Diverse NADase effector families mediate interbacterial antagonism via the type VI secretion system

Abstract

The bacterial type VI secretion system (T6SS) mediates antagonistic cell-cell interactions between competing Gram-negative bacteria. In plant-beneficial bacteria, this pathway has been shown to suppress the growth of bacterial pathogens; however, the identification and mode of action of T6SS effector proteins that mediate this protective effect remain poorly defined. Here, we identify two previously uncharacterized effectors required for interbacterial antagonism by the plant commensal bacterium Pseudomonas protegens. Consistent with the established effector-immunity paradigm for antibacterial T6SS substrates, the toxic activities of these effectors are neutralized by adjacently encoded cognate immunity determinants. Although one of these effectors, RhsA, belongs to the family of DNase enzymes, the activity of the other was not apparent from its sequence. To determine the mechanism of toxicity of this latter effector, we determined its 1.3 Å crystal structure in complex with its immunity protein and found that it resembles NAD(P) ؉ -degrading enzymes. In line with this structural similarity, biochemical characterization of this effector, termed Tne2 (Type VI secretion NADase effector family 2), demonstrates that it possesses potent NAD(P) ؉ hydrolase activity. Tne2 is the founding member of a widespread family of interbacterial NADases predicted to transit not only the Gram-negative T6SS but also the Gram-positive type VII secretion system, a pathway recently implicated in interbacterial competition among Firmicutes. Together, this work identifies new T6SS effectors employed by a plant commensal bacterium to antagonize its competitors and broadly implicates NAD(P) ؉ -hydrolyzing enzymes as substrates of interbacterial conflict pathways found in diverse bacterial phyla.

The RES domain toxins of RES‐Xre toxin‐antitoxin modules induce cell stasis by degrading NAD+

“…For example, the M. tuberculosis TNT toxin kills infected macrophages by depleting them for NAD + (Sun et al, 2015), while the P. aeruginosa Tse6 toxin acts bacteriostatically on target cells by degrading NAD + and NADH (Whitney et al, 2015). Another factor with NAD(P) + -hydrolyzing activity is the recently described Tne2 toxin from P. protegens Pf-5, which plays an important role in interbacterial killing by this pathogen (Tang et al, 2018). Interestingly, all these NADases have so-called immunity proteins that bind to and block access of NAD + in a similar way to antitoxins to protect the bacteria from self-poisoning.…”

Expanding the molecular weaponry of bacterial species

“…The finding of T6SS effectors with no observable antibacterial activity has also been documented for Acinetobacter baumannii (9,10). It is possible that the role of these effectors is dependent on environmental conditions, as was determined for Tge2 (8), or that they have eukaryotic targets. Furthermore, these effectors may play other roles (e.g.…”

“…NADase effector families Tne1 and Tne2 mediate interbacterial antagonism. The T6SS of Gram-negative bacteria (solid black arrows) secretes toxic NADase effectors from both Tne families into adjacent bacteria (7,8). Similarly, Gram-positive bacteria encode Tne2 orthologs possessing domains that suggest they are secreted by the T7SS (dashed black line); however, this remains to be experimentally shown (see text for details).…”

“…Despite the structural similarities between Tse6 and Tne2 (7,8), these two effectors also present considerable differences, specifically in their respective activating loops and NAD ϩ -binding pockets. Moreover, the cognate immunity protein for each effector, Tsi6 and Tni2, respectively, present prominent differences in their overall folds.…”

“…In the current work, Tang et al (8) searched for proteins containing PAAR domains in the genome of the plant commensal Pseudomonas protegens and identified two hypothetical T6SS effectors, RhsA (PFL_6096) and Tne2 (PFL_6209). RhsA shared homology with proteins of the rearrangement hotspot family of T6SS effectors, which commonly function as nucleases.…”