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Lipopolysaccharide transport regulates bacterial sensitivity to a cell wall-degrading intermicrobial toxin [1]

['Kristine L. Trotta', 'Department Of Biochemistry', 'Biophysics', 'University Of California San Francisco', 'San Francisco', 'California', 'United States Of America', 'Beth M. Hayes', 'Johannes P. Schneider', 'Biozentrum']

Date: 2023-07

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.

Bacteria live alongside other microbes that they must compete against for space and resources. To fight rivals, bacteria use a suite of toxin arsenals that are simultaneously potent and specific. Here, we investigate how bacterial competition toxins can selectively target certain bacterial species over others. Our study takes a unique genetic approach to investigate how bacteria are susceptible to attack by the opportunistic human pathogen Pseudomonas aeruginosa and its toxin that destroys the protective cell wall layer. Key to our approach was a genetic screening strategy that systematically tested essential genes, which are otherwise challenging to perturb and test in living bacteria. We were surprised to find that essential genes related to lipopolysaccharide, a bacterial surface layer distinct from the cell wall, were involved in regulating survival against the cell wall toxin. Our findings suggest that disparate cellular components may be more functionally intertwined than previously understood. We also discovered that slowed cellular growth impacts the protective strategies triggered by toxin attack, pointing to systematic behaviors that could influence competition outcomes. Overall, our work provides new insights into the multiple scales of functional specificity that underscore how bacteria cope with external attacks.

Funding: Work performed in the Chou Lab was funded by: NIH NIAID award T32AI060535 (KLT), a UCSF Moritz-Heyman Discovery Fellowship (KLT), the Pew Biomedical Scholars Program (SC), and the Chan-Zuckerberg Biohub (SC). Work performed in the Basler lab was supported by the Swiss National Science Foundation (grant BSSGI0_155778 to JPS, JW, MB) and the European Research Council consolidator grant (865105 - “AimingT6SS” to JPS, JW, MB). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability: Sequencing datasets from this study are accessible at the NCBI Sequence Read Archive under accession PRJNA917770. All other relevant data are within the manuscript and its Supporting Information files.

Copyright: © 2023 Trotta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

The cell wall is a complex and dynamic substrate that is actively regulated to protect the cell [ 27 – 32 ], yet Eco is highly susceptible to lysis by Tae1 in vivo. We therefore hypothesized that Tae1 activity promotes H1-T6SS-mediated lysis in Eco through a unique strategy to overcome neutralization by the recipient cell. In this study, we investigated the Eco cellular features that drive its intrinsic sensitivity to H1-T6SS and the Tae1 toxin during interbacterial competition with Pae. Many T6SS effectors target essential cell features, so we screened the entire complement of essential Eco genes (plus some conditionally essential PG genes) for Tae1 susceptibility determinants. This approach complements previous genetic screens for T6SS recipient fitness which focused on nonessential gene candidates [ 33 , 34 ]. While cell wall-related genes indeed impacted Eco susceptibility to Tae1, we also discovered a strong relationship between survival and another component of the cell envelope, lipopolysaccharide (LPS). Perturbation of LPS synthesis genes msbA and lpxK rendered Eco conditionally resistant to lysis by Tae1 from Pae. Our work revealed that LPS-related resistance was mediated through cell-biological processes that were independent of the biochemical Tae1–PG interaction. Our findings suggest that beyond biochemical specificity and adaptive stress responses lies a role for essential homeostatic processes in defining T6SS effector toxicity in vivo.

However, biochemical specificity is not sufficient to explain the toxicity and organismal selectivity of T6SS effectors in vivo. Bacteria antagonized by T6SSs (‘recipients’) can actively regulate effector toxicity through adaptive stress responses. Eco upregulates its envelope stress responses Rcs and BaeSR after exposure to the Vibrio cholerae (V52) T6SS effectors TseH (a PG hydrolase)[ 21 ] and TseL (a lipase)[ 22 ], suggesting that Eco could counter cell envelope damage by re-enforcing its surface [ 23 ]. Similarly, Bacillus subtilis triggers protective sporulation in response to a Pseudomonas chlororaphis (PCL1606) T6SS effector, Tse1 (a muramidase)[ 24 ]. Additional recipient-cell coordinators of T6SS effector toxicity include reactive oxygen species [ 25 ] and glucose-dependent gene expression [ 26 ]. These studies demonstrate that T6SS effector toxicity in vivo may also depend on downstream adaptive features of recipient cells.

Key to Pae H1-T6SS toxicity are its seven known effectors, each with a unique biochemical activity [ 6 , 11 – 15 ]. The T6S amidase effector 1 (Tae1) from Pae plays a dominant role in H1-T6SS-dependent killing of Eco by degrading peptidoglycan (PG), a structural component of the cell wall that is critical for managing cell shape and turgor [ 16 , 17 ]. Early efforts to understand Tae1 toxicity focused on its in vitro biochemical activity against PG, which offered key insights about how H1-T6SS targets select bacterial species. Tae1 specifically digests γ-D-glutamyl-meso-2,6-diaminopimelic acid (d-Glu-mDAP) peptide bonds, which are commonly found in PG from Gram-negative bacteria [ 8 , 18 ]. Tae1 toxicity is further restricted to non-kin cells through a Pae cognate immunity protein, T6S amidase immunity protein 1 (Tai1), which binds and inhibits Tae1 in kin cells [ 11 , 19 , 20 ].

Many bacteria live in mixed-species microbial communities where they compete with each other for limited space and resources [ 1 ]. Intermicrobial competition is mediated by a diverse array of molecular strategies that can exclude or directly interfere with other microbes, both near and far [ 2 ]. Nearly 25% of Gram-negative bacteria encode a type VI secretion system (T6SS) [ 3 ], which antagonizes neighboring cells by injection of toxic protein effectors into a recipient cell [ 4 – 6 ]. The opportunistic human pathogen Pseudomonas aeruginosa (Pae) harbors an interbacterial T6SS (H1-T6SS) [ 7 ] that can kill the model bacterium Escherichia coli (Eco) [ 8 – 10 ]. Studies of H1-T6SS-mediated competition between these genetically tractable species have provided fundamental insights into the molecular basis of T6SS function and regulation.

Results

Adaptation of native T6SS competitions to study Eco susceptibility to Tae1 We developed an in vivo screen for genetic interactions between the cell wall-degrading H1-T6SS effector Tae1 from Pae and the model target bacterium Eco. Our screen had two fundamental design requirements: (1) the ability to distinguish between general (T6SS-dependent) and specific (Tae1-dependent) genetic interactions, and (2) the capacity to test a broad array of target cell features. We adapted an established interbacterial competition co-culture assay between H1-T6SS-active Pae and Eco, the outcome of which is sensitive to the specific contribution of Tae1[8]. In this assay Eco exhibits a greater fitness advantage when competed against Pae missing tae1 (PaeΔtae1) relative to an equivalent control strain (PaeWT) (Fig 1A). We hypothesized that the Pae:Eco co-culture assay could be leveraged to quantitatively compare recipient cell fitness against both Tae1 (toxin-specific fitness) and the H1-T6SS (Tae1-independent fitness) in interbacterial competition. PPT PowerPoint slide

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TIFF original image Download: 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 log 2 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. https://doi.org/10.1371/journal.ppat.1011454.g001 To screen broadly for Eco determinants, we adopted an established Eco CRISPR interference (CRISPRi) platform that generates hypomorphic mutants through intermediate gene expression knockdowns (KDs)[35]. In contrast to knock-outs or transposon mutagenesis studies, CRISPRi is systematically amenable to essential genes and thus provided an opportunity to make unique insights about genes that are typically challenging to screen for. This includes many essential (or conditionally essential) genes related to peptidoglycan (PG) metabolism, whose KDs we predicted would impact Tae1 toxicity. In this CRISPRi system, inducible sgRNA expression is coupled with constitutive dCas9 expression to conditionally repress transcription at specific loci with and without induction (“induced” and “basal” CRISPRi, respectively) (S1A Fig). In total, our CRISPRi collection was composed of 596 Eco strains with KDs representing most cellular functions as defined by the NCBI clusters of orthologous genes (COG) system (S1B Fig). Our collection also included 50 negative control strains with non-targeting sgRNAs, including rfp-KD, to ensure CRISPRi alone did not impact inherent Eco susceptibility to Pae (S2A Fig). For the interbacterial competition screen, we co-cultured Pae with the pooled Eco CRISPRi collection to test competitive fitness across all KD strains in parallel (Fig 1B). To compare Tae1-dependent and -independent fitness determinants, we conducted screens against H1-T6SS-active Pae strains that either secrete Tae1 (PaeWT; ΔretSΔpppA) or are Tae1-deficient (PaeΔtae1; ΔretSΔpppAΔtae1). As negative controls, we also competed the Eco collection against a genetically H1-T6SS-inactivated Pae strain (Paeinactive; ΔretSΔpppAΔicmF) and included a condition in which the collection was grown without Pae present (Ecoctrl). Experiments were performed under both induced and basal CRISPRi conditions to distinguish between general Eco fitness changes and those due to transcriptional knockdown. We used high-throughput sequencing to quantify KD strain abundance at the beginning and end of each six-hour competition. To understand the contribution of each KD to Eco survival against Pae in the presence or absence of H1-T6SS or Tae1, we calculated log 2 fold-change (L2FC) values for each KD strain after competition and normalized against abundance after growth without competition (Ecoctrl)[36,37]. Across four biological replicates per condition, L2FC values were reproducible (S3A Fig; median Pearson’s r between all replicates = 0.91). L2FC was used as a proxy for competitive fitness of KD strains across different competition conditions. To determine if our screen was sensitive to the effects of Tae1, H1-T6SS, and CRISPRi, we conducted a principal component analysis of L2FC values for each strain under every competition condition (Fig 1C). We observed clear separation of datasets by CRISPRi induction (induced versus basal) across the first principal component (PC1; 85.41%), indicating that KD induction was a major contributor to the performance of the KD library in the pooled screen. We also observed clustering of datasets according to Pae competitor (PC2; 2.66%). These results indicate that each Pae competitor yielded a distinct effect on the fitness of the CRISPRi library and demonstrates that our screen was sensitive to the presence (PaeWT) or absence (PaeΔtae1) of Tae1 delivery from H1-T6SS. From these data we conclude that our screen successfully captured the unique impacts of CRISPRi, Tae1, and H1-T6SS on pooled Eco CRISPRi libraries during interbacterial competition.

msbA-KD disrupts LPS biosynthesis and imparts Tae1 resistance To investigate the hypothesis that non-PG components of the cell envelope may also shape Tae1 toxicity, we focused downstream studies on Tae1-resistant KDs related to the synthesis of LPS, an essential lipidated surface sugar that offers protection and structure to the OM [40]. Candidate KDs targeted highly-conserved, essential genes in Kdo 2 -Lipid A synthesis and transport (lpxA, lpxK, kdsB, waaA, msbA) (Fig 3A). Kdo 2 -Lipid A synthesis is the most-upstream arm of LPS biosynthesis with rate-limiting control over the entire pathway [41,42]. In our screen, the strongest resistance phenotypes we observed were in KDs targeting lpxK (lpxK_-1as and lpxK_32as) (Table 1). LpxK is a kinase that phosphorylates the Lipid-A intermediate tetraacyldisaccharide 1-phosphate to form Lipid IV A [43,44]. In Eco, lpxK is in an operon with msbA (Fig 3B), which encodes the IM Kdo 2 -Lipid A flippase MsbA [45,46]. A KD of msbA (msbA_40as) also conferred resistance to PaeWT in our screen (Table 1). We first experimentally validated pooled screen results by individually testing lpxK-KD and msbA-KD fitness in binary competitions against Pae. We regenerated and validated KD strains for lpxK (lpxK_-1as; “lpxK-KD”) and msbA (“msbA-KD”) for use in these experiments (S6 Fig). Consistent with our screen, msbA-KD gained Tae1-specific resistance in H1-T6SS-mediated competitions (Fig 3D), exhibiting loss of sensitivity to PaeWT relative to PaeΔtae1. In contrast, we could not validate Tae1 resistance for lpxK-KD (Fig 3E). Like rfp-KD (Fig 3C), lpxK-KD maintains sensitivity to PaeWT relative to PaeΔtae1. The gene expression of msbA and lpxK are co-dependent, so we were surprised that msbA-KD and lpxK-KD did not equally reproduce Tae1 resistance. However, CRISPRi-dependent phenotypes could be controlled by factors such as transcriptional polar effects or off-target CRISPRi effects. To address their phenotypic disparities, we quantified transcriptional KD efficacy and specificity for lpxK-KD and msbA-KD with qRT-PCR. For msbA-KD with CRISPRi induced, we found repression of msbA (29-fold), lpxK (15-fold), and ycaQ (3.6-fold) expression (S6A Fig). Thus, owing to downstream polar effects, our msbA-KD strain is a KD of both LPS candidate genes, msbA and lpxK. Conversely, lpxK-KD only repressed lpxK (71-fold) and ycaQ (11-fold) (S6B Fig), but not msbA. Therefore, msbA-KD and lpxK-KD yield distinct transcriptional consequences despite targeting the same operon using CRISPRi. Next, we investigated phenotypic consequences of inducing CRISPRi in msbA-KD and lpxK-KD by comparing their cellular morphologies with cryo-electron tomography. Disruption of msbA and lpxK typically leads to structural deformation in the Eco cell envelope from aberrant accumulation of Kdo 2 -Lipid A intermediates in the IM [44,46,47]. Unlike rfp-KD negative control cells (Fig 3E), msbA-KD cells developed irregular buckling in the IM and OM (Fig 3F, red arrows). We also observed vesicular or tubular membrane structures within the cytoplasm (Fig 3F, blue arrows). Such structural abnormalities are consistent with physical crowding of Kdo 2 -Lipid A intermediates in the IM that are relieved by vesicular internalization. On the other hand, while lpxK-KD had a distended IM and vesicles (Fig 3G red and blue arrows), the OM appeared smooth and regular. This phenotypic divergence points to two distinct KD effects: defects in the IM (both msbA-KD and lpxK-KD) and defects in the OM (msbA-KD only). Together with our transcriptional analyses, these results demonstrate that msbA-KD and lpxK-KD have unique consequences for LPS integrity and Tae1 susceptibility despite targeting the same operon. We focused the remainder of our study on the validated msbA-KD strain which damages the IM and OM.

PG synthesis is suppressed in msbA-KD but sensitive to Tae1 activity Given that we did not find any effects on direct Tae1–cell wall interactions in msbA-KD, we next explored indirect resistance mechanisms. The PG sacculus is dynamically synthesized, edited, and recycled in vivo to maintain mechanical support to the cell during growth and stress [27,50]. We hypothesized that Tae1 hydrolysis could also impact PG synthesis activity in Eco by generating a need to replace damaged PG with new substrate. The ability to repair PG could thus be a valuable determinant of Tae1 susceptibility. To determine if PG synthesis is sensitive to Tae1 exposure, we measured the incorporation of the fluorescent D-amino acid HADA into rfp-KD cell walls both with and without exogenous Tae1 expression. When normalized against control cells (empty), PG synthesis in rfp-KD cells increased by 22% in response to Tae1WT and decreased by 6.5% in response to Tae1C30A (Fig 5A and Table 2). These data show that PG synthesis is stimulated by Tae1 exposure, and this response is dependent on toxin activity. PPT PowerPoint slide

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TIFF original image Download: Table 2. PG synthesis activity is sensitive to CRISPRi and Tae1 overexpression. Descriptive statistics for normalized percent change in HADA fluorescence in rfp-KD and msbA-KD as related to Figs 5 and S10. Data shown: average of 600 single-cell measurements ±s.d. https://doi.org/10.1371/journal.ppat.1011454.t002 PG synthesis is also coordinated to other essential processes in Eco, and sensitive to their genetic or chemical perturbations [31,51]. We investigated if msbA-KD impacts the dynamic PG synthesis response to Tae1. Tae1WT exposure yielded a 26.5% increase in PG activity in msbA-KD, and no significant change in activity with Tae1C30A (Fig 5B and Table 2). These results indicate that PG synthesis is still actively regulated in msbA-KD in accordance with relative Tae1 activity. However, when normalized against baseline rfp-KD activity, all PG synthesis measurements for msbA-KD were significantly diminished (S10 Fig and Table 2). This observation indicates that PG synthesis activity is globally suppressed as a consequence of CRISPRi in msbA. Thus, we conclude that PG dynamism in Eco is sensitive to Tae1 hydrolysis of PG, and that msbA-KD alters the global capacity for PG synthesis activity without altering its sensitivity to Tae1. Furthermore, these data suggest a reactive crosstalk between LPS and PG synthesis activities in vivo.

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1011454

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