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. 2023 Jun 26;19(6):e1011454.
doi: 10.1371/journal.ppat.1011454. eCollection 2023 Jun.

Lipopolysaccharide transport regulates bacterial sensitivity to a cell wall-degrading intermicrobial toxin

Kristine L Trotta  1 , Beth M Hayes  1 , Johannes P Schneider  2 , Jing Wang  2 , Horia Todor  3 , Patrick Rockefeller Grimes  1 , Ziyi Zhao  1 , William L Hatleberg  4 , Melanie R Silvis  3 , Rachel Kim  1 , Byoung Mo Koo  3 , Marek Basler  2 , Seemay Chou  1
Affiliations

Lipopolysaccharide transport regulates bacterial sensitivity to a cell wall-degrading intermicrobial toxin

Kristine L Trotta et al. PLoS Pathog. .

Abstract

Gram-negative bacteria can antagonize neighboring microbes using a type VI secretion system (T6SS) to deliver toxins that target different essential cellular features. Despite the conserved nature of these targets, T6SS potency can vary across recipient species. To understand the functional basis of intrinsic T6SS susceptibility, we screened for essential Escherichia coli (Eco) genes that affect its survival when antagonized by a cell wall-degrading T6SS toxin from Pseudomonas aeruginosa, Tae1. We revealed genes associated with both the cell wall and a separate layer of the cell envelope, lipopolysaccharide, that modulate Tae1 toxicity in vivo. Disruption of genes in early lipopolysaccharide biosynthesis provided Eco with novel resistance to Tae1, despite significant cell wall degradation. These data suggest that Tae1 toxicity is determined not only by direct substrate damage, but also by indirect cell envelope homeostasis activities. We also found that Tae1-resistant Eco exhibited reduced cell wall synthesis and overall slowed growth, suggesting that reactive cell envelope maintenance pathways could promote, not prevent, self-lysis. Together, our study reveals the complex functional underpinnings of susceptibility to Tae1 and T6SS which regulate the impact of toxin-substrate interactions in vivo.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Adaptation of native T6SS competitions to study Eco susceptibility to Tae1.
a) Tae1 from Pseudomonas aeruginosa (Pae) degrades the Escherichia coli (Eco) cell wall to promote H1-T6SS-mediated lysis. Left: PaeWT (dark grey) outcompetes Eco (light grey) using H1-T6SS to deliver a cocktail of toxic effectors, including Tae1 (triangle) which degrades peptidoglycan (orange). Center: Tae1 hydrolyzes d-Glu-mDAP peptide bonds in the donor stem peptides of 4,3-crosslinked peptidoglycan. Right: Pae Δtae1 is less effective at outcompeting Eco using H1-T6SS. b) Method for a genetic screen to test Eco gene function toward fitness against Tae1 from Pae. Left: Pae strains (dark grey) were engineered with modified H1-T6SS activities including: constitutively active PaeWT (ΔretSΔpppA), Tae1-deficient Pae Δtae1(ΔretSΔpppAΔtae1), and T6SS-inactive Paeinactive (ΔretSΔpppAΔicmF). Each Pae strain was mixed with a pool of Eco KD (knockdown) strains engineered to conditionally disrupt a single gene (CRISPRi induced vs. basal). Center: each Pae strain was cocultured with an Eco CRISPRi strain pool for 6 hours. The Eco CRISPRi strain pool was also grown for 6 hours without Pae (Ecoctrl)as a negative control. Genomic sgRNA sequences harvested from competitions were amplified into Illumina sequencing libraries. Right: sgRNA barcode abundances after 6 hours were used to calculate a normalized log2 fold-change (L2FC) for each Eco KD strain under each condition. Above a -log10 p-value cutoff, a positive L2FC value indicates a KD strain which is resistant to a given condition relative to WT Eco; a negative L2FC value indicates a KD strain which is sensitive to a given condition relative to WT Eco. c) Interbacterial competition and CRISPRi induction have distinct effects on the composition of the Eco CRISPRi strain library. Principal component analysis of Eco library composition after competition against PaeWT (blue), Pae Δtae1 (purple), or Paeinactive (green), with induced (solid circles) or basal (hollow circles) CRISPRi induction. Four biological replicates per condition.
Fig 2
Fig 2. CRISPRi reveals toxin-specific and non-specific determinants of Eco fitness against H1-T6SS.
a-b) CRISPRi knockdowns promote Eco survival against PaeWT (a) and PaeΔtae1(b). Volcano plots showing log2-fold change (L2FC) values for each KD strain after interbacterial competition (induced CRISPRi). Data shown: mean from four biological replicates. Statistical test: Wald test. Vertical dotted lines indicate arbitrary cutoffs for L2FC at x = -1.58 and x = 1.58 (absolute FC x = -3 or x = 3). Horizontal dotted line indicates statistical significance cutoff for log10 adjusted p-value (≤ 0.05). Red points represent KDs with L2FC ≥ 1.58 or ≤ -1.58 and log10-adj. ≤0.05. Dark purple points represent non-targeting negative control KDs (n = 50). Lavender points represent KDs that do not meet cutoffs for L2FC or statistical test. c) T6SS competitions identify CRISPRi strains with distinct fitness changes against T6SS and Tae1. Venn diagram of total KDs significantly enriched OR depleted after competition against PaeWT (n = 23), PaeΔtae1(n = 17), and Paeinactive(n = 5).
Fig 3
Fig 3. msbA-KD disrupts LPS biosynthesis and imparts Tae1 resistance.
a) Tae1 resistance emerges in KDs that target the lipopolysaccharide (LPS) biosynthesis pathway. Schematic representation of the LPS biosynthesis pathway and its distribution across the Eco cell envelope. Genes with KDs that render Eco resistant to PaeWT are involved in the biosynthesis of Kdo2-Lipid A (lpxA, lpxK, kdsA, waaA, msbA). Note that Tae1 (grey triangle) targets peptidoglycan (PG), which is physically separate from Kdo2-Lipid A synthesis in the IM. b) The Kdo2-Lipid A biogenesis genes msbA and lpxK are integral members of the ycaI-msbA-lpxK-ycaQ operon in Eco. msbA (red) and lpxK (purple) are co-expressed at the transcriptional level. c-e) msbA-KD loses sensitivity to Tae1 in interbacterial competition against Pae but lpxK-KD does not. Interbacterial competitions between Pae (PaeWT, PaeΔtae1, Paeinactive) and rfp-KD (c; grey), msbA-KD (d; red), or lpxK-KD (e; purple). Data shown are average fold-change in Eco colony forming units (CFUs) after 6 hours of competition (geometric mean 3 biological replicates ± s.d). Statistical test: unpaired two-tailed t-test; p-value ≤0.05 displayed in bold font. f-h) Kdo2-Lipid A mutants develop structural damage to membranes. Cryo-EM tomographs of rfp-KD (f), msbA-KD (g), and lpxK-KD (h) with CRISPRi induced, highlighting cross-sections of the cell envelope (including IM and OM; black arrows). Deformed membranes (red arrows) and novel intracellular vesicles (blue arrows) are demarcated in (g) and (h). Scale bar: 100nm.
Fig 4
Fig 4. Resistance to Tae1 in msbA-KD is independent of cell wall hydrolysis.
a-c) msbA-KD populations have a Tae1-dependent growth advantage. OD600 growth curves of msbA-KD (red) and rfp-KD (black) with CRISPRi induced, overexpressing (a)pBAD24::pelB-tae1WT (Tae1WT), (b) pBAD24::pelB-tae1C30A (Tae1C30A), or (c) pBAD24 (empty). Data shown: average of 3 biological replicates ± s.d. Dotted vertical line indicates plasmid induction timepoint (at OD600 = 0.25). d) The muropeptide composition of msbA-KD PG is identical to control rfp-KD. HPLC chromatograms of muropeptides purified from msbA-KD (red) and rfp-KD (grey) expressing pBAD24 (empty). Inset: major muropeptide species in Eco include tetrapeptide monomers (M4; r.t. ~10 minutes) and 4,3-crosslinked tetra-tetra dimers (D44; r.t. ~15.5 minutes). Tae1 digests D44 peptides (black arrow). Data shown: representative from 3 biological replicates. e) Tae1WT digests PG from both msbA-KD and rfp-KD PG in vivo. HPLC chromatograms of muropeptides purified from msbA-KD (red) and rfp-KD (grey) expressing pBAD24::pelB-tae1WT (Tae1WT). Black arrow indicates D44 peptide partially digested by Tae1. Data shown: representative from 3 biological replicates. f) Tae1 is equally efficient at digesting PG in msbA-KD and rfp-KD. Percent loss of D44 peptide after 60 minutes of periplasmic Tae1WT or Tae1C30A expression. Data shown: average of 3 biological replicates (± s.d.). Statistical test: two-tailed unpaired t-test; p-value ≤0.05 displayed in bold font.
Fig 5
Fig 5. PG synthesis is suppressed in msbA-KD but sensitive to Tae1 activity.
a-b) PG synthesis activity is sensitive to Tae1 overexpression. Single-cell fluorescence intensity measurements for rfp-KD (a; grey) or msbA-KD (b; red) after incorporating the fluorescent d-amino acid HADA into PG after 60 minutes of overexpressing pBAD24::pelB-tae1WT (Tae1WT), pBAD24::pelB-tae1C30A (Tae1C30A), or pBAD24 (empty), with CRISPRi induced. Data shown: 600 cells (200 cells x 3 biological replicates), with average ± s.d. Statistical test: unpaired two-tailed t-test; p-value ≤0.05 displayed in bold font.
Fig 6
Fig 6. Blocks to growth and protein synthesis accompany Tae1 resistance in msbA-KD.
a) msbA-KD cells resist lysis from PaeWT while growing slowly without dividing. Representative frames from time-course imaging of rfp-KD (left column; grey) and msbA-KD (right column; grey) co-cultured with PaeWT (green), with CRISPRi induced. Green foci in PaeWT indicate accumulations of GFP-labelled ClpV, which signal a H1-T6SS firing event. Red arrow indicates lysed cell. Data shown are merged phase contrast and fluorescence channels. Scale bar: 2μm. b) Protein synthesis activity is attenuated in msbA-KD. Single-cell fluorescence intensity measurements for rfp-KD (grey) or msbA-KD (red) cells after incorporating fluorescently-labelled O-propargyl-puromycin (OPP) into nascent peptides during overexpression of pBAD24::pelB-tae1WT (Tae1WT), pBAD24::pelB-tae1C30A (Tae1C30A), or pBAD24 (empty), with CRISPRi induced. All data normalized to average OPP signal in rfp-KD + empty. Data shown: 100 cells/condition, with average ± s.d. Statistical test: unpaired two-tailed t-test; p-value ≤0.05 displayed in bold font.
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