelled B. bacteriovorus strains constitutively expressing Bd0064mCherry (red). Normal predation process (T0-T300 min after mixing predator and prey) occurs for HD100 wild type and 90% of Δbd0875 predators but 10% are devoid of an invaded of a Δbd0875 predator. Images from within one experiment and representative of three biological repeats b Examples of two types of bdelloplasts from live/dead staining of E. coli preyed upon by Δbd0875: (i) wild type predation with live (false coloured yellow) predator inside dead (false coloured magenta) prey (ii) empty dead (false coloured magenta) bdelloplasts without invaded predator, Scale bar 2 µm. Images representative of three biological repeats. c Frequency of dead empty E. coli prey bdelloplasts in predatory cultures of MIDAS gene deletion predator strains versus wild type HD100 monitored using live/dead staining. ^aAnalysis of Δbd1483 was performed alongside a B. bacteriovorus HD100 control but in a separate experiment to the analysis of Δbd0767, Δbd0875, Δbd3132 and Δbd0767Δbd0875Δbd3132. Percentage of uninvaded bdelloplasts for Δbd0875 and Δbd0767Δbd0875Δbd3132 triple mutant were not significantly different from each other (p = 0 9041), but both of these mutant strains were significantly different from all other strains including versus HD100 (p values 0.0029 Δbd0875 and 0.0009 Δbd0767Δbd0875Δbd3132) (see Supplementary Tables 5a–c for full details). Data were derived from three biological repeats and over 1000 bdelloplasts per predator strain (Total and number of bdelloplasts counted per biological repeat are shown Supplementary Table 5d and as follows HD100 n = 1135, Δbd0767 n = 1181, Δbd0875 n = 1121, Δbd3132 n = 1223 Δbd0767Δbd0875Δbd3132 n = 1162; HD100^a n = 1002 and Δbd1483 n = 1090. Mean (+SD) percentage empty bdelloplasts for each strain is shown in Supplementary Table 5a. P values from one-way ANOVA are shown in Supplementary Table 5b and two-tailed unpaired t-test in Supplementary Table 5c. d Anti-mCherry (secondary antibody with Alexa Fluor 488 (green)) detection of positions of Bd0875-mCherry MIDAS adhesin protein expressed by a B. bacteriovorus HD100 strain chromosomally also WT for Bd0875. Fluorescence was detected on invaded prey (large, black) bdelloplasts and between invading, (small, black), predators and prey, but not on unattached predator at times of attack-phase (AP), (T5-10 min). A strain of B. bacteriovorus Bd2740mCherry MAT protein, with known surface expression during attack-phase (black outlined panel) was used as a Positive Control for detection of mCherry (green) in attack phase, even when Bd0875mCherry was not being expressed. No in vivo direct mCherry fluorescence from Bd0875mCherry was detected from the periplasm, hence surface antibody detection of mCherry was used here. Scale bar = 2 µm. Data derived from two biological repeats. e Typical range of different protein positions detected, at invasion junction, (as fluorescent predator is entering under the transparent outer-membrane of the prey), for Bd0875 adhesin proteins during predator invasion of prey (black) at T 25 min post mixing, using anti-mCherry (detected with secondary antibody with Alexa Fluor 555 (shown as red)). Bd0875-mCherry was expressed in B. bacteriovorus HD100 WT strain that was also cytoplasmically labelled with constitutively expressed cytoplasmic protein Bd0064mCerulean (blue) to illuminate the predator cell inside prey. An additional Control bdelloplast image, with contrasting externally attached (right hand side) and invaded (lefthand side) predators, is shown for comparison of invaded and just attached B. bacteriovorus. Scale bar = 2 µm. Range of protein positions illustrated from one biological repeat. Source data and full-sized images are provided in a Source data file accompanying this paper.

Fig. 2. Bd0875 protein has novel cap region absent in other B. bacteriovorus MIDAS proteins.

a PROMALS3D structure-based sequence alignment of the four tested B. bacteriovorus proteins with MIDAS domain showing unique regions of Bd0875 protein region highlighted in pink; formatted using ESPRIPT. b 3D alignment of MIDAS protein Bd1483 (Alphafold model), mapped onto the Bd0875 protein (Alphafold Q6MPH9and Q6MMY6); this illustrates the MIDAS adhesin domain (von Willebrand adhesin vwa) and that an additional cap region (pale blue) on the MIDAS domain (projecting upwards) is unique to Bd0875, the only MIDAS protein that gives a prey-invasion defect. Key to labelling: - The large run of pink circled amino acids in 2a corresponds to the main cap domain from 2b with a cap-adjacent small region encoded by the short run of pink circled amino-acids and the small domain (yellow in 2b) comprised of amino-acids 1-56 and 326-378 of Bd0875 sequence. Regions of alpha helices (α), beta sheets (β) and 3₁₀ helices (η), are annotated and numbered. The blue starred amino acids are those conserved in the MIDAS domain and the green numbered cysteines in the Bd0875 sequence are predicted to form disulphide bonds 1 to1, 2 to 2, 3 to 3, 4 to 4. Further Alphafold details are in Data Availability Statement.

Fig. 3. Key MIDAS motif amino acids are required for Bd0875 protein function.

ai Alphafold model of predicted Bd0875 structure (using Q6MMY6, signal peptide and lipobox of amino acids 1-22 omitted), showing Von-Willebrand adhesin (vwa) domain (blue), including Bd0875 cap region, plus hinge and small beta-rich domain formed from the N and C terminus of the Bd0875 protein sequence (yellow) with aii blue central domain showing numbered, conserved MIDAS motif amino acids on zoomed in adhesin structure; these are described and studied more in legend c. Further Alphafold details are in Data Availability Statement. b Percentage of dead empty E. coli prey bdelloplasts in cultures preyed upon by wild type HD100 and MIDAS gene deletion and gene complemented predator strains, verified using live/dead staining. (Supplementary Table 4) Mean (+SD) percentage empty bdelloplasts for each strain is shown in Supplementary Table 5e. P values from one-way ANOVA corrected for multiple comparisons are shown in Supplementary Table 5f. Total and number of bdelloplasts counted per biological repeat are shown in Supplementary Table 5g. Data for bd0875 complementation of Δbd0875 and Δbd0767Δbd0875Δbd3132 triple mutant were not significantly different from each other (p > 0.9999) but each was significantly different from complementation tests and controls for all other strains (Supplementary Table 5f). Data were derived from three biological repeats and over 1000 bdelloplasts per predator strain. Total number of bdelloplasts counted per biological repeat are shown supplementary Table 5g and as follows- HD100 n = 1181; Δbd0875 n = 1151; Δbd0767Δbd0875Δbd3132 n = 1354; Δbd0875:bd0875 n = 1136 and Δbd0767Δbd0875Δbd3132:bd0875 n = 1090. Source data are provided in a Source data file accompanying this paper. c Percentage of dead empty E. coli prey bdelloplasts in cultures preyed upon by wild type HD100 or MIDAS gene deletion strains undergoing bd0875 complementation tests with wild type gene or point mutants of gene bd0875 encoding: D66A MIDAS domain metal-binding active site, E213A MIDAS domain active site-proximal or Y349A small domain. P values from one-way ANOVA corrected for multiple comparisons are shown in Supplementary table 5i. HD100 control significantly different to data for deletion of Δbd0875 (p = <0.0001) and complementation of Δbd0875 with bd0875:D66A (p = <0.0001). For bd0875:E213A, data were significantly different to wild type HD100 (p = 0.0009). However bd0875:Y349A data were not significantly different to wild type HD100 (p = >0.9999). Data derived from three biological repeats and over 1000 bdelloplasts per strain as follows- HD100 n = 1002; ΔBd0875 n = 1014; ΔBd0875:Bd0875 n = 1187; ΔBd0875:Bd0875 D66A n = 1074; ΔBd0875:Bd0875 E213A n = 1073 and ΔBd0875:Bd0875 Y349A n = 1141. and shown in Supplementary Tables 5h-j where the significance of other comparisons can be viewed. Source data are provided in the Source data file.