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. 2024 Sep 4;3(3):403-416.
doi: 10.1002/mlf2.12138. eCollection 2024 Sep.

Catalytically active prokaryotic Argonautes employ phospholipase D family proteins to strengthen immunity against different genetic invaders

Feiyue Cheng  1 , Aici Wu  1   2 , Zhihua Li  1   2 , Jing Xu  1   2 , Xifeng Cao  1   2 , Haiying Yu  3 , Zhenquan Liu  2   3 , Rui Wang  1 , Wenyuan Han  4 , Hua Xiang  2   3 , Ming Li  1   2
Affiliations

Catalytically active prokaryotic Argonautes employ phospholipase D family proteins to strengthen immunity against different genetic invaders

Feiyue Cheng et al. mLife. .

Abstract

Prokaryotic Argonautes (pAgos) provide bacteria and archaea with immunity against plasmids and viruses. Catalytically active pAgos utilize short oligonucleotides as guides to directly cleave foreign nucleic acids, while inactive pAgos lacking catalytic residues employ auxiliary effectors, such as nonspecific nucleases, to trigger abortive infection upon detection of foreign nucleic acids. Here, we report a unique group of catalytically active pAgo proteins that frequently associate with a phospholipase D (PLD) family protein. We demonstrate that this particular system employs the catalytic center of the associated PLD protein rather than that of pAgo to restrict plasmid DNA, while interestingly, its immunity against a single-stranded DNA virus relies on the pAgo catalytic center and is enhanced by the PLD protein. We also find that this system selectively suppresses viral DNA propagation without inducing noticeable abortive infection outcomes. Moreover, the pAgo protein alone enhances gene editing, which is unexpectedly inhibited by the PLD protein. Our data highlight the ability of catalytically active pAgo proteins to employ auxiliary proteins to strengthen the targeted eradication of different genetic invaders and underline the trend of PLD nucleases to participate in host immunity.

Keywords: Argonaute; DNA interference; PLD protein; genome editing; phage defense.

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Conflict of interest statement

Ming Li, Feiyue Cheng, Aici Wu, and Zhihua Li have filed a related patent.

Figures

Figure 1
Figure 1
Haloarchaeal pAgo proteins associate with a phospholipase D (PLD) family protein. (A) Maximum likelihood phylogeny of all identified pAgos in available haloarchaeal genomes. The pAgos that associate with an AgaP are shaded in blue. (B) Representative ago‐agaP operons, with the size (bp, base pair) of each intergenic region indicated. (C) The ago‐agaP operon in Natrinema pellirubrum DSM 15624, with genes encoding a hypothetical protein shown in white. The primers used for reverse‐transcription‐PCR (RT‐PCR) are indicated. (D) Profiling the transcription pattern of the ago‐agaP operon using RT‐PCR. Genomic DNA, untreated total RNA (with DNA contaminants), and DNA‐digested total RNA from N. pellirubrum cells were separately used as templates for control. cDNA was generated by reverse transcription of the DNA‐free RNA sample using random primers (see Materials and Methods). The primers used for each assay are indicated below the corresponding gel. M, dsDNA size marker.
Figure 2
Figure 2
AgaP exhibits toxicity in the absence of its associated pAgo and features a catalytic motif. (A) Transformation of Haloarcula hispanica cells with a plasmid carrying ago (pA), agaP (pP), or both genes (pAP). (B) The resulting colonies formed by H. hispanica transformants on a selective medium. (C) Mutational analysis performed on AgaP. Vector, the empty pWL502. WT denotes a wild‐type AgaP. The mutated residues are indicated in panel D. Data are presented as mean value ± SD (n = 3). (D) Multiple sequence alignment using N. pellirubrum AgaP (Np_AgaP) and four well‐characterized PLD proteins. The numbers indicate the position of the residues subjected to mutation analysis, with the critical catalytic residues highlighted in red. (E) Prediction of the structure of AgaP and its comparison to Nuc. The structure of Agap was predicted using Nuc (PDB: 1BYR) as a model. The predicted catalytic residues of AgaP (highlighted in orange) align with those of Nuc (in red). The images were visualized using PyMOL. CFU, colony‐forming unit.
Figure 3
Figure 3
AgaP is indispensable for the immunity against plasmid DNA. (A) Schematic representation of the H. hispanica mutants. The ago gene or the ago‐agaP operon from N. pellirubrum was integrated into H. hispanica chromosome at the genomic location of pyrF (HAH_2085), which had been deleted to create an auxotrophic H. hispanica strain. (B) Transformation efficiency of H. hispanica cells by pWL502. The photographs display the colonies formed on selective plates. Data are presented as mean value ± SD (n = 3). The p values were obtained from a two‐sided t‐test. (C) Comparison of cell growth between the WT strain and mutant strains on plates. The transformants were diluted and plated on nonselective (AS‐168) and selective (yeast extract‐subtracted AS‐168) medium, respectively. The ago +/agaP M mutant encodes a catalytically dead AgaP, with both H105 and K107 mutated to alanine. The agoM/agaP+ variant encodes a catalytic mutant Ago, with D630 mutated to alanine.
Figure 4
Figure 4
AgaP enhances the pAgo‐based immunity against virus. (A) Plaque forming unit (PFU) of the same HHPV‐2 dilution on lawns of H. hispanica WT, ago +, ago +/agaP +, or ago +/agaP M cells. Data are presented as mean value ± SD (n = 3). (B) The size of HHPV‐2 plaques formed on different H. hispanica cells. agaP M encodes a catalytically dead AgaP mutant (H105A/K107A). (C) PFU of the same HHPV‐2 dilution on lawns of H. hispanica WT, ago +/agaP +, or ago M/agaP + cells. The HHPV‐2 used in panel A and C were prepared from different batches. Data are presented as mean value ± SD (n = 3). (D) PFU of the same HHPV‐2 dilution on lawns of WT H. hispanica or a derivate encoding a wild‐type N. pellirubrum pAgo (ago +) or its catalytically dead mutant ago M (D630A). Data are presented as mean value ± SD (n = 4). p values were obtained from a two‐sided t‐test. PFU, plaque‐forming unit.
Figure 5
Figure 5
The NpAgo system effectively suppresses HHPV‐2 propagation in H. hispanica cells. (A) Schematic illustration of the experiment procedure. Cultures were serially sampled every 12 h to monitor cell density (panel B), after which the samples were centrifuged, and the supernatants were collected for plaque assays to determine the titer of free virus particles (panel C). The precipitated cells collected at indicated time points were subjected to total DNA extraction and Illumina sequencing to determine the ratio of intracellular viral DNA (panel D). (B) The growth curve of WT or ago +/agaP + H. hispanica cells infected (V+) or uninfected (V−) by HHPV‐2. (C) The titer of free virus particles released from H. hispanica cells during the growth curve. Data are presented as mean value ± SD (n = 3). (D) The ratio of intracellular viral DNA to total DNA in infected WT or ago +/agaP + cells.
Figure 6
Figure 6
NpAgo alone enhances gene editing in Escherichia coli. (A) Schematic representation of the experimental design for testing NpAgo‐assisted gene editing in E. coli. E. coli MG1655 was modified by inserting a promoter‐lacking kanR gene and a gfp gene into chromosome. The editing plasmid pE contains a truncated kanR (kanR‐t) with a constitutive promoter, the λ‐Red system, under the control of an l‐arabinose‐inducible P BAD promoter, and homology arms. Recombination between chromosomal DNA and the editing plasmid results in edited cells that are able to grow and form colonies on selective media due to the introduction of the constitutive promoter to drive the transcription of kanR. The target site is indicated by a red line. (B) Transformation of modified E. coli MG1655 cells with editing plasmid and DNA guides. E. coli cells expressing only NpAgo or both NpAgo and AgaP were transformed with pE and DNA guides. The expression of Ago (and AgaP) was induced by l‐arabinose. (C) The number of E. coli cells transformed by pE with or without DNA guides and survived on a kanamycin‐containing medium. FW, forward guide; RV, reverse guide. Data are presented as mean value ± SD (n = 4). (D) Evaluation of the effect of NpAgo on homologous recombination. E. coli cells transformed with pE and the ago‐expressing plasmid serially cultivated. l‐Arabinose was added to the medium to induce the expression of λ‐Red and NpAgo (or NpAgo and AgaP). Cultures were plated onto selective or nonselective media after serial dilution, and the ratios of survivors on selective plates to those on nonselective plates were calculated and plotted based on three independent biological replicates. Data are presented as mean value ± SD (n = 3). p values were obtained from two‐tailed Student's t‐tests.
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