Mechanisms of Pseudomonas aeruginosa resistance to type VI secretion system attacks

Tejada-Arranz, Alejandro; Plack, Annika; Antelo-Varela, Minia; Kaczmarczyk, Andreas; Klotz, Alexander; Jenal, Urs; Basler, Marek

doi:10.1038/s41467-025-65777-x

Download PDF

Article
Open access
Published: 28 November 2025

Mechanisms of Pseudomonas aeruginosa resistance to type VI secretion system attacks

Nature Communications volume 16, Article number: 10744 (2025) Cite this article

4253 Accesses
1 Citations
56 Altmetric
Metrics details

Subjects

Abstract

The Type VI Secretion System (T6SS) is a molecular nanomachine that injects toxic effector proteins into the environment or neighboring cells, playing an important role in interbacterial competition and host antagonism during infection. Pseudomonas aeruginosa encodes three T6SSs. One of them, the H1-T6SS, delivers toxins in response to attacks mediated by the T6SS of aggressor bacteria, suggesting that P. aeruginosa can resist T6SS assaults. The mechanisms of resistance are poorly characterized. Here, we perform a CRISPRi screen to identify pathways involved in resistance to T6SS effectors of Acinetobacter baylyi ADP1 and Vibrio cholerae 2740-80. We show that members of the GacA/GacS regulon, such as the mag operon or aas, and GacA-independent factors, like the outer membrane protein OprF, confer resistance to different types of T6SS toxins. Interestingly, some of these T6SS protection mechanisms lead to higher antibiotic susceptibility, suggesting complex evolutionary links between T6SS and antibiotic resistance.

Distribution of the four type VI secretion systems in Pseudomonas aeruginosa and classification of their core and accessory effectors

Article Open access 21 January 2025

The type VI secretion system and associated effector proteins

Article 14 November 2025

Collective protection against the type VI secretion system in bacteria

Article Open access 24 April 2023

Introduction

P. aeruginosa is a gram-negative, ubiquitous, opportunistic pathogen that can colonize open wounds and the respiratory airways, and is particularly problematic for patients suffering from cystic fibrosis¹. P. aeruginosa possesses three Type VI Secretion Systems (T6SS), molecular nanoweapons that deliver toxic effectors to neighboring cells². The first one, called H1-T6SS, plays a predominantly antibacterial role, while the H2- and H3-T6SSs are important for internalization and colonization of a eukaryotic host, although they also deliver trans-kingdom effectors that target both eukaryotic and bacterial cells^2,3,4,5. The H3-T6SS also delivers effectors that help iron acquisition⁶ and biofilm formation⁷.

Generally, T6SSs are composed of a membrane complex and a baseplate that trigger the assembly of an Hcp tube that is loaded with a tip complex, composed of a VgrG trimer and PAAR proteins, as well as a set of effectors that can be fused to Hcp, VgrG or PAAR or can be loaded separately^8,9. The membrane complex includes TssM and other structural components that are essential for T6SS function, span the inner and outer membranes and anchor the secretion system to the cell envelope⁸. The Hcp tube is surrounded by a sheath that, upon contraction, propels the Hcp tube as well as the tip complex and all loaded effectors to the extracellular space or into a neighboring cell. Bacteria are typically protected from their own effectors by a set of dedicated immunity proteins that are encoded adjacently to the effectors¹⁰. These immunity proteins are highly specific, with few examples of cross-protection to related effectors with similar mechanisms of action¹¹.

Interestingly, the H1-T6SS of P. aeruginosa was reported to respond to incoming attacks from neighboring bacteria, regardless of whether they are sister cells or members of a different species^12,13. Specifically, the H1-T6SS comprises a sensor module, composed of TagQRST, that senses membrane damage and triggers the assembly of a retaliatory H1-T6SS through post-translational modifications¹². Furthermore, it was proposed that this retaliation strategy is a poor evolutionary strategy, as it prevents P. aeruginosa from attacking first and forces it to resist incoming T6SS attacks¹⁴. Here, we hypothesize that P. aeruginosa must be equipped with specific mechanisms that protect against attacks with foreign T6SS effectors, at least until the H1-T6SS is able to strike back.

Acinetobacter baylyi ADP1 and Vibrio cholerae 2740-80 are gram-negative environmental bacteria that each encode one antibacterial T6SS cluster with multiple effectors that are constitutively expressed and thus highly active in vitro. A. baylyi ADP1 WT contains four described effectors¹⁵: Tpe1 (a predicted metallopeptidase without identified antimicrobial activity), Tae1 (a PG-targeting effector), Tse2 (of unknown mechanism of action) and Tle1 (a lipase). V. cholerae 2740-80 WT, a representative strain for AAA effector-type, non-toxigenic environmental V. cholerae¹⁶, also has four effectors¹⁷: TseH and VgrG3 (two PG-targeting effectors), TseL (a lipase) and VasX (a pore-forming toxin). Thus, they have different types of effectors, and the resistance mechanisms of P. aeruginosa against these two organisms might differ.

In this work, we competed H1-T6SS-deficient P. aeruginosa PAO1 carrying a CRISPRi plasmid library targeting all P. aeruginosa genes with T6SS-positive or –negative A. baylyi ADP1 or V. cholerae 2740-80. Deep sequencing of the plasmid library revealed genes required to resist incoming T6SS attacks. Interestingly, most of these genes belong to the regulon of the GacS/GacA two-component system (TCS), including the previously identified arc1 and arc3 clusters and pa3267, aas¹⁸, as well as the mag operon. The outer membrane protein (OMP) OprF also contributes to T6SS resistance. Our data suggest that these pathways are general protection mechanisms against multiple effectors and thus differ from specific immunity proteins. In addition, we identified a link between these protection mechanisms and antibiotic resistance.

Results

P. aeruginosa resists T6SS attacks in the absence of its H1-T6SS

The H1-T6SS of P. aeruginosa can promptly and efficiently retaliate to incoming T6SS attacks, eliminating the competing bacterium before it can efficiently kill P. aeruginosa^12,14,19. Therefore, we constructed a mutant lacking tssM1, a structural membrane component of the H1-T6SS, that is unable to assemble its H1-T6SS and retaliate to incoming attacks. We observed that the H1-T6SS is indeed important to efficiently resist attacks from A. baylyi ADP1 (Fig. 1A and Supplementary Fig. 1A), but not from V. cholerae 2740-80 (Fig. 1B and Supplementary Fig. 1B) in a 10:1 attacker:prey ratio. The H2- and H3-T6SSs of P. aeruginosa play no role in resistance against T6SS attacks (Fig. 1A, B and Supplementary Fig. 1A, B). Under the tested conditions, where P. aeruginosa is outnumbered by the attacking species (10:1 attacker: prey ratio), no significant difference in attacker recovery was observed (Supplementary Fig. 1A, B). Nevertheless, H1-T6SS-dependent killing of A. baylyi and V. cholerae can be observed when competed against P. aeruginosa in a 1:1 ratio or when P. aeruginosa outnumbers the other species in a 10:1 ratio (Supplementary Fig. 1C–F, where P. aeruginosa is the attacker and A. baylyi and V. cholerae the prey).

**Fig. 1: *P. aeruginosa* (Pa) is more resistant to T6SS from *A. baylyi* (Ab) ADP1 and *V. cholerae* (Vc) 2740-80 than *E. coli* (Ec).**

Furthermore, although the P. aeruginosa ΔtssM1 strain is more susceptible than its parental strain to attacks from the T6SS of A. baylyi ADP1, it is still far more resistant than E. coli (Figs. 1A, C and Supplementary Fig. 1A, G), as evidenced by the almost complete elimination of E. coli when competed in a 10:1 attacker: prey ratio and the strong killing observed in a 1:1 ratio, in a manner that is dependent on the presence of the known A. baylyi ADP1 and V. cholerae 2740-80 effectors (Fig. 1C). This suggests that P. aeruginosa possesses protective mechanisms against T6SS attacks.

Identification of T6SS-protective pathways

To identify new T6SS-protective pathways, we introduced the dCas9 protein from Streptococcus pasteurianus into P. aeruginosa ΔtssM1 strain and performed a CRISPRi screen for genes important for T6SS protection using a sgRNA library similarly to others^20,21. Specifically, we competed this T6SS-deficient P. aeruginosa strain carrying the sgRNA plasmid library with T6SS-active or inactive mutants (lacking tssM) of A. baylyi ADP1 or V. cholerae 2740-80. We performed two rounds of 3 h long incubations of predator and prey at 1:1 (for A. baylyi) and 10:1 (for V. cholerae) ratios (Fig. 2A). The sgRNA loci of the recovered P. aeruginosa cells were PCR-amplified (before competition and after the first and second rounds of competition), and the amplicons were sequenced by Illumina sequencing. Samples from competition with T6SS-active or inactive strains were compared to identify the pathways that, when knocked down, lead to higher sensitivity to T6SS attacks.

**Fig. 2: CRISPRi screen for *P. aeruginosa* knockdowns that are sensitive to T6SS attacks.**

After one and two rounds of competition, plasmids encoding sgRNAs targeting a number of P. aeruginosa genes were depleted (Fig. 2B, C and Supplementary Fig. 2A, B). The same sgRNAs were identified in both cases, although differences were more pronounced after two rounds of competition. This included known T6SS resistance-related genes such as gacA, gacS, aas, arc1A or arc3A¹⁸ as well as novel genes like the mag operon or oprF.

GacA is a key regulator involved in T6SS resistance

Interestingly, many of the genes that we identified were reported to be under the control of the GacA/GacS TCS, including arc1A, arc3A and aas¹⁸. To determine whether the other genes that we identified are also part of the regulon of the GacA/S TCS, we compared the proteome of a ΔgacA strain to that of its parental strain. We found that the members of the mag operon are also downregulated in the ΔgacA strain (Fig. 2D, and Supplementary Data 1), together with components of the H1- and H2-T6SS, whereas oprF is not differentially expressed in that strain.

Next, we assessed whether a ΔgacA strain was able to resist T6SS attacks from A. baylyi ADP1 and V. cholerae 2740-80. Indeed, the ΔgacA strain was 100-fold more susceptible to T6SS attacks from A. baylyi ADP1 and V. cholerae 2740-80 than a strain lacking the H1-T6SS (Fig. 2E and Supplementary Fig. 2C, D), indicating that GacA-regulated genes other than the H1-T6SS are required for T6SS resistance.

To get more insights into the mechanisms of resistance, we competed the P. aeruginosa ΔgacA strain with A. baylyi strains lacking individual effectors (Fig. 2F and Supplementary Fig. 2E). We previously showed that these A. baylyi strains are able to fire their T6SS as efficiently as the parental strain¹⁵. Interestingly, the ΔgacA strain is killed similarly when single effectors are absent, although Tse2 might play the predominant role, as an A. baylyi strain lacking tse2 kills P. aeruginosa less efficiently (Fig. 2F).

Furthermore, we also competed P. aeruginosa ΔgacA with V. cholerae 2740-80 strains encoding individually inactivated effectors through point mutations that were previously reported^13,22, and we found that the inactivation of each effector leads to similar levels of killing of the ΔgacA strain (Fig. 2G and Supplementary Fig. 2F). Taken together, our results suggest that the GacA regulon contains genes that contribute to the resistance to different effector classes, in addition to lipase effectors as previously reported¹⁸.

The mag operon is important for resistance against PG-targeting effectors

Next, we assessed how individual genes from the GacA regulon contribute to T6SS resistance, and in particular the mag operon that was identified in our CRISPRi screen (Fig. 2B). In P. aeruginosa, the mag operon is composed of six genes, magABCDEF. MagD displays weak homology with type II α2-macroglobulin²³, homologs of eukaryotic α-macroglobulins, whereas the rest of the genes of the operon are important for the folding and stability of MagD²⁴. Eukaryotic α2-macroglobulins are innate immunity proteins that protect against proteases by a trapping mechanism^25,26,27. However, bacterial type II α2-macroglobulins lack key residues that conform the active site of eukaryotic α2-macroglobulins²⁴, and their function is thus unclear.

We constructed a strain lacking both tssM1 and magD, and competed it against different mutants of V. cholerae 2740-80. We performed quantitative killing assays and found that MagD is indeed important for resistance to T6SS attacks and that this phenotype can be complemented (Fig. 3A and Supplementary Fig. 3A). Furthermore, MagD protects mainly against the PG-targeting effector VgrG3 (Fig. 3B and Supplementary Fig. 3B). To confirm this result, we also performed a CPRG-based lysis assay¹⁵. CPRG is a colorimetric substrate that cannot penetrate bacterial membranes and can thus only be converted by the LacZ enzyme upon lysis of LacZ-expressing P. aeruginosa, acting as a proxy for lysis. This assay also indicated a VgrG3-dependent lysis of a ΔmagD mutant (Fig. 3C and Supplementary Fig. 3C).

**Fig. 3: MagD is important for resistance against PG-targeting effectors.**

Additionally, we tested whether magD confers resistance against single effectors of A. baylyi by competition with strains expressing single effectors. We found that magD also confers a limited protection against the PG-targeting Tae1 effector of A. baylyi (Fig. 3D and Supplementary Fig. 3D), and not against other effector classes (Supplementary Fig. 3E, F). Thus, MagD encoded by the mag operon protects against certain PG-targeting effectors from both V. cholerae 2740-80 and A. baylyi ADP1.

Arc1A, Arc3A and Aas contribute to resistance against lipase effectors

Arc1A and Arc3A were previously shown to be important for resistance against the T6SS of B. thailandensis. Specifically, Arc3A protects against lipase effectors, and the effector class that Arc1A protects against was not clearly elucidated¹⁸. PA3267 (Aas) was also previously identified in a transposon screen, but no clear role in T6SS resistance was attributed to it, although it was hypothesized that it is relevant for protection against lipases¹⁸. Our screen identified arc1A, arc3A and aas as important for T6SS resistance. Aas is homologous to the lysophospholipid transporter LplT and the Aas acyltransferase from E. coli, where it was shown to be required for defense against phospholipase attacks²⁸. We found that a P. aeruginosa mutant lacking tssM1 and either arc1A, arc3A or aas is indeed more sensitive to T6SS attacks from V. cholerae 2740-80 WT (Fig. 4A and Supplementary Fig. 3G). The ΔtssM1 Δarc3A displays the strongest defect. A strain lacking all four genes is as sensitive to attacks as the ΔtssM1 Δarc3A strain, suggesting an auxiliary role of arc1A and aas in this process. This sensitivity is reduced when V. cholerae 2740-80 lacks an active TseL effector (Fig. 4A and Supplementary Fig. 3G). This suggests that these genes are important for protection against lipases, and likely also contribute to resistance against other effector classes.

**Fig. 4: Arc1A, Arc3A and Aas are important for resistance to lipase effectors.**

Similarly, P. aeruginosa strains lacking tssM1 and either arc1A or arc3A are also more sensitive than P. aeruginosa ΔtssM1 to attacks from A. baylyi ADP1 that carries only its lipase Tle1 effector (Fig. 4B and Supplementary Fig. 3H). The strain lacking all four genes (tssM1, arc1A, arc3A and aas) was as sensitive to either Tae1 or Tse2 from A. baylyi ADP1 as P. aeruginosa ΔtssM1 (Supplementary Fig. 3I, J). We conclude that arc1A, arc3A and aas are important for resistance to lipase effectors, confirming previous observations about arc3A¹⁸, providing evidence for the previously suggested role of aas¹⁸ and identifying one of the types of effectors that arc1A can protect against.

OprF is required for T6SS resistance and is genetically linked to GacA/S

OprF is the main outer membrane porin of P. aeruginosa, and it has been reported to perform many different roles, including channeling small solutes, maintenance of outer membrane integrity, binding and adhesion, biofilm formation, or outer membrane vesicle biogenesis²⁹. OprF is folded in two conformers: a minor population (around 5%) that is folded into a one-domain structure with a channel that acts as a porin; and a major population (around 95%) that is folded into two domains, including a PG-anchoring domain. It was suggested that this conformation is responsible for anchoring of the OM to the PG^30,31, and that a ΔoprF strain displays defects in the OM^32,33. To understand how sgRNA-mediated inhibition of oprF expression resulted in lower resistance to T6SS attacks, we constructed a P. aeruginosa ΔtssM1 ΔoprF strain and competed it against V. cholerae 2740-80 and A. baylyi ADP1 attackers. We show that when the V. cholerae 2740-80 attacker strain carries a full effector set, the ΔoprF strain is killed (Fig. 5A and Supplementary Fig. 4A). Inactivation of single or multiple effectors indicates that VgrG3 is the main driver of this killing. When A. baylyi ADP1 is used as attacker, P. aeruginosa ΔtssM1 ΔoprF is also more susceptible than its parental strain, independently of the presence of any individual effector, indicating that no single effector type is responsible for the killing of P. aeruginosa lacking oprF (Fig. 5B and Supplementary Fig. 4B). Importantly, the ΔoprF strain displays a growth defect in LB, which is exacerbated in LB without salt (Supplementary Fig. 4C), suggesting that the strain is sensitive to osmotic shock as previously shown³².

**Fig. 5: OprF is important for T6SS resistance.**

Furthermore, complementation in trans with a wild-type copy of oprF in a pPSV35 plasmid fully restores both growth and resistance to T6SS attacks (Fig. 5C and Supplementary Fig. 4D, E). The R296E oprF variant, that was previously shown to be unable to bind PG in E. coli^34,35, and fails to complement morphology defects derived from envelope instability (Supplementary Fig. 4E) restores protection against T6SS attacks to the same extent (Fig. 5C and Supplementary Fig. 4F), suggesting that the contribution of the OM anchoring function of OprF to T6SS resistance may be minimal.

Interestingly, we observed different colony sizes when the P. aeruginosa ΔtssM1 ΔoprF strain was grown in LB without salt, suggesting the appearance of suppressors (Fig. 6A). We isolated four of these clones and analyzed their growth, cell morphology and ability to compete with V. cholerae 2740-80. These suppressors displayed wild-type-like growth on LB without salt (Fig. 6B), as well as wild-type cellular morphology (Fig. 6C), as shown by the absence of cell ghosts that are frequent in the ΔtssM1 ΔoprF strain. However, the suppressors were still susceptible to T6SS attacks from V. cholerae 2740-80 at a 1:1 ratio (Fig. 6D and Supplementary Fig. 4G).

**Fig. 6: Δ*oprF* suppressors grow normally and recover wild-type morphology but fail to resist T6SS attacks.**

We performed whole genome sequencing of these suppressors and found that all of them carry the same inactivating 431 bp deletion within the gacS gene, suggesting that the GacA/S is inactive in these strains. We validated this by deletion of gacA in a ΔoprF strain, which restores the growth defect of this strain in LB without salt (Fig. 6E). Thus, GacA/S is involved in causing the growth and morphology defect of the ΔoprF strain, which explains why these suppressors are still sensitive to T6SS attacks, as they lack a functional GacA/S that is important for resistance to such attacks (Fig. 2). Our results show that there is a genetic link between oprF and the GacA/S TCS.

T6SS resistance mechanisms are effector-type specific

While the previous study from Ting et al.¹⁸. identified three operons, arc1, arc2 and arc3, involved in protection against T6SS from Burkholderia thailandensis, only two of them, arc1 and arc3 appeared in our CRISPRi screen. Moreover, the two further resistance factors that we identified, the mag operon and oprF, were not identified in the previous transposon screen. To verify whether these mechanisms are important for resistance to other organisms, we also tested the susceptibility of P. aeruginosa ΔtssM1, ΔgacA, ΔtssM1 ΔmagD, ΔtssM1 Δaas and ΔtssM1 ΔoprF to B. thailandensis attacks (Fig. 7 and Supplementary Fig. 5). Although magD is dispensable for resistance to the T6SS of B. thailandensis, both gacA and oprF are important, albeit to a lesser extent than in the case of A. baylyi ADP1 or V. cholerae 2740-80. Thus, some T6SS-protective mechanisms, such as those in the GacA regulon, are specific to certain effector types that may be carried by different strains, and others, like OprF-mediated mechanisms, appear to be generic protection mechanisms.

**Fig. 7: T6SS resistance mechanisms are species- or effector-specific.**

T6SS resistance strategies can affect antibiotic susceptibility

To determine whether T6SS protection mechanisms might be important for antibiotic resistance, we explored the susceptibility of T6SS-sensitive strains to 30 antibiotics from different classes, including inhibitors of translation, cell wall synthesis, folate synthesis, membrane integrity, DNA replication and transcription, among others. We performed growth curves of the ΔgacA, ΔtssM1, ΔtssM1 Δarc1A, Δarc3A Δaas, ΔmagD and the ΔoprF strains in the presence of antibiotics and compared the cumulative OD_{600 nm} for each condition, relative to wild-type and to the untreated condition (Fig. 8A and Supplementary Figs. 6–8). Interestingly, all tested strains displayed altered antibiotic susceptibility profiles compared to wild-type. Specifically, the ΔtssM1 strain displays increased growth in the presence of nadifloxacin, which is extended to other fluoroquinolones by the additional deletion of arc1A, arc3A and aas (Fig. 8A, B). These effects were exacerbated in the ΔgacA strain that does not express these and other genes that may be implicated in this phenotype. P. aeruginosa ΔmagD is more resistant to chlorhexidine, an antimicrobial compound targeting the bacterial cell membrane (Figs. 8A, C). Lastly, the P. aeruginosa ΔoprF strain is more resistant to antibiotics like the membrane-targeting colistin (Figs. 8A, D) and translation-targeting gentamicin, and more sensitive to the translation inhibitor tetracycline (Figs. 8A, E) and beta-lactams, such as piperacillin or azlocillin (Fig. 8A). This suggests that T6SS resistance mechanisms alter the antibiotic susceptibility profile of P. aeruginosa.

**Fig. 8: T6SS resistance mechanisms influence antibiotic susceptibility.**

Discussion

P. aeruginosa is able to promptly sense and retaliate to T6SS attacks from other species¹². However, the mechanisms that allow P. aeruginosa to withstand the initial attack (Fig. 1) are not well understood. Only one recent report¹⁸ identified three operons involved in resistance to T6SS attacks from B. thailandensis, called arc1, arc2 and arc3, and determined that arc3 is particularly important to resist the delivery of lipase effectors, likely through the detoxification of lysophospholipids that are generated as a result of the activity of these lipases. However, the effector types that arc1 and arc2 confer protection against, and whether there are other mechanisms involved in the resistance to other effector types, are not known. Here, we performed a CRISPRi screen to identify further factors involved in T6SS resistance in P. aeruginosa, using the laboratory models A. baylyi ADP1 and V. cholerae 2740-80 (AAA effector-type¹⁶ strain) as aggressor species. These two strains contain different effector sets^15,17: both of them have at least one lipase (Tle1 in A. baylyi and TseL in V. cholerae) and at least one PG-targeting effector (Tae1 in A. baylyi and VgrG3 and TseH in V. cholerae); A. baylyi encodes two other effectors of unknown activity (Tpe1 – a predicted metallopeptidase - and Tse2); and V. cholerae encodes one membrane-targeting pore-forming effector VasX.

In our screen, we identified known factors, such as the arc1 and arc3 clusters, as well as gacS and gacA of the GacA/S TCS that regulates the expression of said clusters¹⁸ in addition to the expression of the H1- and H2-T6SS^36,37 (Fig. 2B, C, E, G). We also identified further factors that contribute to resistance, the mag operon, that is also regulated by GacA, underscoring the importance of this transcriptional regulator for the survival of P. aeruginosa in polymicrobial communities. Furthermore, we found other factors that are not regulated by GacA (Fig. 2D and Fig. 5), such as the OMP OprF.

Through quantitative killing assays and CPRG-based lysis assays, we could validate that these factors are indeed involved in protection against T6SS attacks (Figs. 2–5). In agreement with Ting et al.¹⁸, we also observed that arc3 is important specifically to resist to lipases (Fig. 4). Here, we also observe that arc1A as well as aas are required for full resistance against the lipases from both V. cholerae 2740-80 and A. baylyi ADP1 (Fig. 4). Previously, aas was hypothesized to protect against lipases, however, arc1 role in protection against B. thailandensis effectors remained unclear¹⁸. This suggests that the specific catalytic mechanism of the lipases in question might be relevant for the mode of action of the corresponding resistance mechanisms.

Our results also show that the GacA regulon is necessary for the resistance against effector classes other than lipases (Fig. 2F, G). We found that the mag operon, that is regulated by GacA, is particularly important to resist against certain PG-targeting effectors, such as VgrG3 from V. cholerae 2740-80 and Tae1 from A. baylyi ADP1 (Fig. 3). Interestingly, the central gene of this operon, magD, displays homology with human α2-macroglobulins²³, innate immunity factors that defend against proteases through a trapping mechanism^25,26,27. However, the catalytic residues of this protein family are not conserved in bacterial type II α2-macroglobulins that magD belongs to²⁴, raising questions about the true function of this protein family in bacteria. Here, we found that MagD contributes to protection against some T6SS PG-targeting effectors (VgrG3 and Tae1, but not TseH), although the exact molecular mechanism remains to be elucidated. Previous studies suggest that TseH is a NlpC/P60 family cysteine endopeptidase³⁸, whereas VgrG3 has conserved residues with the catalytic sites of lysozyme-like chitosanases³⁹, and therefore hydrolyzes the polysaccharidic chains of the PG^40,41. The mechanism of Tae1 of A. baylyi has not been clearly elucidated, and it is unclear whether it is an amidase or a peptidase¹⁵. This raises the possibility MagD protects against PG-targeting effectors in a mechanism-dependent manner. Alternatively, the lack of a protective role against TseH might be explained by a low activity of TseH against P. aeruginosa under our experimental conditions, as the toxicity of TseH is highly dependent on the prey species (high activity against Aeromonas but no activity against immunity-deficient V. cholerae or E. coli³⁸) and on abiotic factors such as the Mg²⁺ and Ca²⁺ concentration⁴². Interestingly, MagD was previously found to co-immunoprecipitate with TssM1²³, a structural transmembrane component of the H1-T6SS of P. aeruginosa, suggesting that these two components might co-localize where a foreign T6SS attack was detected and contribute to the defense against the injected effectors (in the case of MagD) and the assembly of a counterattack H1-T6SS machine (in the case of TssM1).

P. aeruginosa also possesses other resistance mechanisms that are not known to be regulated by GacA (Fig. 2D), such as the OMP OprF. OprF is the most abundant OMP in P. aeruginosa, and carries out multiple functions, including an outer membrane porin as well as anchoring the outer membrane to the PG²⁹. Here, we found that OprF contributes to resistance to multiple T6SS effector classes (Fig. 5A, B). We confirmed this phenotype by complementation with a wild-type or mutant variant of OprF that no longer binds PG (Fig. 5C), and found that this variant is similarly able to promote survival against both V. cholerae 2740-80 and A. baylyi ADP1 suggesting that other functions of OprF besides OM anchoring must be important for survival to T6SS attacks.

Our study also provides evidence for the genetic relationship between oprF and the gacA/S TCS. A strain lacking oprF displays a growth defect in LB that is more pronounced in LB without salt (Supplementary Fig. 4C). In LB without salt, this strain spontaneously reverts to wild-type-like growth at high frequency (Fig. 6A, B) and, although these suppressors also display wild-type-like morphology (Fig. 6C), they are still unable to survive T6SS attacks from V. cholerae 2740-80 (Fig. 6D). Our whole genome sequencing analysis showed that these suppressors carry a loss-of-function deletion of gacS, which explains why these strains are susceptible to T6SS attacks. Indeed, the GacA/S TCS is responsible for the growth defect of the ΔoprF strain, since deletion of gacA restores this defect (Fig. 6E). Further research is needed to determine why the expression of the GacA regulon leads to a growth defect in the ΔoprF genetic background.

Next, we also assessed whether our identified resistance mechanisms also play a role against B. thailandensis E264. As expected from a transposon screen performed by Ting et al.¹⁸, magD does not play a role in defending from attacks from B. thailandensis, despite B. thailandensis encoding a PG-targeting amidase (Tae2)⁴³. As discussed above, this may be due to differences in the mechanism of Tae2 compared to other PG-targeting effectors, or to low activity levels of this particular effector against P. aeruginosa. However, both GacA and OprF are important for defense against the T6SS of B. thailandensis (Fig. 7). Interestingly, the arc2 operon identified by Ting et al.¹⁸ did not appear in our screen. Taken together, our results show that P. aeruginosa possesses an array of T6SS resistance mechanisms, frequently under the control of the GacS/A TCS, that help it survive attacks from a variety of aggressor species that it can encounter in the environment. However, these resistance mechanisms might only protect against effectors with specific modes of action carried by specific species (Fig. 9).

**Fig. 9: *P. aeruginosa* possesses multiple resistance mechanisms against T6SS attacks.**

Interestingly, other general stress response factors, such as those previously found to protect E. coli and P. aeruginosa^38,44, were not identified in our screen. In the case of P. aeruginosa, those factors were identified upon overexpression of TseL in the host periplasm, a condition that is likely more deleterious for P. aeruginosa than a single T6SS attack, where only a few molecules of TseL may be injected at the same time⁴⁴. This would suggest that members of the GacA regulon mount a first layer of defense and that, when they are overwhelmed, general stress response mechanisms of the cell come into play. Performing a similar screen in a ΔgacA genetic background, where dedicated protection mechanisms are not expressed, might uncover such general stress response mechanisms.

We also wondered whether these T6SS protection mechanisms could be involved in resistance to other antimicrobials, such as antibiotics. We screened a panel of 30 antibiotics against the wild-type, ΔgacA, ΔtssM1, ΔtssM1 Δarc1A Δarc3A, Δaas, ΔmagD and ΔoprF and strains (Fig. 8 and Supplementary Figs. 6–8) and found that, surprisingly, strains lacking these T6SS resistance mechanisms display a slight growth advantage in the presence of selected antibiotics as compared to wild-type, suggesting that these T6SS protection mechanisms may worsen the toxicity of antibiotics. Of note, tssM1, arc1A, arc3A, aas and other members of the gacA regulon increase sensitivity to nadifloxacin and other fluoroquinolones. Part of this effect may be attributed to the ability of arc3A and aas to detoxify lysophospholipids that may be generated as a consequence of reactive oxygen species (ROS)^45,46 that are produced upon fluoroquinolone treatment⁴⁷, although further research is required to pinpoint the specific mechanism and other genes of the GacA regulon that may impact fluoroquinolone resistance. Interestingly, a ΔmagD mutant grows better in the presence of chlorhexidine, suggesting a further impact of MagD on the P. aeruginosa envelope. As a consequence, we hypothesize that there are trade-offs to obtaining T6SS protection mechanisms that may lead to higher antibiotic susceptibility in P. aeruginosa. Similar trade-offs were also identified in E. coli that evolved resistance against T6SS attacks from V. cholerae⁴⁸.

It is known that colistin kills gram-negative bacteria by targeting LPS and disrupting the OM^49,50. Most mechanisms of resistance to colistin have been linked to LPS modification⁴⁹. More research is needed to clarify why an oprF mutant, that displays defects in OM stability, is more resistant to colistin. On the other hand, a ΔoprF strain is also more susceptible to antibiotics, such as the ribosome-targeting tetracycline, or the beta-lactams piperacillin or azlocillin. These phenotypes cannot be explained by the porin function of oprF²⁹, since the strain should be more resistant to these antibiotics if oprF was required for drug uptake. Overall, our findings serve as proof-of-concept that the study of the mechanisms of protection against the T6SS can lead to insights about antimicrobial resistance in bacteria.

In conclusion, in this work, we identified multiple protection mechanisms in P. aeruginosa against T6SS attacks from A. baylyi ADP1 and V. cholerae 2740-80, which are also linked with resistance to classical antimicrobials. Interestingly, some T6SS resistance mechanisms may lead to increased sensitivity to antibiotics, suggesting that these mechanisms may not be generally beneficial. Further research will be needed to determine whether these protection mechanisms play a role in the survival of P. aeruginosa strains in naturally occurring polymicrobial communities, and particularly in the context of clinical infections, and in the presence of antibiotics.

Methods

Bacterial strains and growth conditions

Bacterial strains used in this study can be found in Supplementary Data 2. Bacteria were grown in LB at 37 °C unless otherwise stated. E. coli DH5α λpir was used as a cloning strain, and E. coli SM10 λpir was used for the conjugation of P. aeruginosa and V. cholerae. Antibiotics used were streptomycin (100 μg/mL for V. cholerae and A. baylyi on solid media; and 50 μg/mL for A. baylyi in liquid media), gentamicin (20 μg/mL for P. aeruginosa and E. coli), kanamycin (50 μg/mL for A. baylyi), carbenicillin (150 μg/mL for E. coli and V. cholerae and 100 μg/mL for P. aeruginosa), irgasan (20 μg/mL for P. aeruginosa) and trimethoprim (200 μg/mL for B. thailandensis and 50 μg/mL for E. coli).

Mutant construction

All plasmids used in this study are listed in Supplementary Table 1, and primers in Supplementary Data 3. P. aeruginosa mutants were constructed as previously described using the pEXG2 suicide plasmid⁵¹. Briefly, 800 bp homology arms were amplified by PCR using primers (a list of all primers can be found in Supplementary Information 11) with 20 bp stretches that were homologous to the HindIII and XbaI restriction sites of the pEXG2 plasmid. PCR products were inserted into linearized pEGX2 plasmid by Gibson assembly⁵² (NEB, E2611L). The resulting vectors were transformed into chemically-competent E. coli DH5α λpir, selected on LB plates with 20 μg/mL gentamicin and verified by colony PCR and Sanger sequencing. The purified plasmids were then transformed into chemically competent E. coli SM10 λpir, and the resulting strains were used for conjugation of P. aeruginosa PAO1. Selection of P. aeruginosa transconjugants was performed on LB with 20 μg/mL gentamicin and 20 μg/mL irgasan. The pEXG2 backbone was then curated by restreaking on LB plates without salt supplemented with 20 μg/mL irgasan and 10% sucrose, which were incubated for 36 h at room temperature. Colonies were screened by colony PCR with flanking primers.

pPSV35-derived plasmids were introduced into P. aeruginosa by electroporation of 20–200 ng of plasmid into 1 mL of overnight stationary phase cultures washed twice with sterile water using a Bio-Rad GenePulser with the following settings: 25 μF, 400 Ω, 1.8 kV. All steps were performed at room temperature. 1 mL of LB medium was added immediately after the pulse and cells were incubated 1.5–2 h at 37 °C with shaking before plating on LB plates containing 20 μg/mL gentamicin.

The chromosomal insertion of dCas9 in the attnT7 site was performed as previously described⁵³. Briefly, an 18–22 h old culture of P. aeruginosa ΔtssM1 in LB without salt was washed twice with sterile water and resuspended in 100 μL sterile water. The bacterial suspension was electroporated with two plasmids at the same time, pUC18T: TNT7: dCas9Spas and pTNS2 that contains the Tn7 integrase and is unable to replicate in P. aeruginosa. The electroporation was performed as described above. The cells were recovered for 1 h in LB at 37 °C and 100 μL were plated on LB agar plates with 20 μg/mL gentamicin to select for transconjugants that integrated dCas9 into the chromosome. The next day, colonies were grown in LB no salt and, as above, electroporated with plasmid pFLP2 containing the flippase that removes the backbone of the pUC18T plasmid. After recovery, 100 μL were plated on LB agar plates with 100 μg/mL carbenicillin and incubated overnight at 37 °C. The resulting colonies were restreaked on LB no salt + 10% sucrose to counterselect pFLP2. The successful insertion of dCas9 was verified by PCR and the resulting strain was phenotypically validated by expression of an ftsZ-targeting sgRNA that led to cell filamentation as described²¹.

V. cholerae mutants were constructed in a similar manner, using the pWM91 suicide plasmid and the XbaI and HindIII restriction sites. V. cholerae transconjugants were selected on LB plates with 150 μg/mL carbenicillin and 100 μg/mL streptomycin. The pWM91 backbone was curated by restreaking on LB plates without salt supplemented with 100 μg/mL streptomycin and 10% sucrose.

A. baylyi mutants were constructed by natural transformation as previously described¹⁵, using a kanamycin resistance, streptomycin counterselectable, cassette and 800 bp homology arms.

B. thailandensis were conjugated with pMLBAD plasmid as previously described⁵⁴.

Quantitative killing assays

Overnight cultures of P. aeruginosa, V. cholerae, E. coli and B. thailandensis were grown in LB at 37 °C and 200 rpm without antibiotics unless P. aeruginosa or B. thailandensis contained plasmids. A. baylyi overnight cultures were grown at 30 °C in similar conditions without antibiotics. Day cultures were launched in LB at the following dilutions: 1:100 for P. aeruginosa, 1:50 for A. baylyi and B. thailandensis, 1:200 for E. coli and 1:400 for V: cholerae. After 2.5–3 h, when all cultures reached an OD_600nm of 0.7–1.4, cultures were spun down at 12,000 g for 2 min at room temperature. The cultures were concentrated to OD 10 with LB and 1:10 prey:attacker mixes were prepared. Five μL of the resulting mixes were spotted on LB agar plates, in technical triplicates, and allowed to dry. The plates were incubated at 37 °C for 3-4 h and then the spots were excised and resuspended in 500 μL liquid LB by vortexing. Ten-fold serial dilutions were performed and 5 μL of the resulting suspensions were spotted on selective plates. As selective agents, we used irgasan 20 μg/mL for P. aeruginosa, streptomycin 100 μg/mL for V. cholerae and A. baylyi, gentamicin 20 μg/mL for E. coli and 200 μg/mL trimethoprim for B. thailandensis. Plates were incubated overnight at 37 °C and the surviving CFUs were counted the next day and technical triplicates were averaged. The experiments were performed with at least three biological replicates and plotted with Prism.

CRISPRi library electroporation

A P. aeruginosa ΔtssM1 strain containing dCas9 at the attnT7 site was grown overnight in LB without salt and 4 mL were electroporated with 2 μL of the CRISPRi library as described above. Details on the library composition and experimental validation can be found in ref. ⁵³. After electroporation, the bacterial suspension was resuspended in 1 mL LB and transferred to a 1 L Erlenmeyer flask containing 100 mL LB and allowed to recover for 1–1.5 h at 37 °C while shaking. Next, 20 μg/mL gentamicin was added and the culture was incubated overnight at 37 °C while shaking. This protocol led to an electroporation efficiency of 3.8·10⁶ cells/mL, which allows for achieving a good coverage of the ca. 83,000 sgRNA library.

CRISPRi screening competition

Three 100 mL day cultures in LB + 20 μg/mL gentamicin+1 mM IPTG were launched by inoculation of 1 mL at OD_600nm 1 from the library electroporation culture. In parallel, 20 mL day cultures were launched from overnight cultures of V. cholerae and A. baylyi strains. The cultures were incubated for around 3 h until an OD_600nm of 0.7–1.4 was reached. Five mL of each P. aeruginosa culture were taken, pelleted and frozen at −80 ^oC until further processing (time 0 before the competition started). For each replicate, the cultures were concentrated to OD 10 and mixed in a 10:1 V. cholerae:P. aeruginosa ratio or in a 1:1 A.baylyi:P. aeruginosa ratio and 90 μL of the resulting mixes were spotted on LB + 1 mM IPTG plates. P. aeruginosa alone was also spotted (no co-incubation control). The spots were allowed to dry and were then incubated at 37 °C for 3 h. Then, the spots were excised and resuspended in 7 mL LB by vortexing and pipetting. The resuspended bacteria were spun down for 10 min at 3,900 g and resuspended in 7 mL LB. Five mL were pelleted and frozen at −80 ^oC until further processing (samples after the first round of competition). The remaining 2 mL were used to measure the OD_600nm and inoculate new day cultures in 100 mL LB + 20 μg/mL gentamicin + 1 mM IPTG with 1 mL OD 1. New day cultures of V. cholerae and A. baylyi were launched similarly to earlier. The same process was repeated for a second round of competition and the resulting samples were again pelleted and frozen at -80 °C until further processing.

CRISPRi sequencing library preparation

Pellets were thawed and resuspended in 500 μL 50 mM NaOH. The samples were incubated for 10 min at 50 °C and 1 μL was used as template for a PCR reaction using Phusion DNA Polymerase (M0530S, NEB) as described⁵³ using primer pairs oATA402 + 403, oATA404 + 405, oATA406 + 407 and oATA408 + 409 for a fourth of the samples each. The PCR cycles were performed as follows: 98 °C for 3 min, 35 cycles of 98 °C denaturation for 10 s, 63 °C annealing for 10 s and 72 °C elongation for 10 s, followed by a final extension at 72 °C for 1 min and hold at 10 °C until further processing. The PCR products were run on a 2% agarose gel and purified from gel using a NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel). The DNA concentration of the samples was measured and samples were diluted to 1 ng/μL in 10 μL, of which 5 μL were used as template for the indexing PCR.

Indexing was performed with the kit IDT for Illumina Nextera DNA UD Index Set C (ref 20026934) with a PCR mix as follows: 5 μL DNA template, 5 μL primer mix, 5 μL Phusion GC Buffer, 0.5 μL 10 mM dNTPs, 0.25 μL Phusion DNA polymerase (M0530S, NEB) and 9.25 μL H₂O. The following program was used: 98 °C for 3 min, followed by 8 cycles of 98 °C denaturation for 10 s, 55 °C annealing for 10 s and 72 °C elongation for 10 s, and then a final extension at 72 °C for 1 min and hold at 10 °C until further processing. The PCR products were purified using 1.8 volumes of NEBNext Sample Purification Beads (E7767S, NEB). Samples were resuspended in 20 μL H₂O and a quality check was run on a TapeStation 4150 device (Agilent) using DNA ScreenTape chips (Agilent) as recommended by the manufacturer. Sequencing was performed as previously described ⁵⁴ by Illumina sequencing at the Genomics Facility Basel (ETH Zürich, University of Basel) using a NextSeq SR 81/10 kit.

Data analysis was performed using the European Galaxy server⁵⁵ using the previously described MAGeCK pipeline⁵⁶ using median normalization, FDR-adjusted threshold of 0.25, FDR as p-value adjustment method and 10 as remove zero threshold. The median method as used for the gene log-fold change. Data was plotted using Prism.

Sample preparation for proteomics

Overnight cultures of P. aeruginosa were grown at 37 °C and 200 rpm, in biological triplicate. Day cultures in LB were launched (1:100 dilution of the overnight) and incubated for 2.5 3 h at 37 °C and 200 rpm, until they reached an OD_600nm of 0.7–1.4. They were concentrated to an OD of 10 and 20 μL were spotted on LB plates. The spots were allowed to dry and were then incubated at 37 °C for 3 h. The spots were then resuspended in 500 μL phosphate-buffered saline (PBS), pelleted by centrifugation at 12,000 g for 2 min and stored at -80 °C until further processing. Cell pellets were resuspended in lysis buffer (5% Sodium dodecyl sulfate (SDS), 10 mM tris(2-carboxyethyl) phosphine (TCEP), 100 mM Triethyloammonium bicarbonate (TEAB)) followed by incubation at 95 °C for 10 min. Cells were disrupted by ultrasonication using the PIXUL system (Active Motif) for 20 min with default settings (pulse 50 cycles, PRF 1 kHz, burst rate 20 Hz)) and the protein content was determined by tryptophan-based fluorescence assay (Infinite M Plex, Tecan). Sample alkylation was performed by addition of 20 mM iodoacetamide (IAA) and incubation at 25 °C for 30 min with gentle shaking. 10 μg of protein lysate was digested (trypsin 1/100, w/w; Promega) and purified using the SP3 protocol⁵⁷. Peptide concentration was determined using an UV-based assay (Infinite M Nano, Tecan). Samples were then resuspended at a final concentration of 250 ng/μL.

SWATH-MS data acquisition

Samples were separated on a Dionex UltiMate 3000 system (ThermoFisher Scientific) coupled online to an Orbitrap Exploris 480 mass spectrometer (ThermoFisher Scientific). In-house packed 20 cm, 75 μm ID capillary column with 1.9 μm Reprosil-Pur C18 beads (Dr. Maisch, Ammerbuch, Germany) was used. The column temperature was maintained at 60 °C using an integrated column oven interfaced online with the mass spectrometer. Formic acid (FA) 0.1% was used to buffer the pH in the two running buffers used. The total gradient time was 60 min and went from 2% to 12% acetonitrile (ACN) in 5 min, followed by 45 min to 35%, and 10 min at 50%. This was followed by a washout by 95% ACN, which was kept for 20 min, followed by re-equilibration of 0.1% FA buffer. Flow rate was kept at 300 nL/min. Spray voltage was set to 2500 V, funnel RF level at 40, and heated capillary at 275 °C. For DIA experiments full MS resolutions were set to 120,000 and full MS normalized AGC target was 300% with an IT of 45 ms. Mass range was set to 350–1400. Normalized AGC target value for fragment spectra was set at 1000%. In all, 63 windows of 9 Da were used with an overlap of 1 Da. Resolution was set to 15,000 and IT to 22 ms. Normalized CE was set at 28%. All data were acquired in centroid mode using positive polarity and advanced peak determination was set to on.

SWATH-MS data analysis

For data processing and protein identification, raw data were imported into SpectroNaut (16.1.220730.53000, Biognosys) and analyzed with directDIA. Searches were carried out against a FASTA file including the proteomes of P. aeruginosa (UP000002438). Cysteine carbamidomethylation was set as fixed modification. Methionine oxidation, methionine excision at the N terminus were selected as variable modifications. Results were filtered for a 1% false discovery rate (FDR) on spectrum, peptide, and protein levels. Relative protein abundances and pairwise comparisons were calculated using the MSstats package⁵⁸ with default settings selected.

CPRG lysis assay

For this assay, lacZ-deficient attacker strains were used, and P. aeruginosa prey strains carried an IPTG-inducible lacZ on the pME3856 plasmid. Overnight cultures of P. aeruginosa and V. cholerae were grown in LB at 37 °C and 200 rpm without antibiotics. A. baylyi overnight cultures were grown at 30 °C in similar conditions. Day cultures were launched in LB at the following dilutions: 1:100 for P. aeruginosa, 1:50 for A. baylyi and 1:400 for V. cholerae. After 2.5–3 h, when all cultures reached an OD_600nm of 0.7–1.4, cultures were spun down at 12,000 g for 2 min at room temperature. The cultures were concentrated to OD 1 and mixed in a 1:2 prey:attacker ratio. Three μL of the resulting suspensions were spotted in wells of a flat-bottom 96-well plate that had been extemporaneously prepared with 150 μL LB with 1% agar containing 0.1 mM IPTG (isopropyl-β-D-thiogalactoside) and 20 μg/mL CPRG (red-β-D-galactopyranoside) and pre-dried for 20 min at room temperature next to the Bunsen burner. The plates were incubated in an Epoch2 plate reader (BioTek) at 30 °C, and the OD_575nm was measured every 10 min. The curves were plotted with Prism.

Flow cytometry

Bacteria with fluorescently labeled T6SS sheath (TssB1-mNG) were grown to mid-exponential phase at 37 °C from an overnight culture in LB. Prior to measurement, the samples were diluted 100x in PBS and were analyzed in a BD Fortessa flow cytometer, with 950 V blue laser in low flow rate mode. Data were analyzed using FlowJo v10, and the median fluorescence intensity (MFI) was plotted using Prism.

Growth curves

Overnight cultures of P. aeruginosa were diluted to OD_600nm 1 in LB, and used to inoculate wells, in triplicate, of a flat-bottom 96-well plate containing LB or LB without NaCl, with or without antibiotics, at a starting OD_600nm of 0.05. They were incubated at 37 °C in an Epoch2 plate reader (BioTek) with orbital shaking at 240 cpm and the OD_600nm was measured every 10 min. If antibiotic treatment was performed, antibiotics were added after 1.5 h of incubation at 20 µM. The resulting curves were plotted in Prism.

Fluorescence microscopy

Overnight cultures of P. aeruginosa were diluted 100-fold in LB and incubated for around 3 h at 37 °C and 200 rpm. They were then pelleted on a tabletop centrifuge at 12.000 g for 2 min at room temperature and resuspended to OD 10 with LB containing 4 μg/mL FM4-64 (T13320, ThermoFisher Scientific) and incubated for 10 min at room temperature in the dark. One μL of the cell suspensions were then placed on agar pads (1/3 LB, 2/3 PBS with 1% agarose) and imaged on a Nikon Ti-E inverted motorized microscope with Perfect Focus System and Plan Apo 100x Oil Ph3 DM (NA 1.4) objective lens. Spectra X light engine (Lumencor) and ET-mCherry (Chroma 49008) filter sets were used to excite and filter fluorescence and a sCMOS camera pco.edge 4.2 (PCO, Germany, pixel size 65 nm) and VisiView software (Visitron Systems, Germany) were used. Imaging was carried out at 30 °C by an Okolab T-unit (Okolab).

Whole genome sequencing

Genomic DNA was extracted from overnight bacterial cultures in 1.5 mL LB following the instructions of the GenElute Bacterial Genomic DNA kit (NA2110, Sigma). DNA concentration was measured using the Qubit dsDNA Quantitation Broad Range Assay kit (Q32850, ThermoFisher Scientific) and barcoding was performed according to the manufacturer’s instructions using the Rapid Barcoding Kit 24 V14 (SQK-RBK114.24, Oxford Nanopore Technologies). Samples were sequenced on a MinION flow cell (R10.4.1, FLO-MIN114, Oxford Nanopore Technologies). Basecalling was performed using Dorado software from Nanopore, and the genomes were compared using the Snakemake evo-genome-analysis pipeline that is publicly available at https://github.com/mmolari/evo-genome-analysis/. This pipeline uses the raw reads from each sample and maps it to the reference using Minimap2⁵⁹, extracting genomic changes including single nucleotide polymorphisms, gaps, insertions, clips and rearrangements.

Statistics

When only two means were compared, we performed unpaired t-tests. When multiple means were compared, we used ordinary one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison test or two-way ANOVA with Sidak’s multiple comparison test, using GraphPad Prism version 9.3.1. Unless otherwise stated, data are represented as mean ± standard deviation (SD). *p-value < 0.05, **p-value < 0.005, ***p-value < 0.0005, ****p-value < 0.00005, ns – p-value > 0.05.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All mass spectrometry raw data files associated with this manuscript are accessible at MassIVE (https://massive.ucsd.edu) under accession number MSV000096798. Genome and CRISPRi screen sequencing data for this study have been deposited in the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB84063. Source data are provided as a Source Data file and in the Zenodo repository with accession number 14811405 (https://zenodo.org/records/14811406). Source data are provided with this paper.

References

Malhotra, S., Hayes, D. & Wozniak, D. J. Cystic fibrosis and Pseudomonas aeruginosa: the host-microbe interface. Clin. Microbiol. Rev. 32, e10038–18 (2019).
Sana, T., Berni, N. & Bleves, S. The T6SSs of Pseudomonas aeruginosa Strain PAO1 and their effectors: beyond bacterial-cell targeting. Front. Cell Infect. Microbiol. 6, 61 (2016).
Article PubMed PubMed Central Google Scholar
Swart, A. L. et al. Pseudomonas aeruginosa breaches respiratory epithelia through goblet cell invasion in a microtissue model. Nat. Microbiol. 9, 1725–1737 (2024).
Article PubMed Google Scholar
Sana, T. G. et al. Internalization of Pseudomonas aeruginosa strain PAO1 into epithelial cells is promoted by interaction of a T6SS effector with the microtubule network. mBio 6, 61 (2015).
Wettstadt, S., Wood, T. E., Fecht, S. & Filloux, A. Delivery of the Pseudomonas aeruginosa phospholipase effectors PldA and PldB in a VgrG- And H2-T6SS-dependent manner. Front. Microbiol. 10, 01718 (2019).
Lin, J. et al. A Pseudomonas T6SS effector recruits PQS-containing outer membrane vesicles for iron acquisition. Nat. Commun. 8, 14888 (2017).
Yang, Y. et al. H3-T6SS of Pseudomonas aeruginosa PA14 contributes to environmental adaptation via secretion of a biofilm-promoting effector. Stress Biol. 2, 1–14 (2022).
Article CAS Google Scholar
Wang, J., Brodmann, M. & Basler, M. Assembly and subcellular localization of bacterial type VI secretion systems. Annu. Rev. Microbiol 73, 621–638 (2019).
Article PubMed CAS Google Scholar
Cherrak, Y., Flaugnatti, N., Durand, E., Journet, L. & Cascales, E. Structure and activity of the type VI secretion system. Microbiol. Spectr. 7, 1128 (2019).
Yang, X., Long, M. & Shen, X. Effector–immunity pairs provide the T6SS nanomachine its offensive and defensive capabilities. Mol. J. Synth. Chem. Nat. Prod. Chem. 23, 1009 (2018).
Zhang, H., Gao, Z. Q., Wei, Y., Xu, J. H. & Dong, Y. H. Insights into the cross-immunity mechanism within effector families of bacteria type VI secretion system from the structure of StTae4-EcTai4 complex. PLoS One 8, e73782 (2013).
Basler, M., Ho, B. T. & Mekalanos, J. J. Tit-for-tat: Type VI secretion system counterattack during bacterial cell-cell interactions. Cell 152, 884–894 (2013).
Article PubMed PubMed Central CAS Google Scholar
George, M., Narayanan, S., Tejada-Arranz, A., Plack, A. & Basler, M. Initiation of H1-T6SS dueling between Pseudomonas aeruginosa. mBio 15, e00355-24 (2024).
Smith, W. P. J. et al. The evolution of tit-for-tat in bacteria via the type VI secretion system. Nat. Commun. 11, 884–894 (2020).
Ringel, P. D., Hu, D. & Basler, M. The role of type vi secretion system effectors in target cell lysis and subsequent horizontal gene transfer. Cell Rep. 21, 3927–3940 (2017).
Article PubMed CAS Google Scholar
Unterweger, D. et al. The Vibrio cholerae type VI secretion system employs diverse effector modules for intraspecific competition. Nat. Commun. 5, 3549 (2014).
Article ADS PubMed Google Scholar
Crisan, C. V. & Hammer, B. K. The Vibrio cholerae type VI secretion system: toxins, regulators and consequences. Environ. Microbiol. 22, 4112–4122 (2020).
Ting, S. Y. et al. Discovery of coordinately regulated pathways that provide innate protection against interbacterial antagonism. Elife 11, 74658 (2022).
Hersch, S. J., Manera, K. & Dong, T. G. Defending against the type six secretion system: beyond immunity genes. Cell Rep. 33, 108259 (2020).
Article PubMed CAS Google Scholar
Todor, H., Silvis, M. R., Osadnik, H. & Gross, C. A. Bacterial CRISPR screens for gene function. Curr. Opin. Microbiol. 59, 102–109 (2021).
Tan, S. Z., Reisch, C. R. & Prather, K. L. J. A robust CRISPR interference gene repression system in Pseudomonas. J. Bacteriol. 200, e00575-17 (2018).
Liang, X. et al. An onboard checking mechanism ensures effector delivery of the type VI secretion system in Vibrio cholerae. Proc. Natl. Acad. Sci. USA 116, 23292–23298 (2019).
Article ADS PubMed PubMed Central CAS Google Scholar
Robert-Genthon, M. et al. Unique features of a Pseudomonas aeruginosa α2-macroglobulin homolog. mBio 4, e00309-13 (2013).
Zouhir, S. et al. Assembly of an atypical α-macroglobulin complex from Pseudomonas aeruginosa. Sci. Rep. 8, 527 (2018).
Rehman, A. A., Ahsan, H. & Khan, F. H. -2-Macroglobulin: a physiological guardian. J. Cell Physiol. 228, 1665–1675 (2013).
Article PubMed CAS Google Scholar
Marrero, A. et al. The crystal structure of human α2-macroglobulin reveals a unique molecular cage. Angew. Chem. Int Ed. Engl. 51, 3340–3344 (2012).
Article PubMed CAS Google Scholar
Meyer, C., Hinrichs, W. & Hahn, U. Human α2-macroglobulin—another variation on the venus flytrap. Angew. Chem. Int Ed. Engl. 51, 5045–5047 (2012).
Article PubMed CAS Google Scholar
Lin, Y. et al. The phospholipid-repair system LplT/Aas in Gram-negative bacteria protects the bacterial membrane envelope from host phospholipase A2 attack. J. Biol. Chem. 293, 3386–3398 (2018).
Article PubMed PubMed Central CAS Google Scholar
Chevalier, S. et al. Structure, function and regulation of Pseudomonas aeruginosa porins. FEMS Microbiol. Rev. 41, 698–722 (2017).
Article PubMed CAS Google Scholar
De Mot, R. & Vanderleyden, J. The C-terminal sequence conservation between OmpA-related outer membrane proteins and MotB suggests a common function in both gram-positive and gram-negative bacteria, possibly in the interaction of these domains with peptidoglycan. Mol. Microbiol. 12, 333–334 (1994).
Article PubMed Google Scholar
Koebnik, R. Proposal for a peptidoglycan-associating alpha-helical motif in the C-terminal regions of some bacterial cell-surface proteins. Mol. Microbiol. 16, 1269–1270 (1995).
Article PubMed CAS Google Scholar
Rawling, E. G., Brinkman, F. S. L. & Hancock, R. E. W. Roles of the carboxy-terminal half of Pseudomonas aeruginosa major outer membrane protein OprF in cell shape, growth in low-osmolarity medium, and peptidoglycan association. J. Bacteriol. 180, 3556–3562 (1998).
Article PubMed PubMed Central CAS Google Scholar
Gotoh, N., Wakebe, H., Yoshihara, E., Nakae, T. & Nishino, T. Role of protein F in maintaining structural integrity of the Pseudomonas aeruginosa outer membrane. J. Bacteriol. 171, 983 (1989).
Article PubMed PubMed Central CAS Google Scholar
Ishida, H., Garcia-Herrero, A. & Vogel, H. J. The periplasmic domain of Escherichia coli outer membrane protein A can undergo a localized temperature dependent structural transition. Biochimica et. Biophysica Acta (BBA) - Biomembranes 1838, 3014–3024 (2014).
Article PubMed CAS Google Scholar
Deghelt, M. et al. Peptidoglycan-outer membrane attachment generates periplasmic pressure to prevent lysis in Gram-negative bacteria. Nat. Microbiol. 10, 1963–1974 (2025).
Brencic, A. & Lory, S. Determination of the regulon and identification of novel mRNA targets of Pseudomonas aeruginosa RsmA. Mol. Microbiol. 72, 612–632 (2009).
Article PubMed PubMed Central CAS Google Scholar
Allsopp, L. P. et al. RsmA and AmrZ orchestrate the assembly of all three type VI secretion systems in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 114, 7707–7712 (2017).
Article ADS PubMed PubMed Central CAS Google Scholar
Hersch, S. J. et al. Envelope stress responses defend against type six secretion system attacks independently of immunity proteins. Nat. Microbiol. 5, 706–714 (2020).
Article PubMed PubMed Central CAS Google Scholar
Dong, T. G., Ho, B. T., Yoder-Himes, D. R. & Mekalanos, J. J. Identification of T6SS-dependent effector and immunity proteins by Tn-seq in Vibrio cholerae. Proc. Natl. Acad. Sci. USA 110, 2623–2628 (2013).
Article ADS PubMed PubMed Central CAS Google Scholar
Monzingo, A. F., Marcotte, E. M., John Hart, P. & Robertus, J. D. Chitinases, chitosanases, and lysozymes can be divided into procaryotic and eucaryotic families sharing a conserved core. Nat. Struct. Biol. 3, 133–140 (1996).
Article PubMed CAS Google Scholar
Saito, J. I. et al. Crystal structure of chitosanase from bacillus circulans MH-K1 at 1.6-Å resolution and its substrate recognition mechanism. J. Biol. Chem. 274, 30818–30825 (1999).
Article PubMed CAS Google Scholar
Tang, M.-X. et al. Abiotic factors modulate interspecies competition mediated by the type VI secretion system effectors in Vibrio cholerae. ISME J 1–11 https://doi.org/10.1038/s41396-022-01228-5 (2022)
Russell, A. B. et al. A widespread bacterial type VI secretion effector superfamily identified using a heuristic approach. Cell Host Microbe 11, 538–549 (2012).
Article PubMed PubMed Central CAS Google Scholar
Kamal, F. et al. Differential cellular response to translocated toxic effectors and physical penetration by the type VI secretion system. Cell Rep. 31, 107766 (2020).
Article PubMed CAS Google Scholar
Wang, B. et al. ROS-induced lipid peroxidation modulates cell death outcome: mechanisms behind apoptosis, autophagy, and ferroptosis. Arch. Toxicol. 97, 1439–1451 (2023).
Article PubMed CAS Google Scholar
Engel, K. M., Schiller, J., Galuska, C. E. & Fuchs, B. Phospholipases and reactive oxygen species derived lipid biomarkers in healthy and diseased humans and animals - a focus on lysophosphatidylcholine. Front. Physiol. 12, 732319 (2021).
Drlica, K. & Zhao, X. Bacterial death from treatment with fluoroquinolones and other lethal stressors. Expert Rev. Anti Infect. Ther. 19, 601–618 (2021).
Article PubMed CAS Google Scholar
MacGillivray, K. A. et al. Trade-offs constrain adaptive pathways to the type VI secretion system survival. iScience 26, 108332 (2023).
Yin, C., Alam, M. Z., Fallon, J. T. & Huang, W. Advances in development of novel therapeutic strategies against multi-drug resistant pseudomonas aeruginosa. Antibiotics 2024 13, 119 (2024).
CAS Google Scholar
Sabnis, A. et al. Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane. Elife 10, e65836 (2021).
Rietsch, A., Vallet-Gely, I., Dove, S. L. & Mekalanos, J. J. ExsE, a secreted regulator of type III secretion genes in Pseudomonas aeruginosa. Proc. Natl Acad. Sci. USA 102, 8006–8011 (2005).
Article ADS PubMed PubMed Central CAS Google Scholar
Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).
Article PubMed CAS Google Scholar
Kaczmarczyk, A., Klotz, A., Manfredi, P. & Jenal, U. Genome-wide high-density CRISPR interference screens reveal condition-specific metabolic vulnerabilities in Pseudomonas aeruginosa PAO1. bioRxiv 2025.08.12.669819 https://doi.org/10.1101/2025.08.12.669819 (2025).
Plum, M. T. W. et al. Burkholderia thailandensis uses a type VI secretion system to lyse protrusions without triggering host cell responses. Cell Host Microbe 32, 676–692 (2024).
Article PubMed CAS Google Scholar
Abueg, L. A. L. et al. The Galaxy platform for accessible, reproducible, and collaborative data analyses: 2024 update. Nucleic Acids Res. 52, W83–W94 (2024).
Article Google Scholar
Li, W. et al. Quality control, modeling, and visualization of CRISPR screens with MAGeCK-VISPR. Genome Biol. 16, 281 (2015).
Article PubMed PubMed Central CAS Google Scholar
Hughes, C. S. et al. Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nat. Protoc. 14, 68–85 (2019).
Article PubMed CAS Google Scholar
Choi, M. et al. MSstats: an R package for statistical analysis of quantitative mass spectrometry-based proteomic experiments. Bioinformatics 30, 2524–2526 (2014).
Article PubMed CAS Google Scholar
Li, H. Minimap2: pairwise alignment for nucleotide sequences. Bioinformatics 34, 3094–3100 (2018).
Article PubMed PubMed Central CAS Google Scholar

Download references

Acknowledgements

This work was supported by the National Center of Competence in Research AntiResist funded by the Swiss National Science Foundation (51NF40_180541) to MB and UJ. We thank Mattia Zampieri for providing the antibiotic collection used in this study. We thank Marco Molari, Valentin Druelle and Leonardo Rocha for providing help with Nanopore sequencing sample preparation and data analysis. We thank the members of the Biozentrum FACS Core Facility (FCF) for help with flow cytometry and maintaining the Nanopore sequencing equipment. We thank the members of the Biozentrum Proteomics Core Facility (PCF) for help with mass-spectrometry data acquisition and analysis.

Author information

Minia Antelo-Varela
Present address: Proteomics Unit, Gulbenkian Institute for Molecular Medicine, Lisbon, Portugal
Alexander Klotz
Present address: Department of Biosystems Science and Engineering, ETH Zurich, Basel, Switzerland

Authors and Affiliations

Biozentrum, University of Basel, Basel, Switzerland
Alejandro Tejada-Arranz, Annika Plack, Minia Antelo-Varela, Andreas Kaczmarczyk, Alexander Klotz, Urs Jenal & Marek Basler

Authors

Alejandro Tejada-Arranz
View author publications
Search author on:PubMed Google Scholar
Annika Plack
View author publications
Search author on:PubMed Google Scholar
Minia Antelo-Varela
View author publications
Search author on:PubMed Google Scholar
Andreas Kaczmarczyk
View author publications
Search author on:PubMed Google Scholar
Alexander Klotz
View author publications
Search author on:PubMed Google Scholar
Urs Jenal
View author publications
Search author on:PubMed Google Scholar
Marek Basler
View author publications
Search author on:PubMed Google Scholar

Contributions

Conceptualization: A.T.A. and M.B.; Methodology: A.T.A.; Software: A.T.A., M.A.V., and A.K.l.; Validation: A.T.A. and A.P.; Formal analysis: A.T.A. and M.A.V.; Investigation: A.T.A., A.P., and M.A.V.; Resources: A.K.a., A.K.l., U.J., and M.B.; Data curation: A.T.A.; Writing – original draft: A.T.A.; Writing – review & editing: A.T.A. and M.B.; Visualization: A.T.A. and A.P.; Supervision: A.T.A. and M.B.; Project administration: A.T.A. and M.B.; Funding acquisition: M.B. and U.J.

Corresponding author

Correspondence to Marek Basler.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Communications thanks the anonymous reviewers for their contribution to the peer review of this work. A peer review file is available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Description of Additional Supplementary Files

Supplementary Data 1

Supplementary Data 2

Supplementary Data 3

Reporting summary

Transparent Peer Review file

Source data

Source Data

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Reprints and permissions

About this article

Cite this article

Tejada-Arranz, A., Plack, A., Antelo-Varela, M. et al. Mechanisms of Pseudomonas aeruginosa resistance to type VI secretion system attacks. Nat Commun 16, 10744 (2025). https://doi.org/10.1038/s41467-025-65777-x

Download citation

Received: 20 January 2025
Accepted: 22 October 2025
Published: 28 November 2025
Version of record: 28 November 2025
DOI: https://doi.org/10.1038/s41467-025-65777-x

Subjects

Abstract

Similar content being viewed by others

Introduction

Results

P. aeruginosa resists T6SS attacks in the absence of its H1-T6SS

Identification of T6SS-protective pathways

GacA is a key regulator involved in T6SS resistance

The mag operon is important for resistance against PG-targeting effectors

Arc1A, Arc3A and Aas contribute to resistance against lipase effectors

OprF is required for T6SS resistance and is genetically linked to GacA/S

T6SS resistance mechanisms are effector-type specific

T6SS resistance strategies can affect antibiotic susceptibility

Discussion

Methods

Bacterial strains and growth conditions

Mutant construction

Quantitative killing assays

CRISPRi library electroporation

CRISPRi screening competition

CRISPRi sequencing library preparation

Sample preparation for proteomics

SWATH-MS data acquisition

SWATH-MS data analysis

CPRG lysis assay

Flow cytometry

Growth curves

Fluorescence microscopy

Whole genome sequencing

Statistics

Reporting summary

Data availability

References

Acknowledgements

Author information

Authors and Affiliations

Contributions

Corresponding author

Ethics declarations

Competing interests

Peer review

Peer review information

Additional information

Supplementary information

Source data

Rights and permissions

About this article

Cite this article

Share this article

Search

Quick links