# Strain-specific variation in the complement resistome of Pseudomonas aeruginosa

Manon Janet-Maitre, Mylène Robert-Genthon, François Cretin, Sylvie Elsen, Ina Attrée

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Manon Janet-Maitre, Mylène Robert-Genthon, François Cretin, Sylvie Elsen, Ina Attrée. Strain-specific variation in the complement resistome of Pseudomonas aeruginosa. Infection and Immunity, 2025, 93 (9), pp.00055-25. 10.1128/iai.00055-25. hal-05288479

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# Strain-specific variation in the complement resistome of *Pseudomonas aeruginosa*

Manon Janet-Maitre,¹ Mylène Robert-Genthon,¹ François Cretin,¹ Sylvie Elsen,¹ Ina Attrée¹

**AUTHOR AFFILIATION** See affiliation list on p. 16.

**ABSTRACT** Bloodstream infections caused by *Pseudomonas aeruginosa* are associated with high mortality rates. The complement system, a key component of the innate immune response, plays a major role in eliminating *P. aeruginosa* from human blood. However, the sensitivity of *P. aeruginosa* strains to plasma varies widely, ranging from highly sensitive to persistent or fully resistant. Although most studies use model strains, the species genomic and phenotypic diversities suggest more complex interactions with complement than previously appreciated. In this study, we characterized the plasma resistome of *P. aeruginosa* using Tn-seq in three strains with varying levels of plasma sensitivity. A gain-of-function screen in the plasma-sensitive strain PA14 revealed numerous bacterial factors influencing plasma resistance, including components of the RetS-LadS/Gac/Rsm regulatory pathway and outer membrane porins. In the plasma-resistant strains CHA and YIK, Tn-seq analysis indicated that each strain relies on a distinct, limited set of proteins to evade complement-mediated killing. Despite these differences, we identified common mechanisms across all three strains. These include the production of exopolysaccharides (EPSs), the presence of surface appendages, and modifications in the O-specific antigen. Notably, we identified Ssg and Crc as shared contributors to plasma resistance. Although deletion mutants lacking *ssg* and/or *crc* exhibited reduced survival in plasma, a subpopulation of these mutants was able to persist during prolonged exposure. Overall, this work provides new insights into the complex interplay between *P. aeruginosa* and the human complement system in the context of bloodstream infections and raises concerns regarding the efficacy of therapies that target individual virulence factors.

**KEYWORDS** *Pseudomonas aeruginosa*, complement system, bloodstream infections, LPS, Tn-seq

Bacterial bloodstream infections (BSIs) represent a huge burden for modern healthcare. BSIs caused by *Pseudomonas aeruginosa* exhibit the highest mortality rate (1, 2). This leading nosocomial pathogen causes various primary infections, such as burn wounds, pulmonary and urinary tract infections (3). Using its arsenal of virulence factors, it is able to breach both epithelial and endothelial barriers ([4–7], reviewed in [8]). Once in the bloodstream, *P. aeruginosa* faces the host immune response. In a previous study, we showed the complement system to be the main innate immune component responsible for the elimination of *P. aeruginosa* in the blood (9). The complement system is an enzymatic cascade of more than 30 proteins, whose activation results in the formation of the membrane attack complex (MAC) in the outer membrane of the target pathogen. Complement-mediated killing happens through three main steps including complement activation, assembly of the C5b-9 MAC on the outer membrane, and penetration of the MAC into the bilayer, eventually leading to bacterial lysis (10, 11). *P. aeruginosa* evolved strategies to evade those three steps. The secretion of proteases including AprA and LasB, cleaving C1, C2, and C3 or the secretion of ecotin, a protease

**Editor** Kimberly A. Kline, Université de Geneve, Genève, Switzerland

Address correspondence to Ina Attrée, ina.attrée@ibs.fr, or Manon Janet-Maitre, manon@wustl.edu.

The authors declare no conflict of interest.

See the funding table on p. 16.

**Received** 28 January 2025

**Accepted** 24 June 2025

**Published** 11 August 2025

Copyright © 2025 Janet-Maitre et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
inhibitor, leads to the blockade of complement activation (12–14). *P. aeruginosa* can also
recruit host complement inhibitors to its surface, notably through the exposure of
the elongation factor Tuf or the dihydrolipoamide dehydrogenase Lpd ([15, 16], reviewed
in [8]). These immune evasion proteins can recruit the Factor H family of comple-
ment regulators, resulting in C3b degradation and preventing downstream proteolysis
cascade. Finally, *P. aeruginosa* can modify its surface by producing exopolysaccharides
(EPSs) such as alginate. When acylated, alginates decrease bacterial opsonization by the
C3b molecule (17, 18).

Sensitivity of *P. aeruginosa* to the complement system varies widely. The survival rate of strains in human plasma/serum ranges from full resistance to high sensitivity and persistence and was not found to correlate with the serotype or toxin content (9). Some strains, although mostly sensitive to plasma-dependent killing, persist over a long incubation in plasma (9). Herein, the terms "resistance" and "persistence" will be used as defined in the context of antibiotics, with "resistance" attributed to a heritable resistance factor and "persistence" describing a situation where a tolerant subpopulation is able to survive, whereas the rest of the population is eliminated (19). A gain-of-function screen identified the first molecular factors involved in *P. aeruginosa* persistence in plasma. However, the screen was performed with the clinical strain IHMA879472 (IHMA87), a member of a distant clade of *P. aeruginosa*, recently reclassified as *Pseudomonas paraeruginosa* (20–22).

Several studies documented the important genetic and phenotypic diversity of
bacterial strains within a *P. aeruginosa* species (22–24), with its pangenome accounting
for more than 50,000 genes (22). Given the high intra-species genomic diversity and
variability in plasma sensitivity, this work aims at characterizing strategies used by *P.
aeruginosa* to evade complement killing in the blood, using genome-wide transposon
mutagenesis (Tn-seq) on three strains belonging to different phylogenetic groups (9,
23–25). We found strain-specific sets of factors with only little overlap between strains.
However, some common strategies used by *P. aeruginosa* to evade plasma killing could
be drawn. We also report that targeting some factors may not eliminate resistant
bacteria from human plasma but generate a persistent subpopulation. This work not
only provides insight into a more global comprehension of *P. aeruginosa*-complement
system interplay but also raises questions on the targeted antimicrobial or anti-virulence
strategies developed on a unique bacterial strain.

RESULTS

Choice of strains and global Tn-seq analysis

The three strains used in this work belong to the two major clades of *P. aeruginosa* and have different characteristics in terms of plasma sensitivity, serotype, and toxin content (Fig. 1). The strains CHA, a hyper producer of the EPS alginate, isolated from the lungs of a cystic fibrosis patient (26, 27), and YIK, a recent bacteremia isolate (28), are both resistant to human plasma (~100% and ~91% survival, respectively, Fig. 1A). On the contrary, the laboratory strain PA14 (29) is highly sensitive to plasma and forms a persistent subpopulation, similarly to IHMA87 used in a previous study (Fig. 1A, [9, 30, 31]). PA14, CHA, and YIK harbor the type III secretion system (T3SS) with different toxin contents (Fig. 1B). IHMA87 synthesizes the ExIBA two-partner secretion system instead of the T3SS and is part of the third, the most distant phylogenetic group of *P. aeruginosa* (22, 23), recently reclassified as *P. paraeruginosa* (20).

We first generated transposon libraries in the three described strains, each containing
above 300,000 mutants using the Himar-1 transposon carried by pBTK24. Between 74%
and 91% of *P. aeruginosa* genes contained transposon insertions (between 5821-6125
genes in the genome), consistent with previous studies reporting an average of 400-800
essential genes per strain (Table S1, [33, 34]). The screen on IHMA87 (21) was done in
parallel, as a comparison for PA14. Transposon mutant libraries were challenged for 3 h in
90% pooled plasma from healthy donors (output) or heat-inactivated plasma (HIP),
unable to kill *P. aeruginosa*, as a control (input). Survivor cells were then recovered in a
<figure><img src="image_3.png" /><figcaption>FIG 1 *P. aeruginosa* strains show variable survival in plasma. (A) Bacterial survival after a 3 h incubation in human pooled plasma was calculated relative to T0 using colony-forming units (CFU) measurements. Data were log-transformed for one-way ANOVA statistical analysis followed by Tukey's test. All differences in survival between strains were found to be statistically significant except between IHMA87 and PA14. ns: non-significant. (B). Characteristics and origin of strains used in this study. ND (not determined).*: Recently reclassified as *P. paraeruginosa*. **: Reactivity against both serotype antibodies (9, 32). *: Gene ID found in supplemental tables.</figcaption></figure>

rich medium prior to genomic DNA extraction, bank preparation, and Illumina-based sequencing. The screening setup enables the identification of mutants with altered survival phenotypes by measuring changes in mutants' abundance across the genome, that is, decreased survival for plasma-resistant strains (CHA and YIK) or enhanced survival for plasma-sensitive strains (PA14). As expected, the overall survival of the pool of PA14 transposon mutants was about 3 logs higher than that of the parental strain, as already noted for the sensitive strain IHMA87 (Fig. S1, [21]).

Bioinformatics analysis of the sequencing reads was performed for both coding and intergenic regions due to the possible polar effect of the inserted transposon. Depending on the orientation and insertion site of the transposon, which has an outward-directed *P*<sub>tac</sub> promoter, the insertion can either inactivate the target gene or overexpress the neighboring gene(s), as shown in (33). The analysis of the Tn-seq data showed that plasma resistance of CHA and YIK relies on a few determinants as the screen identified 45 and 37 hits, respectively, using Log<sub>2</sub>(fold change (FC))>1 and adjusted *P*-val (*P*<sub>adj</sub>) <0.01 as thresholds (Fig. 2A through D; Tables S2, S3, S4 and S5).

Conversely, more than 600 hits were found to improve PA14 survival in plasma (682 hits with Log<sub>2</sub>(FC) >1, *P*<sub>adj</sub> <0.01, Tables S6 to S8). As observed in the volcano plot Fig. 2E and F, few transposon insertions negatively affected PA14 survival in human plasma
<figure><img src="image_4.png" /><figcaption>**FIG 2** Bioinformatic analysis of Tn-seq data both in genes and intergenic regions. Volcano plot representation of the data obtained for CHA (A, B), YIK (C, D), and PA14 (E, F) both in genes (A, C, E) and intergenic regions (B, D, F). Sequencing data were analyzed using the Galaxy platform (35) and visualized using GraphPad Prism 9. Pathways are highlighted in different colors. Dotted line represents a *P*<sub>val</sub> = 0.01.</figcaption></figure>

given the already high sensitivity of the parental PA14 strain to complement-dependent killing. Members of the RetS-LadS/Gac/Rsm regulatory system, involved in *P. aeruginosa* phenotypic switch between planktonic and biofilm lifestyles, had improved plasma resistance including *retS* and *rsmA* with Log₂(FC) of 18.7 and 17.2, respectively (Table S6, [36, 37]). Transposon insertion disrupting the gene of several outer membrane porins, for example, *oprD* and *oprP*, also drastically improved PA14 resistance to plasma with Log₂(FC) of 12.1 and 8.0, respectively. The role of OprF, another outer membrane porin, as a C3 binding acceptor molecule has been previously reported (38). This result suggests that other outer membrane porins may also serve as C3 acceptors. Additionally, disruption of genes involved in metabolisms of nucleotide (pyrimidines: *pyrC*, *pyrE*, and
purines: *purH*), amino acids (e.g., *glyS*, *hisM*), energy production (*sucC*), biotin biosynthesis (*bioA*), etc., led to increased plasma resistance, in line with the data from the strains IHMA87 (21).

Surprisingly, no hit was shared between the two plasma-resistant strains CHA and YIK (Fig. S2), although the presence and the synteny of most genes were conserved at the genomic level. When comparing the plasma-sensitive strain PA14 with the data obtained for IHMA87, 99 common genes showed $Log_2(FC) >1$, $P_{adj} <0.01$ (Fig. S2 and [21]). However, except for *retS*, which was one of the top hits for both PA14 and IHMA87 (Tables S6 to S8), the other hits had highly variable $Log_2(FC)$ between the two strains, suggesting that their contribution to protection against the plasma-dependent killing is variable among the strains. The limited redundancy found between the different strains is coherent with what was observed in a similar study on *Klebsiella pneumonia* (39). Although the individual gene sets were not strictly shared between the strains tested, we identified common strategies used to evade complement-mediated killing across strains.

## Contribution of EPS to survival in plasma is strain-dependent

*P. aeruginosa* can produce three types of EPS: Psl, Pel, and alginate. Both Psl and alginate were previously described to protect *P. aeruginosa* from opsonization by C3b ([17, 18, 40], reviewed in [8]). Here, we confirm EPSs, and particularly Psl, to have a central role in *P. aeruginosa* survival in plasma, but in a strain-dependent manner.

Psl is a neutral exopolysaccharide, which is important in *P. aeruginosa* attachment and maintenance of the biofilm architecture. Although Psl biosynthetic genes are encoded in the same locus, the detailed role of individual genes in Psl synthesis is still unclear (41). Briefly, Psl is thought to be produced by the Psl biosynthesis machinery. PslA is proposed to assemble oligosaccharide repeating units that are then polymerized by PslE. PslG controls the size of the oligosaccharide. Finally, Psl is exported to the bacterial surface through the transporter PslD (Fig. 3A; [41, 42]). Psl was shown to reduce bacterial opsonization including reduced C3b, C5, and C7 binding at the bacterial surface (40). However, the role of Psl in MAC-dependent bacterial killing is still under debate. Jones and Wozniak (43) showed a slight beneficial effect of Psl on the survival of *P. aeruginosa* mucoid strains to serum killing, which was not observed by Mishra et al. (40).

In the non-mucoid, plasma-resistant strain YIK, transposon insertions in three genes within the *psl* locus drastically reduced bacterial survival in plasma ($pslE - Log_2(FC) = -5.33$, *pslH - Log_2(FC) = -2.95*, *pslD - Log_2(FC) = -1.86*, (Fig. 2C, D and 3B; Tables S4 and S5). We engineered a *pslE* deletion mutant ($\Delta pslE$) in YIK and assessed its survival rate in human plasma. On average, 20 times fewer bacteria were recovered after the exposure of YIK$\Delta pslE$ to plasma compared with the parental strain, supporting the role of Psl in complement evasion in YIK (Fig. 3C).

Furthermore, the RetS-LadS/Gac/Rsm regulatory system, which participates in the phenotypic switch between planktonic and biofilm growth and regulates the expression of EPS loci (37, 44), was found as a hit in several strains. For example, in YIK, the deletion of *gacS* led to a 2-fold decrease in bacterial counts compared with the parental strain when challenged with plasma (Fig. 3C). Similarly, in the plasma-sensitive strain PA14, a transposon insertion mutant in the gene *retS*, isolated directly as the screen output (PA14::Tn*retS*), displayed a 1,000-fold increased survival ($Log_2(FC) = 18.66$) (Fig. 2E, F, 3D and E), in line with our results in the strain IHMA87 ($Log_2(FC) = 9.11$, [21]). The PA14 strain is a natural mutant in *ladS* and lacks several *psl* genes (45, 46). In addition, alginate is not typically produced by PA14 under standard conditions (47). Therefore, although RetS has a pleiotropic role, we hypothesize that the Tn insertion in *retS* probably activates the synthesis of Pel EPS, thereby improving PA14 survival in plasma.

Surprisingly, transposon insertion mutants in either Pel, Psl, or alginate biosynthetic pathways in hyper-mucoid strain CHA showed unaltered plasma resistance (Fig. S3; Tables S2 and S3). Accordingly, a nonmucoid *algD* mutant, in which the last step of the alginate biosynthetic pathway is disrupted (Fig. 3F), displayed comparable plasma resistance to the parental strains (Fig. 3G). Previous studies reported that alginates
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<figure><img src="image_11.png" /><figcaption>**FIG 3** Exopolysaccharides contribute to survival in plasma in a strain-dependent manner. (A) Scheme of Psl production and export in *P. aeruginosa*. (B) Tn-seq profile of *pslE* genomic environment in YIK showing the number of normalized reads in input (HIP) and output (plasma). (C) Bacterial survival after a 3 h incubation in human pooled plasma of YIK, YIKΔ*pslE*, and YIKΔ*gacS*. (D) Tn-seq profile of *retS* genomic environment in PA14, showing the number of normalized reads in input (HIP) and output (plasma). (E) Bacterial survival after a 3 h incubation in human pooled plasma of PA14 and PA14Tn::*retS*. (F) Schematic of alginate synthesis and export in *P. aeruginosa*. (G) Bacterial survival after a 3 h incubation in human pooled plasma of CHA and CHAΔ*algR* and CHAΔ*algD*. Note that the survival of CHA Δ*algD* is ~100%. A log transformation was performed on the data set and a one-way ANOVA, followed by Tukey's test. The dashed line represents the expected survival for the strain shown. ns: non-significant, \*: *p*<sub>val</sub> <0.05, \*: \*: *p*<sub>val</sub> <0.01.</figcaption></figure>

and especially acetylated alginates decreased C3b-mediated bacterial opsonization and therefore increased bacterial survival in clinical strains isolated from cystic fibrosis patients (17, 18), which was not confirmed in our work using the alginate hyper-producer
strain CHA. It is worth noting that transposon-insertion mutants in *algU* or *algR* showed decreased abundance following plasma challenge (Tables S2 and S3). Both genes, *algU* encoding the alternative sigma factor and *algR* encoding for a global regulator, are key players in *P. aeruginosa* virulence (48–51). However, the resistance to plasma of an *algR* deletion mutant was found unaffected when tested individually, as opposed to tested in-pool during the Tn-seq experiment (Fig. 3G). Due to the large size of AlgU and AlgR regulons involved in the response to envelope stress and virulence (49, 51), both proteins may affect CHA plasma resistance in an alginate-independent manner. Hence, although alginates can increase plasma resistance in sensitive strains (e.g. IHMA87 [21]), they are not crucial EPSs for evasion in other strains.

Together, our work shows that EPSs, mostly Pel and Psl, provide potent but strain-
dependent protection against the complement system in plasma.

Surface appendages favor survival in plasma

Bacterial flagella and pili are potent immune activators (52). In this work, we found that the type IV pili (T4P) and the flagellum are surface components providing an advantage to strains CHA and YIK in plasma (Fig. 4; Tables S2 to S5). In the screen with the strain CHA, the transposon mutants in 18 T4P-related genes had a reduced abundance in plasma compared with the inactivated plasma (Tables S2 and S3). In addition, in the strain YIK, Tn mutants were underrepresented for 12 flagella-related genes (Tables S4 and S5).

To validate the data, we created a *flgC* deletion mutant in YIK and a *pilQ* deletion mutant in CHA and challenged them individually in human plasma. In accordance with Tn-seq sequencing reads (Fig. 4A), the deletion in *flgC*, encoding a component of the flagellar basal body (53), resulted in a 4-fold drop in survival of YIK (Fig. 4B). Surprisingly, the deletion in CHA of a gene essential for pili synthesis, *pilQ* (54, 55), did not confirm the Tn-seq data (Fig. 4C and D). However, considering the Log₂(FC) and *P*-values of several distinct T4P-related hits in Tn-seq data sets, it is unlikely that pili genes were false positives. One hypothesis could be that the identified transposon mutants had a competitive disadvantage in the pooled Tn-seq screen compared with the challenge of an individual deletion mutant.

Several studies point toward a protective role of flagella and pili against serum-
mediated killing. In *Streptococcus pyogenes*, pili were shown to bind haptoglobin, an
immunosuppressive serum protein, and sequestration provided a survival advantage in
serum (56). Tad pili, one type of T4P in *Vibrio vulnificus*, protect bacteria from comple-
ment-mediated killing in serum (57). In addition to pili, flagellar components were also
found to be required for serum resistance, such as FliG in *Escherichia coli* (58).

Altogether, the flagellum contributes to YIK resistance to human plasma, potentially
by binding serum components, such as opsonins or other inhibitors, thereby reducing
complement-mediated lysis of *P. aeruginosa*.

ssg and crc mutants persist in the plasma

Previous studies showed that MAC insertion in the outer membrane is impacted by
the length of O-specific antigens (OSA - [59]). *P. aeruginosa* LPS is synthesized in two
forms, either bound with a D-Rhamnose homopolymer known as Common Polysacchar-
ide Antigen (CPA) or OSA, a heteropolymer of sugar repeats with repeat composition
determined by the *wbp*<sub>OSA</sub> locus (60, 61).

The length of newly generated OSA is regulated by Wzz1 and 2, before its attachment
to the nascent lipid A (59, 62, 63). In the mucoid and plasma-resistant strain CHA,
transposon insertions in wzy and wzz represented the two best hits and led to elimi-
nation of mutants from plasma (Log₂(FC) = -5.75 and Log₂(FC) = -3.71, respectively)
(Tables S2 and S3; Fig. 5A and B), which was confirmed by the complete elimination
of clean deletion mutants of either wzy or wzz in CHA (Fig. 5C). Similarly, a transposon
insertion in the promoter of wzz, probably leading to the overexpression of the wzz
gene, was enriched in plasma in the sensitive strain IHMA87 (21). Although wzz was
<figure><img src="image_12.png" /><figcaption>FIG 4 The strain-specific role of surface appendages in survival in plasma. (A) Tn-seq profile of the *flgC* genomic environment in YIK showing a number of normalized reads in input (HIP) and output (plasma). (B) Bacterial survival after 3 h incubation in human pooled plasma of YIK and YIKΔ*flgC*. (C) Tn-seq profile of *pilQ* genomic environment in CHA showing number of normalized reads in input (HIP) and output (plasma). (D) Bacterial survival after a 3 h incubation in human pooled plasma of CHA and CHAΔ*pilQ*. The dashed line represents the expected survival for the strain shown. The data were log-transformed, and a t-test was performed. ns: non-significant, \*: *P*<sub>val</sub> <0.05.</figcaption></figure>

not a significant hit in the PA14 Tn-seq data set, due to the high sensitivity of PA14 to human plasma, the zoomed Tn-seq profile suggests a similar role of *wzz* in PA14 plasma resistance (Fig. 5D). Altogether, our data are in agreement with the literature.

In addition to *wzz* and *wzy*, two other genes with a potential role in LPS synthesis were identified in the screen: *ssg* and *crc*. The inactivation of *ssg*, a conserved gene encoding a putative glycosyltransferase, in the YIK strain led to a drastic survival drop (Log₂(FC) = -4.31, Fig. 5E; Tables S4 and S5), whereas *crc*, with a Log₂(FC) = -2.86 was the second top hit in CHA (Fig. 5C; Tables S2 and S3).

A *crc* deletion mutant tested individually showed a drop in plasma resistance, reaching less than 1% survival after a 3 h challenge compared with the fully resistant parental strain. In the Tn-seq data, *crc* was neither found as a significant hit in the YIK strain nor was *ssg* in CHA. Therefore, we re-examined those genes in the original Tn-seq data sets. Although the data were not significant due to fewer insertion mutants in the input library, a similar trend could be observed in both strains (Fig. 6A through D).

We therefore engineered *ssg* and *crc* deletions in both strains. The four mutants displayed survival rates between 1.6% and 4.1% (Fig. 6E). Surprisingly, although deletions in *wzz* and *wzy* led to full bacterial elimination, *crc* and *ssg* mutants became more sensitive but were not fully eliminated after a 3 h challenge in plasma (Fig. 6E). We
<figure><img src="image_13.png" /><figcaption>**FIG 5** LPS is crucial for survival in plasma. Tn-seq profile of wzy and wzz genomic environment in CHA (A and B) showing a number of normalized reads in input (HIP) and output (plasma). (C) Bacterial survival after a 3 h incubation in human pooled plasma of CHA, CHAΔwzy, CHAΔwzz, and CHAΔcrc. nd: non detected. Data were log-transformed, and a one-way ANOVA and Tukey's test were performed. (D) Tn-seq profile of wzz genomic environment in PA14 in input (HIP) and output (plasma). (E) Survival after a 3 h incubation in human pooled plasma of YIK and YIKΔssg. Data were log-transformed, and a t-test was performed. \*: *P*<sub>val</sub> <0.05. \*:\*: *P*<sub>val</sub> <0.01. The dashed line represents the expected survival for the strain shown.</figcaption></figure>

therefore investigated the killing kinetics of *ssg* and *crc* mutants. The killing curves obtained by a challenge of the different mutants in plasma for up to 6 h were reminiscent of those obtained for persistent strains, PA14 or IHMA87 (9), with a rapid decrease in survival within initial 2 h and then a stabilization to the levels of 1%–10% (Fig. 6F and G). The plasma sensitivity after 3 h of a double mutant in YIK was lower than that of either single mutant individually, suggesting distinct roles of *ssg* and *crc* in plasma survival (Fig. 6E).

## Characterization of *ssg* and *crc* mutants for LPS and EPS

Literature mining suggested a possible link between *ssg* and *crc*, OSA, and EPS. A *ssg* mutant in *Pseudomonas alkylphenolia* was found to lack OSA and displayed altered composition of EPS lacking fucose and mannose (64). In *P. aeruginosa*, the *ssg* mutant in the PAO1 strain was impaired in auto-aggregation due to its LPS hydrophobic profile and loss of OSA, whereas in PA14, a *ssg* mutant displayed increased susceptibility to polymyxin B and increased permeability (65–67). *crc* encodes a post-transcriptional global regulator of carbon metabolism (68). Among other targets, *crc* regulates genes involved in LPS biosynthesis (68). A *crc* deletion mutant in PAO1 had an increased proportion of LPS molecules with longer OSA compared with the parental strain.

Given the known importance of LPS in bacterial sensitivity to plasma, we first analyzed LPS content of *Δcrc* and *Δssg* mutants in CHA and YIK genetic backgrounds. The LPS quantity and pattern were indistinguishable between *Δcrc* mutants and their respective parental strains (Fig. 6H), in contrast with the previous report (68). LPS extraction and staining do not enable clear discrimination between high molecular weight OSA and CPA. However, in agreement with the literature, fewer bands were observed in *Δssg* compared with both the WT and the *Δcrc* strains, suggesting the presence of CPA but the absence of OSA in *Δssg* (Fig. 6H, [65–67]). Additionally, the *Δssg*
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<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /><figcaption>FIG 6 *crc* and *ssg* mutants persist in plasma. (A) Tn-seq profile of *crc* (IPC1262_RS08345) genomic environment in CHA showing number of normalized reads in input (HIP) and output (plasma). (B) Tn-seq profile of *ssg* (153039_00322) genomic environment in YIK showing the number of normalized reads in input (HIP) and output (plasma). (C) Tn-seq profile of *crc* genomic environment in YIK and (D) *ssg* genomic environment in CHA. (E) Bacterial survival after a 3 h incubation in human pooled plasma of CHA, *CHAΔssg*, and *CHAΔcrc* and YIK, *YIKΔssg*, *YIKΔcrc*, and *YIKΔcrcΔssg*. Data were log-transformed for statistical analysis by a one-way ANOVA followed by a Welch and Brown-Forsythe test. (F) Survival kinetics of CHA, *CHAΔssg*, and *CHAΔcrc* over a 6-h incubation in human pooled plasma. (G) Survival kinetics of YIK, *YIKΔssg*, and *YIKΔcrc* over a 6-h incubation in human pooled plasma. (F, G) AUC of each biological replicate was calculated, and a one-way ANOVA was applied followed by Tukey's test. \*: *P*<sub>val</sub> <0.05, \*: \*: *P*<sub>val</sub> <0.01, \*: \*: \*: *P*<sub>val</sub> <0.001, \*: \*: \*: \*: *P*<sub>val</sub> <0.0001. ns: non-significant. (H). LPS extraction and silver stain of CHA, *CHAΔssg*, and *CHAΔcrc* and YIK, *YIKΔssg*, and *YIKΔcrc*. The blue box shows long O-specific antigens (OSA) in CHA and *CHAΔcrc*, which are absent in *CHAΔssg*. The red box shows very long OSA in YIK and *YIKΔcrc*, absent from *YIKΔssg*. (I) Congo red binding of colonies of WT, *Δssg*, and *Δcrc* in CHA and YIK genetic backgrounds.</figcaption></figure>
mutants lacked long OSA in CHA (Fig. 6H, blue box) and very long OSA in YIK (Fig. 6H, red box) and exhibited decreased amounts and shifts in the free LPS core, particularly striking in the CHA genetic background (Fig. 6H). In plasma challenge experiments, Δssg formed persisters, in contrast to Δwzz and Δwzy mutants (Fig. 6F and G, reviewed in [8]).

We then investigated the role of *crc* and *ssg* in EPS production using Congo red plates. CHAΔ*crc* and YIKΔ*crc* had apparent Psl production similar to that of the parental strain (Fig. 6I). Interestingly, the colony of CHAΔ*ssg* mutant was dark red and not shiny, indicating increased Psl production and reduced alginate production (69).

We conclude that although *crc* and *ssg* appeared as good targets in two resistant strains, their deletion led to persistence without complete bacterial elimination.

DISCUSSION

In this work, we used Tn-seq in three genetically and phenotypically diverse strains to investigate the convergence of molecular mechanisms interfering with the complement system cascade and subsequent bacterial killing. Data analysis revealed no universally shared factors involved in plasma resistance across strains, consistent with previous findings in *K. pneumoniae* strains exposed to serum (39). However, we could identify common strategies used by the different strains. In addition, the results confirmed for one strain may be useful to understand plasma resistance in other strains as we highlighted for *crc* and *ssg* phenotypic analysis.

*P. aeruginosa* genomic complexity has been highlighted by various studies (22, 23) serving as resources to adapt to different environments and stresses. In addition to the lack of conservation of some potential targets among strains, each strain has its own expression profile of virulence factors, which could also account for part of the diversity in molecular determinants for plasma resistance. In plasma-persistent strains, PA14 and IHMA87, the number of transposon insertions positively affecting global survival was more important, compared with the number of hits negatively affecting the resistance of CHA and YIK. Our results suggest a highly strain-specific defense mechanism against the complement system, involving a myriad of bacterial effectors that constitute a specific cocktail of bacterial factors, shaping a strain's resilience to complement-mediated killing (Fig. 7).

Poulsen and colleagues investigated the core essential genome of nine strains of *P. aeruginosa* in five infection-relevant media including heat-inactivated serum, sputum, and urine (34). They propose that four strains are sufficient to establish a reliable essential genome. In this work, the variability was such that four strains were insufficient to assess the full complexity of the host-pathogen interface in the plasma of *P. aeruginosa*. This underscores the limitations of drawing broad conclusions from single-strain studies in host-pathogen interaction research. Complement system-mediated killing is initiated onto the bacterial surface and results in the formation of a pore in the outer membrane, eventually leading to bacterial lysis and death. As suggested in *Borrelia burgdorferi*, the presence of various outer membrane proteins was responsible for bacterial resistance to MAC-dependent killing (70). This was also evidenced in *K. pneumoniae*, in which the deletion of the gene coding for the outer membrane lipoprotein LppA led to an increased bacterial susceptibility to complement-mediated killing (71). Although the exact subsequent mechanism is still under debate, two main hypotheses are drawn. The first would imply that the formation of the MAC pore on the outer membrane allows the passage of C9, subsequently forming pores in the inner membrane and causing bacterial killing. The other possible molecular mechanism implies that the formation of pores in the outer membrane destabilizes the envelope, leading to the loss of envelope integrity and subsequent bacterial death. Both hypotheses were discussed in reference (72). The main resistance mechanism involves the inhibition of opsonization on the bacterial envelope and degradation of complement components (71). To reduce opsonization, bacteria can secrete EPS or modify their outer membrane composition to reduce the number of opsonin acceptors such as OprF (38). For this reason, we expected an enrichment of outer membrane proteins as the top hits of the screen. Surprisingly, the
<table><caption>**FIG 7** The plasma resistance of each *P. aeruginosa* strain depends on a unique combination of molecular factors. Dots represent the implication of the corresponding gene or pathways in resistance to plasma in the indicated strain either confirmed experimentally on individual mutants (darker color) or in Tn-seq data (lighter dots for CHA, PA14, and IHMA87) in this work or in (21).</caption><thead><tr><th></th><th></th><th>CHA</th><th>YIK</th><th>PA14</th><th>IHMA87</th></tr></thead><tbody><tr><td rowspan="3">EPS</td><td>Alginate</td><td></td><td></td><td>●</td><td>●</td></tr><tr><td>Psl</td><td></td><td>●</td><td></td><td>●</td></tr><tr><td>GacS/RetS</td><td></td><td>●</td><td>●</td><td>●</td></tr><tr><td rowspan="3">LPS</td><td>wzz</td><td>●</td><td></td><td>●</td><td>●</td></tr><tr><td>wzy</td><td>●</td><td></td><td></td><td></td></tr><tr><td>ssg</td><td>●</td><td>●</td><td></td><td></td></tr><tr><td colspan="2">crc</td><td>●</td><td>●</td><td>●</td><td></td></tr><tr><td rowspan="2">Motility</td><td>Flagellum</td><td>●</td><td></td><td>●</td><td>●</td></tr><tr><td>Cup pili</td><td></td><td></td><td>●</td><td>●</td></tr><tr><td colspan="2">PolyP</td><td>●</td><td></td><td>●</td><td>●</td></tr><tr><td colspan="2">Biotin</td><td></td><td></td><td>●</td><td>●</td></tr><tr><td colspan="2">Purine</td><td></td><td></td><td>●</td><td>●</td></tr><tr><td colspan="2">srgA</td><td></td><td></td><td></td><td>●</td></tr></tbody></table>

outer membrane proteins accounted for only 9.3%, 0%, 2.9%, and 3.2% for CHA, YIK, IHMA87, and PA14, respectively (Fig. S4). Conversely, all the strains had a high proportion of inner membrane-associated genes in the list of significant hits of about 25%–30%. These results are in agreement with the work of Heesterbeek et al. (72), which suggests the permeabilization of the inner membrane to be the rate-limiting step in MAC-dependent killing. In addition, the screen reports many cytoplasmic hits, which may exert an indirect function onto the bacterial envelope or metabolism to help the bacterium cope with the complement system-derived envelope stress. Global strategies, highlighted in other species such as *K. pneumoniae* or *Acinetobacter baumannii* (39, 73), were shared across strains including the production of protective exopolysaccharides and the synthesis of OSA. However, the production of motility appendages, which could protect bacteria from the complement-mediated killing effect, potentially sequestering harmful serum proteins, was never highlighted in genome-wide screens. In contrast, other screens identified the maintenance of lipid asymmetry (*mla*) as a central pathway in serum resistance (73). Mutants in the *mla* locus were also previously identified in *P. aeruginosa* PAO1 strains to be more sensitive to serum killing (74). However, in our screen, the survival of mutants in the MLA pathway was found to be negatively impacted by insertions in *mlaE* and *mlaF* only in the strain YIK (Tables S4 and S5, *mlaE* - Log₂(FC) = -1.43, *mlaF* - Log₂(FC) = -1.21).

In this work, we also confirmed that persistence in plasma is a widespread mechanism of *P. aeruginosa* evasion, as resistant strains may become persistent by mutations in their genome. These mutations affecting bacterial surface (e.g., *ssg* mutant) or global metabolism (e.g., *crc* mutant) led to the persistence of a bacterial subpopulation of otherwise plasma-resistant strains. It is worth noting that the complement system is the main immune component responsible for the elimination of *P. aeruginosa* in the blood (9); however, it works in concert with the cellular arm of innate immunity to eliminate invaders. Therefore, genome-wide approaches involving immune cells should be considered for further studies. Given that blood from different donors cannot be pooled, this would require a high number of samples to average the immune variability between individuals. Alternatively, purified innate immune cells together with plasma could be used to screen transposon mutant libraries.
Overall, this work identifies many strain-specific bacterial factors as important in plasma resistance, which constitutes a valuable source of information for future work. This study not only gives insight into a more global comprehension of *P. aeruginosa*/complement system interplay but also raises some concerns about the targeted antimicrobial or anti-virulence strategy, calling for more holistic studies.

# MATERIALS AND METHODS

## Bacterial strains and genetic manipulations

Bacterial cultures were performed in Lysogeny Broth (LB) at 37°C with shaking. LB plates containing irgasan (25 µg/mL) were used to select for *P. aeruginosa*. Antibiotic concentrations used were: 75 µg/mL gentamicin for *P. aeruginosa* and 50 µg/mL gentamicin and 100 µg/mL ampicillin for *E. coli*. All bacterial strains, plasmids, and primers are listed in Tables S9 and S10 Deletion mutants were engineered by amplifying approximately 500–700 bp upstream and downstream flanking regions of the gene of interest, according to (75), from genomic DNA by PCR using appropriate primer pairs (sF1/sR1, sF2/sR2). Subsequently, overlapping fragments were cloned into *SmaI*-digested pEXG2 using Sequence- and Ligation-Independent Cloning (SLIC, [76]). Using triparental mating, allelic exchange vectors were introduced into *P. aeruginosa*, with pRK600 as a helper plasmid. Selection of merodiploid clones resulting from homologous recombination was performed on LB agar plates containing irgasan and gentamicin. Merodiploids colonies were streaked onto NaCl-free LB plates with 10% sucrose (wt/vol) selected for plasmid loss. The resulting clones were screened for gene deletion and antibiotic sensitivity by PCR with appropriate primer pairs.

## Generation of transposon libraries

Libraries were generated as previously described in (21). Briefly, *P. aeruginosa* strains (CHA, YIK, and PA14) were grown overnight from an isolated colony at 37°C with shaking and mixed with *E. coli* SM10 carrying pBTK24 ([33], grown overnight with ampicillin). Biparental conjugation was performed, mixing equal volumes of *P. aeruginosa* and *E. coli* strains. Bacteria were pelleted by centrifugation and resuspended in fresh LB, resulting in a 5-fold concentrated bacterial suspension. Approximately 15 40-µL puddles were spotted onto dry LB plates and incubated at 37°C for 4 h. Bacteria were scraped off the plates and resuspended into LB. Bacterial suspension was then spread onto irgasan- and gentamicin-containing plates and grown at 37°C. Transposon mutant colonies were collected by plate flushing with LB and scraped off using a loop. Glycerol was added to a 20% final concentration. The resulting transposon libraries were aliquoted and stored at -80°C until use.

## Plasma pool preparation

Heparinized human blood from healthy donors was provided by the French National Blood Institute (EFS, Grenoble, France). Fresh blood samples were centrifuged for 10 min at 1,000 × g at room temperature. Plasma-containing supernatants of 10 donors were pooled and filtered through a 0.45 µm membrane. Pooled plasma was then aliquoted and stored at -80°C until needed. Prior to use, pooled plasma was thawed at 4°C and centrifuged for 10 min at 10,000 rpm on a microcentrifuge, and the supernatant was filtered through a 0.22-µm membrane. To prepare heat-inactivated plasma (HIP), pooled plasma was thawed at 4°C and inactivated by a 30 min incubation at 56°C followed by a 10 min centrifugation step at 10,000 rpm and filtration through a 0.22-µm membrane.

## Screen in plasma

Screen in plasma was performed as described in (21). Briefly, transposon mutant libraries were thawed on ice and diluted in 30 mL of LB. Bacteria were grown for 16 h at
30°C. Bacterial culture was diluted to OD₆₀₀nm~0.1 and grown at 37°C under agitation. When cultures reached an OD₆₀₀nm~1, bacteria were harvested and resuspended in PBS supplemented with calcium and magnesium ions (PBS +/-, Thermofisher Scientific). Plasma and HIP were inoculated with a final bacterial concentration of 2.25 x 10⁷ CFU/mL (90% final plasma concentration). The initial count was determined by CFU counting, and samples were incubated for 3 h at 37°C on a rotating wheel. Bacterial survival at the end of the incubation was assayed by CFU counting. PA14 was spread onto LB plates to isolate transposon mutants with increased survival in plasma. After the challenge, bacteria were recovered in LB and allowed to grow overnight. Clonal populations of the corresponding wild-type strains were challenged in parallel for survival comparison. About 10⁹ bacteria were harvested for genomic DNA extraction and library constructions. The experimental procedure was performed in biological duplicates.

## Library construction, sequencing, and data analysis

### Genomic DNA extraction

Bacterial pellets were harvested from the screen and lysed by SDS-NaCl lysis (2% SDS, 0.1M EDTA pH8, 0.6M sodium perchlorate, 0.15M NaCl), followed by two successive steps of phenol-chloroform extractions. Genomic DNA (gDNA) was precipitated in cold ethanol absolute and diluted in the TE buffer (10 mM Tris-HCl pH8, 1 mM EDTA). gDNA was mechanically sheared at 4°C using a QSonica sonicator (Q700, 40% amplitude) for 20 min with 15-s pulses. gDNA fragments, with a size of 150×400 bp, were then concentrated in monarch columns (PCR & DNA cleanup kit, NEB) and controlled on 2% agarose gel.

### Library construction and sequencing

Fragmented gDNA was end-repaired using an End-repair module (NEB#E6050) followed by a dA-tailing with Klenow (NEB#6053). Annealing of short and long adaptors (Table S10) was performed in a thermocycler with a program allowing a decrease of 1°C/cycle, from 95°C to 20°C. Annealed gDNA fragments were then ligated at 16°C with T4 DNA ligase (NEB#M0202). Fragments of 200×400 bp were selected on a 2% agarose gel and purified from the gel (Monarch DNA Gel Extraction Kit, NEB#T1020L). Purified DNA-adaptor fragments were amplified using the Phusion polymerase (NEB#M0530) and a specific pair of primers (PCR1 Tn-spe direct and PCR1 Adaptor comp.). A second amplification step was performed using primers carrying the P5 and indexed-P7 Illumina sequences (NEB#E7335S). At each step, double-stranded DNA (dsDNA) concentration was assessed using the Qubit dsDNA HS assay kit (Q33230). Finally, the library's quality was assessed on the Agilent Bioanalyzer 2100 (HS DNA Chips Ref #5067-4626). and sequencing was performed on an Illumina Nextseq High (I2BC, Saclay Paris).

### Data analysis

Following quality control of the data, sequencing reads were trimmed and aligned to the corresponding genome using Bowtie (77). Read count per feature was calculated by Htseq-count (78), and differential representation of the insertion mutants between the heat-inactivated control and the plasma sample was performed using DESeq2 (79). Concerning the analysis of Tn insertion in the intergenic regions, annotation files were created for each strain attributing the intergenic region to the downstream gene, independent of its orientation. Alignment files produced by Bowtie were then used with these new annotation files and analyzed by Htseq-count and DESeq2. All analyses were performed on the galaxy platform (35). In order to compare data from each strain, a BLAST reciprocal best (80, 81) hit analysis was performed, blasting all the proteins from each strain against PAO1 proteins with a 90% coverage and 80% identity. Reference genomes were downloaded from NCBI; EC2A-CHA: GCF_003698505.1, YIK: JAPTST000000000, UCBPP-PA14: GCF_000014625.1.
## Gene equivalent between strains

Gene equivalents among the strains of interest were determined using Blast Reciprocal Best Hits on Galaxy version 0.3.0 (usegalaxy.eu). Genes were considered equivalent when coverage was higher than 90% and identity higher than 80%.

## Plasma-killing assay

Plasma-killing assays were performed as described in (9). Bacterial cultures were inoculated with an OD<sub>600nm</sub> of 0.1 and grown at 37°C with shaking until they reached an OD<sub>600nm</sub> of 1. Bacteria were harvested and resuspended in PBS +/-, and plasma was inoculated with a final bacterial concentration of 2.25 x 10<sup>7</sup> CFU/mL (90% final plasma concentration). Initial and final bacterial loads were assessed by CFU counting at indicated times.

## LPS extraction and visualization

LPS was extracted by the hot phenol-water method as described previously (82, 83) with some modifications. Briefly, 30 mL of bacterial culture grown in LB to exponential phase (OD<sub>600nm</sub> =1) of CHA or YIK wild-type or mutants were pelleted by centrifugation and resuspended in 1 mL of 20 mM Tris pH8, 20 mM MgCl<sub>2</sub>, 50 mM NaCl buffer. Sonication was performed for 20 min at 4°C (10 s on/10 s off, 40% amplitude) using a QSonica Oasis-180. In order to eliminate proteins and nucleic acids, treatment with DNase (Euromedex 1307, 50 µg/mL) and RNase (Sigma R6513, 40 µg/mL) was carried out for 1 h at 37°C followed by Proteinase K (Roche-311879001) treatment for 1 h at 65°C and an overnight incubation at 37°C. Then, two successive hot phenol-water extractions (Invitrogen 15594-04) were carried out. Briefly, an equal volume was added, incubated for 10 min at 65°C with vigorous shaking every minute, then centrifuged for 10 min at 20,000 x g at 4°C. The aqueous phase was precipitated by 0.5M sodium acetate (pH 5.2) with 10 volumes of cold ethanol and stored at -20°C. After centrifugation 10 min 20,000 x g at 4°C, the pellets were washed with water and dialyzed. The final purified LPS product was lyophilized and stored at -20°C. Freeze-dried samples were resuspended in SDS-PAGE buffer and boiled for 5 min. Samples were loaded onto a NuPAGE Bis-Tris 4%-12% gel (Invitrogen). LPS were stained using the ProQ Emerald 300 lipopolysaccharide gel stain kit (Molecular Probe P20495).

## CONGO red plates

Five microliters of culture at OD<sub>600nm</sub> of 1 were spotted on the BHI agar plate (Brain Heart Infusion Broth - Sigma 53286) containing 5% sucrose and 0.8 g/L Congo red (Sigma C6767). Plates were incubated for 24 h at 37°C and at 4°C before imaging.

## Statistical analysis

Statistical analyses were performed using SigmaPlot and Graphpad Prism 9 software. The tests used are specified in figure legends. Survival data were log<sub>10</sub>-transformed. Figures were performed using GraphPad Prism 9 and Inkscape.

## ACKNOWLEDGMENTS

The authors thank Peter Panchev and Emily Rey for their help with transposon mutant libraries construction, and Joanna Goldberg and Dina A. Moustafa for their valuable discussion.

The work was supported by grants from Agence Nationale de la Recherche (ANR-15-783 C.E. CE11-0018-01), the Laboratory of Excellence GRAL, financed within the University Grenoble Alpes graduate school (Écoles Universitaires de Recherche) CBH-EUR-GS 785 (ANR-17-EURE-0003), the Fondation pour la Recherche Médicale (Team FRM 2017, 786 DEQ20170336705) to I.A. The sequencing part has benefited from the
facilities and expertise of the high throughput sequencing core facility of I2BC (Centre de Recherche de Gif- http://www.i2bc.paris-saclay.fr/). M.J.-M. received a Ph.D. fellowship from the French Ministry of Education and Research.

## AUTHOR AFFILIATION

¹University Grenoble Alpes, Institut de Biologie Structurale-IBS, UMR5075, Team Bacterial Pathogenesis and Cellular Responses, Grenoble, France

## PRESENT ADDRESS

Manon Janet-Maitre, Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA

## AUTHOR ORCIDs

Manon Janet-Maitre http://orcid.org/0000-0001-8954-7245

Ina Attrée http://orcid.org/0000-0002-2580-764X

## FUNDING

<table><thead><tr><td>Funder</td><td>Grant(s)</td><td>Author(s)</td></tr></thead><tbody><tr><td>Agence Nationale de la Recherche</td><td>ANR-15-783 CE11-0018-01, ANR-17-EURE-0003</td><td>Ina Attrée</td></tr><tr><td>Fondation pour la Recherche Médicale</td><td>786 DEQ20170336705</td><td>Ina Attrée</td></tr></tbody></table>

## AUTHOR CONTRIBUTIONS

Manon Janet-Maitre, Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing – original draft, Writing – review and editing | Mylène Robert-Genthon, Investigation, Methodology, Project administration, Validation, Visualization | François Cretin, Investigation, Methodology, Validation | Sylvie Elsen, Investigation, Methodology, Writing – review and editing | Ina Attrée, Conceptualization, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing – review and editing

## DATA AVAILABILITY

The Tn-seq data generated in this work is available at GEO under the series accession number GSE287536.

## ADDITIONAL FILES

The following material is available online.

### Supplemental Material

Supplemental material (IAI00055-25-s0001.docx). Fig. S1 to S4; Tables S1 and S2, S4, S6 and S7, S9 and S10.

Table S8 (IAI00055-25-s0002.xlsx). Tn-seq data for the strain PA14.

Table S5 (IAI00055-25-s0003.xlsx). Tn-seq data for the strain YIK.

Table S3 (IAI00055-25-s0004.xlsx). Tn-seq data for the strain CHA.

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