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Genome-wide screen in human plasma identifies multifaceted complement evasion of Pseudomonas aeruginosa [1]

['Manon Janet-Maitre', 'University Grenoble Alpes', 'Institute Of Structural Biology', 'Team Bacterial Pathogenesis', 'Cellular Responses', 'Grenoble', 'Stéphane Pont', 'Frerich M. Masson', 'Medical Microbiology', 'University Medical Center Utrecht']

Date: 2023-04

Pseudomonas aeruginosa, an opportunistic Gram-negative pathogen, is a leading cause of bacteremia with a high mortality rate. We recently reported that P. aeruginosa forms a persister-like sub-population of evaders in human plasma. Here, using a gain-of-function transposon sequencing (Tn-seq) screen in plasma, we identified and validated previously unknown factors affecting bacterial persistence in plasma. Among them, we identified a small periplasmic protein, named SrgA, whose expression leads to up to a 100-fold increase in resistance to killing. Additionally, mutants in pur and bio genes displayed higher tolerance and persistence, respectively. Analysis of several steps of the complement cascade and exposure to an outer-membrane-impermeable drug, nisin, suggested that the mutants impede membrane attack complex (MAC) activity per se. Electron microscopy combined with energy-dispersive X-ray spectroscopy (EDX) revealed the formation of polyphosphate (polyP) granules upon incubation in plasma of different size in purD and wild-type strains, implying the bacterial response to a stress signal. Indeed, inactivation of ppk genes encoding polyP-generating enzymes lead to significant elimination of persisting bacteria from plasma. Through this study, we shed light on a complex P. aeruginosa response to the plasma conditions and discovered the multifactorial origin of bacterial resilience to MAC-induced killing.

Persistence of bacterial pathogens is a main cause of treatment failure and establishment of chronic bacterial infection. Despite innate immune responses, some bacteria may persist in human blood and plasma. Here we used a genome-wide screen to investigate the molecular determinants influencing Pseudomonas aeruginosa survival in human plasma facing the complement system. Alongside a multifactorial strategy that include surface-attached molecules and bacterial adaptation to stress, we found that intracellular polyphosphates and biotin significantly influence bacterial capacity to deal with membrane attack complex (MAC)-dependent killing. These results underline the need to understand the complex interplay between bacterial pathogens and the human immune system when seeking to develop efficient antibacterial strategies.

Funding: The work described in this paper was supported by grants from the French national agency for research (Agence Nationale de la Recherche; ANR-15-CE11-0018-01), the Laboratory of Excellence GRAL, funded through the University Grenoble Alpes graduate school (Écoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003), the Fondation pour la Recherche Médicale (Team FRM 2017, DEQ20170336705) to I.A., and the European Union's Horizon 2020 research programs H2020-EU-ITN-EJD (CORVOS No #860044 to F.M. and S.H.M.R.). This work availed of the platforms at the Grenoble Instruct-ERIC center (ISBG; UAR 3518 CNRS-CEA-UGA-EMBL) within the Grenoble Partnership for Structural Biology (PSB), supported by FRISBI (ANR-10-INBS-0005- 02) and GRAL, funded through the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche) CBH-EUR-GS (ANR-17-EURE-0003). S.P, J.T and M.JM were recipients of Ph.D. fellowships from the French Ministry of Education and Research. S.S received the Master 2 GRAL fellowship. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are either within the paper and its Supporting Information files or available through the NCBI Gene Expression Omnibus (GEO) under super-series accession number GSE192831, or by using accession numbers GSE192769 and GSE192761 for the Tn-seq and RNA-seq complete data sets, respectively.

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

For this study, we employed a genome-wide approach to obtain insights into P. aeruginosa factors impacting levels of persistence in human plasma. A screen based on transposon-insertion sequencing (Tn-seq) was applied to a recent clinical isolate that was sensitive to plasma but produced between 0.01% and 0.1% of evaders after 6 h in plasma. We hypothesized that transposon mutants for which an increased proportion of evaders was detected or mutants with overall increased plasma tolerance/resistance could reveal novel determinants of bacterial resilience to plasma and pathogen immune escape. In addition to P. aeruginosa factors known to be involved in resistance to CS, such as O-specific antigen (OSA) length and exopolysaccharides, we identified an uncharacterized small periplasmic protein that increases complement tolerance up to 100-fold. Our screen and the associated phenotypic characterization of mutants also revealed that biotin, ATP, and polyphosphate (polyP) levels modulate pathogen sensitivity to MAC-induced lysis.

The CS, which is the main immune component of blood responsible for bacterial elimination, is generally efficient against P. aeruginosa sensitive strains. However, a tiny fraction of the initial bacterial population can persist despite prolonged incubation in blood or plasma [ 11 ]. The kinetics of P. aeruginosa killing by human plasma displays a classical persister-like biphasic profile [ 3 ], with rapid elimination of up to 99.9% of the population, reaching a plateau at an average of 0.1% survival after 2 h. In our previous study, we found that, out of 11 BSI isolates tested, 3 were plasma-resistant, 3 displayed a tolerant phenotype, and 5 were able to form evaders. The formation of evaders may represent a frequent strategy used by P. aeruginosa to escape MAC-mediated bactericidal activity in plasma. The plasma evaders were distinct from antibiotic persisters since a stationary phase population did not give rise to a higher population of evaders, as reported for antibiotic persisters [ 18 ].

P. aeruginosa is a leading nosocomial pathogen that is extremely difficult to eradicate due to its high adaptability to a range of environments and its intrinsic and acquired antibiotic resistance [ 12 ]. Bloodstream infections (BSI) due to P. aeruginosa strains hold the record for highest mortality rate, at about 40% [ 13 , 14 ]. The complement system (CS) is a complex multi-protein cascade and a major innate immune component in human blood responsible for Gram negative bacterial killing. The complement cascade can be activated through three pathways (classical, lectin and alternative) which all converge to the formation of the C3 convertase. C3 proteolysis by the C3 convertase releases C3b, which opsonizes the bacterial surface, in turn forming the C5 convertases. C5b and C6 interaction initiate the binding of C7 and C8 at the membrane [ 15 ]. The final step is the polymerization of multiple copies of C9 to form a C5b-9 pore in the bacterial outer membrane [ 16 ]—the membrane attack complex (MAC)—which leads to inner membrane damage and bacterial lysis [ 17 ].

Human bacterial pathogens employ sophisticated strategies to escape control by the immune system and to resist or tolerate antibiotic treatments. Bacterial persistence toward antibiotics is a major obstacle in the treatment of life-threatening infections. It is frequently associated with the establishment of chronic infections, as it allows the survival of a subpopulation of bacteria that are tolerant to otherwise lethal antibiotic stress [ 1 , 2 ]. Unlike antibiotic resistance, persistence is a non-heritable, fully reversible trait characterized by a biphasic killing curve–with rapid killing of the bulk population and survival of a subpopulation over a long period of time. Among the factors hypothesized to lead to antibiotic persistence [ 3 ], the most cited are low intracellular ATP levels [ 4 , 5 ] and production of the “alarmone” signaling molecule guanosine (penta) tetraphosphate, (p)ppGpp [ 6 , 7 ]. The host immune system also elicits persistence in a number of bacterial species. For example, acidification of macrophage vacuoles after Salmonella internalization induces persister cell formation and could contribute to establishing a bacterial reservoir for infection relapse [ 8 ]. In a similar manner, exposure to human serum triggers the formation of antibiotic persisters as well as so-called “viable but non-culturable” forms of Vibrio vulnificus [ 9 ]. Putrinš et al. [ 10 ] reported that phenotypic heterogeneity in Escherichia coli could serve as a mean to evade serum-mediated and antibiotic-induced killing. In a previous study [ 11 ], we demonstrated that a persister-like sub-population of Pseudomonas aeruginosa evaders forms when generally sensitive bacteria are incubated in human blood or human plasma. In this article, we refer to resistance, tolerance, and persistence as defined in a context of exposure to antibiotics [ 3 ]. Briefly, resistance is due to a heritable resistance factor allowing survival in the face of higher stress conditions. In contrast, tolerance is a phenomenon that allows survival of the population despite an otherwise lethal stress for a longer period of time, but without deploying any resistance mechanism. The third term, persistence, is the capacity of a tolerant sub-population to survive whereas the rest of the population is eliminated. Finally, we used the term resilience to describe several of these phenomena.

Results

Gain-of-function screen in plasma In our previous study, we found that the P. aeruginosa isolate IHMA879472/AZPAE15042 (IHMA87) persisted in human blood and plasma. Although the overall population was mostly sensitive to complement killing, approximately 0.1% of evaders survived prolonged incubation in plasma [11]. To search for mutants generating higher numbers of evaders using a genome-wide approach, we constructed a transposon library of about 300,000 mutants in IHMA87 using the Himar-1 transposon. The transposon library was challenged in native human plasma (output) or heat-inactivated plasma (HIP, input)—devoid of complement activity—for three hours before harvesting (Fig 1A). Bacterial survival was expressed as colony forming unit (CFU) counts for the whole library before and after the challenge. This analysis revealed that overall survival was increased by about 2-log in the mutant library compared to the parental strain. Thus, the screen selected for mutants with increased survival. Resistant, hyper-evader, and tolerant phenotypes could be differentiated based on killing kinetics of isolated mutants during longer incubation in plasma. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Gain-of-function Tn-seq reveals common and novel pathways contributing to P. aeruginosa survival in plasma. A. Schematic representation of the screening method used for this study. A transposon-insertion mutant library was generated in the plasma-sensitive clinical isolate P. aeruginosa IHMA87 and exposed either to human plasma or to HIP (input) for 3 h. Aliquots were plated on LB and individual transposon mutants were stored for further analysis. B. Bioinformatics analyses presented as Volcano plots. Insertions in coding regions (left) and intergenic regions (right) were analyzed separately. Significant hits in genes involved in the same pathway are shown in the same color. C. Survival of isolated transposon mutants following incubation for 3 h in plasma. Survival rates were calculated based on CFU measurements. The dataset was log-transformed, and rates for all mutants were significantly different from rates for the wild-type strain (p-value < 0.005). Log 2 (Fold-Change (FC)) obtained from the Tn-seq analysis for each gene or intergenic region is indicated above the histogram. https://doi.org/10.1371/journal.ppat.1011023.g001 Tn-seq data was analyzed both for insertions in coding regions and in intergenic regions, as the transposon included an outward tac promoter, which could modulate the expression of neighboring genes. In both analyses, transposon insertions strongly increasing bacterial survival were associated with three main pathways: biosynthesis of purine and biotin; and biosynthesis, production, and regulation of the alginates (Fig 1B and Table 1, full results in S1 Table). In addition, several novel determinants worth exploring were identified (Table 1).

Analysis of Tn mutants isolated during the screen In parallel to Tn-seq, ten transposon mutants were isolated during the screen, targeting seven different genes, and their survival was assayed in plasma killing assays. Survival rates for these mutants were between 10- and 200-fold higher than that of the parental strain (Fig 1C), thus validating the screen. Of ten mutants, three mutants displayed hyper-mucoid phenotypes–reflecting alginate overproduction–associated with insertions within mucD and in the intergenic region upstream mucA. P. aeruginosa can produce three main exopolysaccharides; alginates, Psl and Pel [19,20]. Expression of the alginate biosynthetic genes is regulated by the extracytoplasmic function (ECF) sigma factor AlgU. In planktonic growth, AlgU is retained at the bacterial inner membrane by its anti-sigma factor MucA, repressing alginate production [21]. MucD acts upstream of MucA and indirectly acts as a negative regulator of the release of AlgU in response to the presence of misfolded outer membrane proteins [22]. We also isolated a mutant in which the transposon was inserted into the intergenic region upstream of mucE, encoding a positive regulator of alginate production [23,24]. The orientation of the transposon and its mucoid phenotype suggested an overexpression of mucE. As previously reported, alginate overproduction may interfere with the CS by decreasing C3b-dependent opsonization and thus preventing CS activation upon alginate acetylation [25,26]. One of the mutants with increased survival carried a transposon insertion in ladS. LadS is a part of a signaling pathway RetS/LadS/Gac/Rsm regulating Psl exopolysaccharides production. This complex regulatory system which manages P. aeruginosa phenotypic switch from planktonic to biofilm lifestyle is composed of a central two-component regulatory system GacA/GacS modulated by two hybrid sensor kinases, LadS, a positive regulator of the pathway, and RetS, which represses the system. When activated, the GacA/GacS two-component regulatory system represses the Psl production [27–30]. In accordance with our data, LadS-regulated exopolysaccharide Psl were reported to improve bacterial survival of a mucoid strain in serum [31]. However, the role of Psl in complement resistance might be strain-dependent, as assays using the reference strain PAO1 initially showed that Psl reduces bacterial opsonization without affecting C9 insertion or bacterial survival in serum [32]. Survival in plasma was also increased by insertion of the transposon in the promoter of pprB (survival ~50%, Fig 1C). PprB is a regulator acting on type IVb pili/Tad, BapA adhesin and CupE fimbriae expression [33,34], suggesting that type IVb pili or CupE fimbriae could be also involved in P. aeruginosa plasma resilience. As shown in Vibrio cholerae and Neisseria gonorrhoeae, type IV pili contribute to serum resistance by recruitment of the host negative complement regulator C4BP [35,36]. Although none of the ten randomly picked colonies had a transposon insertion in LPS-related genes, the screen did confirm the importance of LPS and more specifically OSA biosynthesis in the bacteria’s interaction with plasma with enrichment of mutants with Tn insertion upstream of wzz1 (P wzz1 , Log 2 (FC) of 12.59, Table 1). Wzz1 is one of the OSA chain length regulators [37–40]. Finally, in four isolated mutants with increased survival, the transposon was inserted into bioA and bioB, encoding enzymes involved in biotin biosynthesis (~12% survival each, Fig 1B). To our knowledge, these genes have not previously been associated with plasma or complement resistance. PPT PowerPoint slide

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TIFF original image Download: Table 1. Mutants enriched in plasma compared to HIP carry insertions in three main pathways. https://doi.org/10.1371/journal.ppat.1011023.t001

Expression of the three-gene operon srgABC improves survival in plasma We focused first on a mutant carrying the insertion in an intergenic region between a three-gene operon IHMA87_01573-IHMA87_01571 of unknown function, (PA3369-PA3371 in PAO1, renamed srgABC) and panM (IHMA87_01574/PA3368), coding for a probable acetyltransferase (Fig 2A). This mutant was significantly enriched in Tn-seq datasets (Log 2 (FC) of 13.2; Fig 2B and Table 1). The mutant isolated during the screen displayed a tolerant phenotype over a 6-h period (S1 Fig). In addition, its survival was increased up to 100-fold compared to the parental strain after 3 h (Fig 2C). In preliminary experiments, upon exposure to plasma, survival of strains bearing deletions in panM or in srgABC was identical to survival of the wild-type strain. Therefore, to get an idea of the overall transposon-induced changes in the mutant, we determined its transcriptomic profile and compared it to the parental strain in LB. RNA-seq data (S2 Table) revealed overexpression (about 20-fold compared to the parental strain) of srgABC (for serum resistance genes). Based on transposon position upstream of srgA, the mutant will hereafter be referred to as Tn::P srg . Other genes were overexpressed in Tn::P srg compared to the wild-type strain (S2 Table), including algU–encoding the ECF sigma factor AlgU—and five other alginate genes (algR and mucABCD operon, Fig 2D). PPT PowerPoint slide

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TIFF original image Download: Fig 2. Overexpression of srgA increases survival 100-fold. A. Schematic representation of the predicted srgABC operon and position of the transposon in the isolated Tn::P srg mutant. Corresponding genes from PAO1 are indicated. Note that the transposon is inserted 13 bp upstream of the predicted transcriptional start site. B. Zoom in on the Tn-seq profiles in the region surrounding the srg operon, showing the number of normalized reads in input (HIP) and output (plasma). C. Role of individual srg genes in the plasma resilience phenotype of the Tn::P srg strain. Deletion of srgA restores sensitivity to plasma. D. Differential gene expression between Tn::P srg and the parental IHMA87 strain represented in a volcano plot showing overexpression of srgABC and alginate-related genes. RNA was extracted from cultures grown on LB, and whole transcriptomes were determined by an RNA-seq pipeline E-F. Survival in plasma and alginate production for P. aeruginosa strains. Alginate synthesis was visualized based on colony morphology and quantified by carbazole assay, as described in Materials and Methods. Alginate overproduction appears as a darker bacterial spot (F). The data shown are from one representative experiment, performed in biological triplicates. https://doi.org/10.1371/journal.ppat.1011023.g002 To establish the relationship between the srg operon, alginate production and bacterial resilience to complement, we inactivated algD, encoding GDP-mannose 6-dehydrogenase, a checkpoint in alginate biosynthesis [44–46] both in the parental strain and in Tn::P srg , and investigated relevant phenotypes. In the assay, we included the alginate-overproducing mutant Tn::mucB isolated during the screen. We also designed a deletion mutant in the gene IHMA87_01134 (PA3819), an uncharacterized gene that was overexpressed (Log 2 (FC) = 2.35, P adj = 2x10-153) in the Tn::P srg strain. For each strain, we established the survival rate by CFU counting, the quantities of alginates using carbazole assay and assessed the colony morphology on plates (Fig 2E and 2F). Tn::P srg displayed 10% survival in human plasma (0.01% for the parental strain), along with an increased alginate production. Although alginate overproduction can lead to higher resistance to human plasma, as seen for the mutant Tn::mucB, the alginates in Tn::P srg did not account significantly for its resistance in plasma. Finally, the deletion of IHMA87_01134 had no impact neither on alginate production nor on plasma resistance. The srg operon encodes three small putative proteins: SrgA (9.9 kDa), SrgB (5.3 kDa) and SrgC (6.5 kDa). Structural predictions indicate SrgA to be a periplasmic protein with a signal peptide cleavage site at position Gly27. Both SrgB and SrgC are predicted to be membrane proteins with one and two transmembrane alpha-helices, respectively. All three srg gene products are highly conserved (98–100% amino acid sequence identity) over the 232 complete P. aeruginosa genomes available in the Pseudomonas genome database 43. The individual contributions of the srg genes to P. aeruginosa tolerance to plasma were evaluated by testing single, double and triple deletion mutants directly in the Tn::P srg background strain. Plasma killing assays performed with these deletion mutants showed that SrgA was required and sufficient for increased survival in plasma (Fig 2C). Overall, these results demonstrate that a srg overexpressing mutant produces more alginates and has a drastically increased plasma resistance in an alginate-independent manner and that SrgA alone is sufficient to modulate the plasma resistance.

Purine and biotin pathway deficiencies lead to increased persistence We then focused on mutants for which the transposon was present in genes and intergenic regions of biotin and purine biosynthesis. Mutants with insertions in the bioBFHCD operon and in the bioA gene–covering all steps of biotin biosynthesis–were significantly overrepresented following plasma challenge (Table 1 and Fig 3A, top). This result suggests that lack of biotin promotes survival in plasma. This finding was unexpected as the biotin pathway has been shown to be essential for survival in human serum for several pathogens, including Klebsiella pneumoniae and Mycobacterium tuberculosis [47–49]. To confirm this result, in addition to the transposon mutants isolated from the screen (Fig 1C; Tn::bioA and Tn::bioB), we engineered a mutant carrying a deletion of bioB (ΔbioB), coding for the biotin synthase and examined its behavior in plasma over a 6-h challenge (Fig 3B). The ΔbioB mutant survived significantly better than the wild-type strain in plasma (over 10% vs. about 0.01% survival, respectively). Moreover, in plasma, IHMA87ΔbioB displayed a biphasic killing curve, implying that the levels of biotin determine the persistence levels. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Inactivation of biotin and purine biosynthetic pathways increases survival rates of IHMA87 in plasma. A. Zoom in on Tn-seq profiles of bio and pur genes and operons, showing normalized numbers of reads in input (HIP) and output (plasma) samples. B. IHMA87 wild-type strain, ΔbioB, and ΔpurD mutants’ survival kinetics in plasma over 6 h incubation, as measured by CFU counting (n = 5). Note the biphasic killing curves for the parental strain and ΔbioB mutant, indicating increased persistence of ΔbioB. The ΔpurD mutant displayed increased tolerance compared to the parental strain. https://doi.org/10.1371/journal.ppat.1011023.g003 Like biotin synthesis, survival in plasma was clearly enhanced for bacteria bearing mutations in the purine biosynthetic pathway (Fig 3A, bottom). De novo purine biosynthesis, together with pyrimidine production, are essential for bacterial nucleotide metabolism and important for bacterial growth and survival in plasma and serum [50–53]. Transposon-insertion mutants in three main operons encoding enzymes involved in purine biosynthesis were overrepresented in the screen (Table 1). The two first hits in the pathway purF, encoding an amido-phospho-ribosyltransferase, and purD, encoding phosphoribosylamine-glycine ligase, were significantly enriched in plasma with a Log 2 (FC) of 17.26 and 14.06, respectively (Table 1). To further study the role of purine biosynthesis in plasma resistance, we designed a deletion mutant of purD (ΔpurD), given its non-operonic structure. Killing kinetics of ΔpurD was studied over 6 h in plasma. In accordance with Hill et al. in an antibiotic context [54], the ΔpurD mutant displayed a tolerant phenotype in plasma (Fig 3B). Interestingly, no similar level of gene enrichment was found for the pyrimidine pathway suggesting that the specific product of the purine pathway, rather than the nucleotide metabolism as a whole, contributed to increased survival in human plasma.

ATP levels influence P. aeruginosa tolerance to plasma Recent studies showed that low intracellular ATP concentration resulted in increased antibiotic persistence [4,5]. Along the same line, bacteria harvested at the stationary growth phase have lower levels of intracellular ATP and form a higher proportion of persisters when treated with antibiotics [55]. As the ATP molecule is a final product of the purine pathway (Fig 4A), we hypothesized that it could also play a role in the emergence of evaders. To investigate this hypothesis, we first measured intracellular ATP levels in ΔpurD both in rich medium and after incubation in HIP. As expected, in both media, the ATP concentration measured for ΔpurD was 5-fold lower than that measured for the parental strain (Fig 4B). To confirm that the low ATP concentration was indeed responsible for higher resilience to human plasma, we modulated ATP levels by supplementing the plasma with exogenous ATP during the plasma killing assay (Fig 4C). The addition of ATP to a concentration—in the range of that in healthy individual blood (Human Metabolome database [56])—when bacteria were exposed to plasma restored a wild-type-like sensitivity, further demonstrating the ATP-dependent phenotype of pur mutants. PPT PowerPoint slide

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TIFF original image Download: Fig 4. ATP and biotin influence bacterial sensitivity to plasma. A. Schematic view of the purine pathway (adapted from KEGG database [42]). Significantly enriched insertions in corresponding genes of the Tn-seq screen are indicated. B. Measurement of intra-bacterial ATP levels in LB and after 2h-incubation in HIP normalized to the CFU counts. C. Trans-complementation of ΔbioB and ΔpurD phenotype by exogenous biotin or ATP, respectively. Biotin (1 μM) and ATP (1.5 mM) were added at the beginning of the incubation of bacteria in plasma. The survival was estimated by CFU counting and the median of all independent experiments is represented by the histogram. Statistical analysis was performed and p-value <0.05 or 0.01 are indicated with ‘*’ and ‘**’, respectively. D. The biotin biosynthetic pathway. Significantly enriched hits in Tn-seq are highlighted in green. https://doi.org/10.1371/journal.ppat.1011023.g004 No apparent links between the biotin biosynthesis pathway (Fig 4D) and ATP synthesis exist. Nevertheless, as biotin is involved in many biological processes, we tested whether the ΔbioB mutant could be rescued by ATP. As shown in Fig 4C, biotin but not ATP supplementation restored a wild-type-like sensitivity to ΔbioB, suggesting a distinct molecular mechanism for evader formation in this mutant. A double mutant purD-bioB exhibited an even higher survival rate in plasma than both simple mutants, further showing that the two pathways are independent and important in bacterial strategy to evade the CS (S2 Fig).

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