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. 2019 Jul 31:10:1718.
doi: 10.3389/fmicb.2019.01718. eCollection 2019.

Delivery of the Pseudomonas aeruginosa Phospholipase Effectors PldA and PldB in a VgrG- and H2-T6SS-Dependent Manner

Sarah Wettstadt  1 , Thomas E Wood  1 , Selina Fecht  1 , Alain Filloux  1
Affiliations

Delivery of the Pseudomonas aeruginosa Phospholipase Effectors PldA and PldB in a VgrG- and H2-T6SS-Dependent Manner

Sarah Wettstadt et al. Front Microbiol. .

Abstract

The bacterial pathogen Pseudomonas aeruginosa uses three type VI secretion systems (T6SSs) to drive a multitude of effector proteins into eukaryotic or prokaryotic target cells. The T6SS is a supramolecular nanomachine, involving a set of 13 core proteins, which resembles the contractile tail of bacteriophages and whose tip is considered as a puncturing device helping to cross membranes. Effectors can attach directly to the T6SS spike which is composed of a VgrG (valine-glycine-rich proteins) trimer, of which P. aeruginosa produces several. We have previously shown that the master regulator RsmA controls the expression of all three T6SS gene clusters (H1-, H2- and H3-T6SS) and a range of remote vgrG and effector genes. We also demonstrated that specific interactions between VgrGs and various T6SS effectors are prerequisite for effector delivery in a process we called "à la carte delivery." Here, we provide an in-depth description on how the two H2-T6SS-dependent effectors PldA and PldB are delivered via their cognate VgrGs, VgrG4b and VgrG5, respectively. We show that specific recognition of the VgrG C terminus is required and effector specificity can be swapped by exchanging these C-terminal domains. Importantly, we established that effector recognition by a cognate VgrG is not always sufficient to achieve successful secretion, but it is crucial to provide effector stability. This study highlights the complexity of effector adaptation to the T6SS nanomachine and shows how the VgrG tip can possibly be manipulated to achieve effector delivery.

Keywords: Pseudomonas aeruginosa; VgrG; bacterial toxin; phospholipase; type VI secretion system.

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Figures

FIGURE 1
FIGURE 1
P. aeruginosa encodes two phospholipases delivered via their cognate VgrGs. (A) vgrG4b (PA3489, green) is encoded directly upstream of pldA (PA3488, bright green). (B) vgrG5 (PA5090, blue) is encoded directly upstream of pldB (PA5089, cyan). Second panel: Representative figures of western blots from secretion assays using PAO1ΔrsmA expressing BlaTEM–1-tagged versions of PldA (A) or PldB (B). The absence (Δ) of the native vgrG4b (A) or vgrG5 (B) are indicated. For complementation experiments of the (A) vgrG4b and (B) vgrG5 mutants, the introduction in trans of (A) vgrG4b (pTrc-vgrG4b) and (B) vgrG5 (pBBR1-mcs5-vgrG5-HA) are marked with (+). Membranes in (A) and (B) were revealed, from top to bottom, using antibodies against BlaTEM–1, the C-terminal extension domain of VgrG4b (αVgrG4bC), the HA-tag, RpoB and Hcp2 as indicated on the right. In the bottom panel, bacterial competition outcomes are shown by plots of recovered cfu of prey strains susceptible to PldA delivery, PAO1ΔrsmAΔpldAtli5a::lacZ (A), or PldB delivery, PAO1ΔrsmAΔpldBtli5b123::lacZ (B), after contact with the attacker strain PAO1ΔrsmA encoding or lacking (Δ) the native vgrG4b (A) or vgrG5 (B). Spots were incubated for 24 h at 25 ∘C in a 1:1 ratio. One-Way ANOVA analysis with Dunnett’s multiple comparisons test was conducted on the data set obtained from recovered prey in the absence of an attacker strain with *⁣*⁣**p < 0.0001.
FIGURE 2
FIGURE 2
VgrG5 secretion and PldB delivery are H2-T6SS dependent. (A) Representative figure of a western blot from a secretion assay using PAO1ΔrsmA. Strains encode native vgrG4b or the vgrG5623-vgrG4b187 chimera (+) as well as an active or inactive (Δ) H2-T6SS (ΔtssB2) or H3-T6SS (ΔtssK3). Antibodies used (from top to bottom) are against VgrG4bC, RpoB and Hcp2 as indicated on the right. A section from a Coomassie gel was included to serve as a loading control (LC) for the supernatant fraction. (B) Representative figure of a western blot from a secretion assay using PAO1ΔrsmA expressing a BlaTEM–1-tagged version of PldB. Strains produce an active or inactive (ΔtssB2) H2-T6SS. Antibodies used (from top to bottom) are against BlaTEM–1, RpoB, Hcp2 and LasB as indicated on the right. (C) Bacterial competition represented by plots of recovered cfu of prey strain PAO1ΔrsmAΔpldBtli5b123::lacZ after contact with the attacker strain that encodes a non-functional H2-T6SS (ΔtssB2) or H3-T6SS (ΔtssK3). As a positive control for PldB-mediated killing, PAO1ΔrsmA was included. Spots were incubated for 24 h at 25 ∘C in a 1:1 ratio. One-Way ANOVA analysis with Dunnett’s multiple comparisons test was conducted on the data set obtained from recovered prey on its own with *⁣*⁣**p < 0.0001.
FIGURE 3
FIGURE 3
The C-terminal domain of a VgrG specifically stabilizes its cognate effector protein. (A) Structural models of the C termini of VgrG4b (green) and VgrG5 (blue) as compared to the transthyretin-like protein from Salmonella dublin (red, pdb: 2gpz). (B) Schematic of the here used chimerae between VgrG5 (blue) and VgrG4b (green). Highlighted are each the gp5-/gp27-like domains of VgrG4b and VgrG5 as well as the C-terminal domain consisting of the TTR-like domains (aa 648 – 770, see Supplementary Figure S3 for more details) as shown with an arc form. (C,D) Mutant strains carry BlaTEM–1-tagged version of PldA (C) or PldB (D) as well as the gene clusters as sketched in the top panels and using the color code as in Figure 1. (C) Strains express one of the following vgrG genes: native vgrG5 (lane 2), native vgrG4b (lane 3), vgrG5623-vgrG4b187 chimera (lane 4) or vgrG4b629-vgrG5169 chimera (lane 5). Representative figures of western blots from whole cell fractions of PAO1ΔrsmA derivates as indicated by (+). (D) vgrG4b629-vgrG5169 chimera replaces the native vgrG4b and the native vgrG5 gene is deleted. Controls for native effector stability are shown with the parental strains in (C) lane 1 and (D) lane 2, while the negative controls lack the cognate vgrG locus in (C) lane 2 or in (D) lane 3. Antibodies used (top to bottom) are against BlaTEM–1, VgrG4bC and RpoB as indicated on the right of each panel.
FIGURE 4
FIGURE 4
Modifying the VgrG C-terminus abrogates delivery of cognate effectors. The vgrG cluster color code is as in Figure 1. The mutant strains used carry the gene sequence for (A) vgrG5623-vgrG4b187 chimera and (B) vgrG4b621-vgrG5169 chimera. Note that mutants in (A) encode the N terminal 623 amino acids of VgrG5 (blue) and the last C-terminal 187 amino acids of VgrG4b (+, green, Supplementary Figure S4B) and mutants in (B) encode the N terminal 621 amino acids of VgrG4b and the last C-terminal 169 amino acids of VgrG5 (+, Supplementary Figure S4C). Bottom panels: bacterial competition experiments and plots of recovered cfu of prey strains as described in Figure 1. Recovery is after contact with the attacker strain that encodes (A) native vgrG5 or the vgrG5623-vgrG4b187 chimera (+) or (B) native vgrG4b or the vgrG4b621-vgrG5169 (+). As a positive control for effector-mediated killing, the parental strains were included (lanes 2), while the negative control lacked the cognate vgrG (lanes 3). Spots were incubated for 24 h at 25 ∘C in a 1:1 ratio. One-Way ANOVA analysis with Dunnett’s multiple comparisons test was conducted on data set obtained from recovered prey on their own with *⁣*⁣**p < 0.0001.
FIGURE 5
FIGURE 5
Substitution of the VgrG5 C-terminus with the VgrG4b C-terminus restores PldA delivery. (A) Schematic depicting a strain carrying the vgrG5623-vgrG4b187 chimeric gene at the native vgrG5. The native vgrG4b is deleted. (B) Representative figure of a western blot from a secretion assay using PAO1ΔrsmA or PAO1ΔrsmA::pldA-blaTEM–1 (+) encoding native vgrG4b or the vgrG5623-vgrG4b187 chimera (+). As a control for PldA-BlaTEM–1 secretion, the parental strain was included (lane 6), while the negative control lacked the native vgrG4b (lane 7). Antibodies used (from top to bottom) are against BlaTEM–1, VgrG4bC, RpoB and Hcp2 as indicated on the right. (C) Bacterial competition represented by plots of recovered cfu of prey strain PAO1ΔrsmAΔpldAtli5a::lacZ after contact with the attacker strain that encodes native vgrG4b or the vgrG5623-vgrG4b187 chimera (+). As a positive control for PldA-mediated killing, PAO1ΔrsmA was included (lane 2), while the negative control lacked vgrG4b (lane 3). Spots were incubated for 24 h at 25 ∘C in a 1:1 ratio. One-Way ANOVA analysis with Dunnett’s multiple comparisons test was conducted on data set obtained from recovered prey on their own with *⁣*⁣**p < 0.0001.
FIGURE 6
FIGURE 6
Production of the VgrG5 N-terminus (VgrG5628) does not support PldB secretion. (A) Schematic depicting a strain carrying the chimeric gene vgrG4b629-vgrG5169 at the native vgrG4b locus and in absence of the native vgrG5 gene. A construct encoding a quadruple HA-tagged version of VgrG5628 is expressed from pBBR1-mcs4 (pBBR4-vgrG5628). (B) Representative figure of a western blot of a secretion assay using PAO1ΔrsmA::pldB-blaTEM–1 in presence of the native vgrG5, encoding the vgrG4b629-vgrG5169 chimera (+) or carrying pBBR4-vgrG5628 (+). Antibodies used (from top to bottom) are against BlaTEM–1, VgrG4bC, HA, RpoB and Hcp2 as indicated on the right. (C) Bacterial competition represented by plot of recovered cfu of prey strain PAO1ΔrsmAΔpldBtli5b3::lacZ after contact with the attacker strain that encoded native vgrG5 or the vgrG4b629-vgrG5169 chimera (+) or pBBR1-mcs4 vgrG5628 (+). As a positive control for PldB-mediated killing, PAO1ΔrsmA was included (lane 2), while the negative control lacked the native vgrG5 locus (lane 3). Spots were incubated for 24 h at 25 ∘C in a 1:1 ratio. One-Way ANOVA analysis with Dunnett’s multiple comparisons test was conducted on data set obtained from recovered prey on their own with *⁣*⁣**p < 0.0001.
FIGURE 7
FIGURE 7
VgrG5 mediates delivery of both PldA and PldB. (A) Plots of recovered cfu of prey strains susceptible for PldA delivery upon bacterial competition assays. Prey strains are as described in Figure 1. PAO1ΔrsmA attacking strains either lacked (Δ) vgrG4b, vgrG5 or both, or expressed vgrG5628 in trans (+). Spots were incubated for 24 h at 25 ∘C in a 1:1 ratio. One-Way ANOVA analysis with Dunnett’s multiple comparisons test was conducted on the data set obtained from recovered prey on their own with *⁣*⁣**p < 0.0001. (B) Representative figure of western blot from a secretion assay using PAO1ΔrsmA. The absence (Δ) of the native vgrG5 or vgrG4b is shown as well as when expressing an HA-tagged version of vgrG5628 in trans (+). Antibodies (from top to bottom) against the C-terminal portion of VgrG4b (VgrG4b), HA, RpoB and Hcp2 were used as indicated on the right.
FIGURE 8
FIGURE 8
Absence of VgrG4b has no impact on PldB delivery. (A) Plots of recovered cfu of prey strains susceptible for PldB delivery upon bacterial competition assays. Prey strains are as described in Figure 1. PAO1ΔrsmA attacking strains either lacked (Δ) vgrG4b, vgrG5 or both. Spots were incubated for 24 h at 25∘C in a 1:1 ratio. One-Way ANOVA analysis with Dunnett’s multiple comparisons test was conducted on the data set obtained from recovered prey on their own with *⁣*⁣**p < 0.0001. (B) Representative figure of a western blot from a secretion assay using PAO1ΔrsmA::pldB-blaTEM–1. Strains lack (Δ) native vgrG4b or vgrG5. The blue arrow on the right indicates the secretion product of PldB-BlaTEM–1, which is not visible when vgrG5 is deleted. Antibodies used (from top to bottom) are against BlaTEM–1, VgrG4bC, RpoB and Hcp2 as indicated on the right.
FIGURE 9
FIGURE 9
VgrG4b can deliver PldA when fused to its C-terminus. (A) The STOP codon of vgrG4b, the intergenic region and START-codon of pldA were deleted resulting in a single chimeric gene product. (B) Representative figure of a western blot from a secretion assay using PAO1ΔrsmA with an active or inactive H2-T6SS (ΔtssB2) encoding VgrG4b-PldA (ΔTAAvgrG4b-ATGpldA). As a positive control for VgrG4b secretion, PAO1ΔrsmA (lane 1) was included, while the negative control lacked vgrG4b (lane 2). Antibodies used (from top to bottom) are against VgrG4bC, RpoB, Hcp2 and LasB, a T2SS substrate (Olson and Ohman, 1992) acting as a loading control for the supernatant, as indicated on the right. (C) Bacterial competition represented by plots of recovered cfu of prey strain PAO1ΔrsmAΔpldAtli5a:: lacZ after contact with the attacker strain encoding WT PldA, WT VgrG4b or the fusion VgrG4b-PldA (ΔTAAvgrG4b-ATGpldA) as indicated. As a positive control for PldA-mediated killing, PAO1ΔrsmA was included (lane 2) while the negative controls lacked pldAtli5a (lane 3) or vgrG4b (lane 4). Spots were incubated for 24 h at 25 ∘C in a 1:1 ratio. One-Way ANOVA analysis with Dunnett’s multiple comparisons test was conducted on the data set obtained from the recovered prey on its own with ∗p < 0.05 and ∗∗p < 0.01.
FIGURE 10
FIGURE 10
Genomic loci containing vgrG or paar genes of P. aeruginosa PAO1. vgrG genes are shown in green, effector genes in red, cognate immunities in cyan, hcp genes in dark gray, paar genes in purple, tap genes in magenta and genes of unknown function in gray. For all examples, the PA numbers of the two outer genes are shown. Effector genes close to their (putative) cognate vgrG or PAAR genes are the following: (A) tse6; (B) tse7; (C) tse5; (D) tle4; (E) tle3; (F) tseF; (G) tle1; (H) pldA; (I) pldB; (J) PA5265; (K) PA0822; (L) tseT.
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