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Published in final edited form as: ACS Chem Biol. 2021 Feb 17;16(3):501–509. doi: 10.1021/acschembio.0c00901

Pseudomonas Virulence Factor Pathway Synthesizes Autoinducers That Regulate the Secretome of a Pathogen

Ashley M Kretsch 1,#, Gina L Morgan 2,#, Katie A Acken 3, Sarah A Barr 4, Bo Li 5,*
PMCID: PMC8164881  NIHMSID: NIHMS1700170  PMID: 33595276

Abstract

Cell-to-cell communication via chemical signals is an essential mechanism that pathogenic bacteria use to coordinate group behaviors and promote virulence. The Pseudomonas virulence factor (pvf) gene cluster is distributed in more than 500 strains of proteobacteria including both plant and human pathogens. The pvf cluster has been implicated in the production of signaling molecules important for virulence; however, the regulatory impact of these signaling molecules on virulence had not been elucidated. Using the insect pathogen Pseudomonas entomophila L48 as a model, we demonstrated that pvf-encoded biosynthetic enzymes produce PVF autoinducers that regulate the expression of pvf genes and a gene encoding the toxin monalysin via quorum sensing. In addition, PVF autoinducers regulate the expression of nearly 200 secreted and membrane proteins, including toxins, motility proteins, and components of the type VI secretion system, which play key roles in bacterial virulence, colonization, and competition with other microbes. Deletion of pvf also altered the secondary metabolome. Six major compounds upregulated by PVF autoinducers were isolated and structurally characterized, including three insecticidal 3-indolyl oxazoles, the labradorins, and three antimicrobial pyrrolizidine alkaloids, the pyreudiones. The signaling properties of PVF autoinducers and their wide-ranging regulatory effects indicate multifaceted roles of PVF in controlling cell physiology and promoting virulence. The broad genome distribution of pvf suggests that PVF-mediated signaling is relevant to many bacteria of agricultural and biomedical significance.

Graphical Abstract

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Extracellular small-molecule signals play essential roles in cell-to-cell communication in bacteria. These molecules, termed autoinducers, are produced by the bacterium itself and accumulate to higher extracellular levels as cell density increases. When their concentration reaches a critical threshold, a “quorum,” autoinducers stimulate a concerted response in the bacterial population by altering global gene expression. This “quorum sensing” mechanism allows bacteria to coordinate group behaviors and regulate a wide variety of phenotypes important for adaptation and survival.1,2 Quorum sensing is a critical mechanism used by many pathogenic bacteria to regulate the production of virulence factors, formation of biofilms, and acquisition of antibiotic resistance and is, therefore, a promising target for antivirulence strategies.3,4 Multiple quorum sensing systems have been identified, including at least three different types of autoinducers produced by the well-studied pathogen Pseudomonas aeruginosa:1 multiple acyl homoserine lactones,5 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas quinolone signal),6–8 and the diffusible signal factor cis-2-dodecenoic acid.9 However, many bacteria do not produce any of these known autoinducers but still conduct cell-to-cell signaling, providing opportunities for the discovery of unique signaling systems.10

The Pseudomonas virulence factor (pvf) is a four-gene cluster that encodes a nonribosomal peptide synthetase, pvfC,11,12 an N-oxygenase, pvf B,13 and two genes of unknown functions, pvfA and pvf D (Figure 1A). This gene cluster is conserved in more than 500 strains of Pseudomonas and other bacteria,12,13 including pathogenic species and species commensal to human and plant hosts. Deletion of pvfC in the entomopathogen Pseudomonas entomophila L48, and its homologue, mgoA, in the phytopathogen Pseudomonas syringae pv. syringae UMAF0158, significantly reduced virulence against Drosophila melanogaster and tomato plants, respectively.15,14 Enzymes encoded by pvf have been implicated in the production of extracellular signaling molecules that regulate the expression of virulence factors,12 including the pore-forming toxin monalysin in L48 (Figure 1B) and the phytotoxin mangotoxin in UMAF0158.16–18

Figure 1.

Figure 1.

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Pseudomonas virulence factor (pvf) gene cluster is responsible for synthesizing PVF signaling molecules. (A) The pvf gene cluster from P. entomophila L48. (B) Enzymes encoded by pvf produce extracellular PVF autoinducers that regulate the expression of the gene encoding the toxin monalysin (mnl). (C) Strains containing the lacZ promoter-reporter to evaluate the regulatory effects of PVF autoinducers on mnl and pvf expression.

Here, we characterize the signaling properties of the molecules produced by pvf-encoded enzymes (PVF signaling molecules) and elucidate their regulatory roles in virulence, competition, and bacteria–host interactions. We use P. entomophila L48 as a model organism because the genome does not harbor the biosynthetic enzymes for any known autoinducers, and it is a model for Drosophila–Pseudomonas interactions.19,20 We demonstrate that pvf-encoded biosynthetic enzymes produce extracellular signaling molecules that autoinduce pvf expression (PVF autoinducers). The signaling activity of PVF autoinducers depends on bacterial cell densities and autoinducer concentration. Proteomic and metabolomic analyses revealed that PVF autoinducers globally regulate the “secretome” of P. entomophila, including both secreted proteins and small molecules that play critical roles in colonization, virulence, nutrient acquisition, and competition with other organisms.

RESULTS AND DISCUSSION

Enzymes Encoded by pvf Produce Extracellular, Autoinducing Signaling Molecules.

We characterized the timing of pvf expression and the autoinducing effect of PVF signaling molecules using reporter strains. The promoter region of pvf was inserted upstream of the lacZ reporter gene and incorporated at the TN7 site of P. entomophila wildtype (WT) and ΔpvfC (WT::Ppvf-lacZ and ΔpvfC::Ppvf-lacZ, respectively, Figure 1C). The pvf reporter activity in WT::Ppvf-lacZ increased rapidly during the late exponential growth phase and more slowly during the stationary phase (Figure 2A), suggesting that pvf expression is cell-density dependent. To verify that pvf induction results from the production of extracellular small molecules, the cell-free spent media of WT or ΔpvfC cultures were extracted using dichloromethane (hereafter referred to as “extract”). The ΔpvfC::Ppvf-lacZ reporter activity increased when the culture was supplemented with WT extract, but not with ΔpvfC extract, suggesting that pvf-encoded biosynthetic enzymes are responsible for producing the extracellular small molecules that induce pvf expression (Figure S1). Supplementing the WT::Ppvf-lacZ culture with WT extract enhanced pvf reporter activity further above WT levels (Figure 2B). To confirm that molecules produced by pvf-encoded enzymes are responsible for autoinduction, we expressed pvfABCD on a plasmid in ΔpvfC (ΔpvfC + pvf)11 and a heterologous host, E. coli Bap1 (E. coli + pvf). The extract of both ΔpvfC + pvf and E. coli + pvf induced pvf reporter activity of WT::Ppvf-lacZ (Figure 2B). These combined data further suggest that PVF signaling molecules are autoinducers produced by pvf-encoded enzymes.

Figure 2.

Figure 2.

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Molecules synthesized by pvf-encoded enzymes autoinduce pvf expression. (A) The Ppvf-lacZ reporter activity in P. entomophila wildtype (WT) is cell-density-dependent. β-Galactosidase activity, reported in Miller Units (MU), of the Ppvf-lacZ reporter was plotted over time (blue). Bacterial growth was measured by cell density (OD600) and plotted on a logarithmic scale over time (black). (B) Extracellular PVF signaling molecules autoinduce the expression of the Ppvf-lacZ reporter in P. entomophila WT. The bar graph shows Ppvf-lacZ reporter activity of WT::Ppvf-lacZ culture at 24 h postinoculation (green), and reporter activity of WT::Ppvf-lacZ culture supplemented with extracts from P. entomophila WT (purple), P. entomophila ΔpvfC + pvf (blue), or E. coli + pvf (orange). (C) Extracellular PVF signaling molecules restore the expression of the Pmnl-lacZ reporter in P. entomophila ΔpvfC. The bar graph shows Pmnl-lacZ reporter activity 24 h postinoculation in P. entomophila WT (green) and ΔpvfC (red), and reporter activity of the ΔpvfC::Pmnl-lacZ culture supplemented with extracts from P. entomophila WT or ΔpvfC (purple), P. entomophila ΔpvfC + pvf (blue), or the non-pvf-expressing P. aeruginosa PAO1 or PAO1 + pvf (orange). Student’s t test against ΔpvfC::Ppvf-lacZ or ΔpvfC::Pmnl-lacZ: *p < 0.05, **p < 0.01.

PVF Signaling Molecules Regulate Expression of mnl.

We characterized the ability of PVF signaling molecules to regulate gene expression using a second set of reporter strains for the expression of the monalysin-encoding gene (mnl), which is regulated by PVF.12 The mnl reporter activity was significantly higher in the WT (WT::Pmnl-lacZ) than ΔpvfC (ΔpvfC::Pmnl-lacZ; Figure S2), confirming that PVF autoinducers upregulate the transcription of mnl. The reporter activity of ΔpvfC::Pmnl-lacZ increased in a dose-dependent manner when the culture was supplemented with increasing amounts of cell-free spent media from WT cultures up to 15% (v/v) of the total culture volume (Figure S2), indicating that regulation of mnl expression depends on the concentrations of extracellular PVF signaling molecules. Similar to pvf reporter activity, mnl reporter activity in ΔpvfC::Pmnl-lacZ was enhanced by extracts from WT, ΔpvfC + pvf, and E. coli + pvf (Figure 2C, S2). We expressed pvfABCD in a second heterologous host, P. aeruginosa PAO1 (PAO1 + pvf), which does not natively harbor pvf. The extract from PAO1 + pvfalso induced mnl reporter activity (Figure 2C). Reporter activity in ΔpvfC::Pmnl-lacZ was not affected by extracts from ΔpvfC, or E. coli and P. aeruginosa expressing an empty vector (Figures 2C, S2), further confirming that pvf-encoded enzymes are responsible for synthesizing extracellular signaling molecules that regulate mnl expression. The combined results of pvf expression at high cell density, the autoinducing effect of PVF signaling molecules, and the concentration-dependent PVF-regulation of mnl expression indicate that PVF signaling molecules are autoinducers that mediate quorum sensing.

PVF Autoinducers Regulate the Secreted Proteome of P. entomophila.

We hypothesized that PVF autoinducers exert wide-ranging regulatory effects on protein production that promote virulence. To test this hypothesis, we conducted multiple comparative proteomic analyses. Because many virulence factors are secreted or associated with the bacterial membrane, we compared the abundance of proteins in the spent media between WT, ΔpvfC, and ΔpvfC + pvf. To identify proteins regulated by PVF autoinducers, we isolated proteins from the spent media and profiled them using gel electrophoresis (Figure S3) as well as liquid chromatography–tandem mass spectrometry and label-free quantification (Figures 3A, S4, and S5). The growth of P. entomophila was not affected by deletion or overexpression of pvf, but a higher total protein concentration was observed in samples from ΔpvfC + pvf (Figure S4). Using analysis of variance (ANOVA), we identified 1047 proteins produced at significantly different levels between WT, ΔpvfC, and ΔpvfC + pvf (p < 0.1, Figures 3A, S5, Data Set 1). Principal component analysis showed that samples from each strain cluster separately (Figure S6), suggesting that these strains exhibit differential levels of secreted proteins. A cutoff of p < 0.01 reduced the number to 506 proteins, among which 209 proteins exhibited at least 2-fold change between WT and ΔpvfC (Figure 3B). Among these, 113 proteins were produced at a significantly higher level and 84 proteins were produced at a significantly lower level in ΔpvfC + pvf compared to ΔpvfC or WT (Figure 3B, dark gray or colored in Figure 3A). This observation suggests that complementation of ΔpvfC with pvf restores protein production in ΔpvfC and enhances the differences of protein production between WT and ΔpvfC. Thus, we focused on the 197 differentially expressed proteins to examine the regulatory effects of PVF autoinducers. Several membrane-associated proteins were identified (Data Set 1), which likely dissociated from the cell membrane during sample preparation; these proteins were analyzed as part of the secretome as they are relevant to the regulatory effects of PVF autoinducers.

Figure 3.

Figure 3.

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Secreted and membrane-associated proteins are differentially produced between P. entomophila WT, ΔpvfC, and ΔpvfC + pvf. (A) Scatter plot of the log2 fold change between the mean intensities of proteins in WT compared with ΔpvfC (WT-to-ΔpvfC abundance ratio, RW:D, x axis) and the ANOVA p value (y axis). Points represent proteins with an ANOVA p < 0.1 (1047 total). Vertical dashed line represents a cutoff of RW:D > 2 or < 0.5. Horizontal dashed line represents a cutoff of p < 0.01. Protein types are shown in different colors: virulence-related (red), type VI secretion system (orange), siderophore receptors (green), and motility-related (blue). (B) A total of 209 proteins exhibited at least 2-fold difference in abundance between WT and ΔpvfC and between ΔpvfC and ΔpvfC + pvf (p < 0.01). Of these, 113 proteins showed higher production in WT and ΔpvfC + pvf than ΔpvfC (green), and 84 proteins showed lower production in WT and ΔpvfC + pvf than ΔpvfC (red). (C) Deletion of pvf increased the swarming motility of P. entomophila. Genetic complementation in ΔpvfC + pvf reduced motility back to WT levels.

Deletion of pvfC decreased the production of a wide range of proteins. Among the highest decrease in protein production are proteins of the type VI secretion system (T6SS, Figures 3A, S5), which exhibited WT-to-ΔpvfC protein abundance ratios (RW:D) ranging from 3 to 390. The T6SS is a membrane-associated protein complex that can target host cells and other bacteria by directly translocating effector proteins into neighboring cells.21 The T6SS resembles a phage tail, with many proteins comprising the baseplate complex, spike, tube, and sheath. Proteins that significantly decreased in abundance in ΔpvfC include the inner tube protein Hcp (RW:D = 47) and four VgrG spike proteins (RW:D = 3, 19, 96, and 390, respectively). Both Hcp and VgrG are essential for T6SS assembly and are also secreted as effectors. All the VgrG proteins identified contain a DUF2345 domain with proposed involvements in recruiting toxin effectors in E. coli and mediating antibacterial and antieukaryotic effects in Klebsiella pneumoniae.22,23 Finally, the production of two secreted Rhs repeat-containing effector proteins, PSEEN0541 and PSEEN5274, also decreased in ΔpvfC compared to WT (RW:D = 71 and 81, respectively). Rhs repeat-containing effector proteins include toxins that have been shown to mediate intercellular competition in Pseudomonas and other bacterial species21,24 and activate inflammasome-mediated death of phagocytic cells.25,26

The pvfC deletion also decreased the production of several secreted virulence-related proteins. Production of AprA, a protease secreted by P. entomophila,27 was decreased in ΔpvfC (RW:D = 74) and increased by 13-fold in ΔpvfC + pvf, compared to WT (Figures 3A, S5). Production of monalysin, the aforementioned pore-forming toxin, only decreased slightly in ΔpvfC (RW:D = 1.7) but increased 310-fold in ΔpvfC + pvf compared to WT (Figure S5), further supporting that PVF autoinducers upregulate the expression of mnl. Both AprA and monalysin are important virulence factors that promote virulence and pathogenicity in flies: monalysin causes gut damage while AprA cleaves secreted premonalysin, yielding the active toxin monalysin.16,27 Further, AprA inhibits fly defense by degrading antimicrobial peptides.16,27 In addition, production of the exoprotein CbpD in ΔpvfC is lower than WT (RW:D = 40). The CbpD homologue in P. aeruginosa is a chitin-binding protein secreted by the type II secretion system, and due to its increased expression during P. aeruginosa infection, CbpD was thought to play a role in pathogenesis by adhering to host epithelial cells.28,29

Conversely, several proteins showed increased production in ΔpvfC compared to WT . The highest increase in abundance was observed for proteins involved in the assembly, regulation, or function of the bacterial flagella (Figures 3A and S5), which allow bacteria to swim and swarm. The flagellar assembly proteins with increased levels in ΔpvfC include the flagellin protein FlaG, a structural component of the flagellar filament (3.3-fold increase compared to WT, RW:D = 0.30), the proximal rod protein FlgF (140-fold increase, RW:D = 0.0071), the hook filament junction protein FlgL (3.9-fold increase, RW:D = 0.26), and the hook capping protein FlgD (5.2-fold increase, RW:D = 0.19). Additionally, two flagella regulatory proteins exhibited a dramatic increase in expression in ΔpvfC compared to WT: FliK, which controls the hook length (120-fold increase, RW:D = 0.0083), and the antisigma factor FlgM (540-fold increase, RW:D = 0.0019). FlgM suppresses expression of flagellar genes involved in the late assembly steps and is itself inhibited by secretion from the cytoplasm,30,31 thus, the increase of FlgM in the secretome is consistent with augmented production of late flagellar proteins like FlaG. However, the flagellar motor protein MotB decreased in ΔpvfC (RW:D = 4.8), suggesting that MotB is differentially regulated by PVF autoinducers compared to the other flagellar proteins.

The increased production of motility proteins in ΔpvfC prompted us to examine phenotypic changes in motility between WT and ΔpvfC. We compared the swarming motility of WT, ΔpvfC, and ΔpvfC + pvf. WT does not exhibit swarming behavior while ΔpvfC swarms to about half the diameter of the plate (Figure 3C), corroborating the increased production of motility proteins in ΔpvfC. When ΔpvfC is complemented with pvf, the swarming defect is reduced back to near WT levels, confirming that PVF autoinducers downregulate motility. To determine whether PVF autoinducers might also reduce motility by downregulating biosurfactant production,32,33 we assessed surfactant activity in a droplet collapse assay. The ability of P. entomophila cultures to collapse a droplet, i.e., produce surfactants, was significantly reduced in ΔpvfC but recovered in ΔpvfC + pvf (Figure S7), suggesting that PVF autoinducers upregulate biosurfactant production instead. To confirm this result, we measured the effect of a pvfC insertional mutation on the expression of the protein EtlB in the biosynthesis of entolysin, a lipopeptide surfactant and hemolytic compound produced by P. entomophila.34 The activity of the translational fusion etlB-lacZ was significantly reduced in the pvfC mutant compared to WT (Figure S8). Thus, PVF autoinducers upregulate biosurfactant production but downregulate production of flagellar components, resulting in net reduced motility.

Finally, deletion of pvf altered the production of several proteins likely involved in the uptake of metal ions. Production of the siderophore transporters BauA (PSEEN2492, RW:D = 7) and BauB (PSEEN2493, RW:D = 460) was decreased in ΔpvfC compared to WT (Figures 3A, S5). In contrast, a putative extracellular ferric iron binding protein (PSEEN4935) was increased by 20-fold in ΔpvfC (RW:D = 0.050), and two putative TonB-dependent outer membrane iron transporters (PSEEN0263 and PSEEN3587) were increased in ΔpvfC by 4.8- and 3.2-fold (RW:D = 0.21 and 0.32, respectively, Figures 3A, S5). Although the roles of these proteins remain to be characterized, our proteomics data suggest that P. entomophila employs multiple metal-uptake pathways that are differentially regulated by PVF autoinducers.

PVF Autoinducers Regulate the Production of Secondary Metabolites.

We also examined regulation of the small-molecule secretome by PVF autoinducers. To identify PVF-regulated secondary metabolites, we conducted comparative metabolomic analyses between WT and ΔpvfC and between WT and Δpvf, a mutant in which the entire pvf cluster was deleted. Metabolites were extracted from the cell-free spent media of WT, ΔpvfC, or Δpvf cultures grown under optimized conditions for pvf expression (Figure S9) and separated by preparative high-performance liquid chromatography (HPLC). The WT metabolome exhibited significantly higher levels of secondary metabolite production than Δpvf or ΔpvfC, among which 14 mass features exhibited a ≥ 16-fold increase (Figures 4A and S10, Table S3, Data Set 2). We purified six of the most abundant species, compounds 1 —6 (Figure 4A), and characterized their structures by liquid-chromatography coupled high-resolution mass spectrometry (LC-HRMS) and NMR analyses (Figure 4B). Further LC-HRMS analyses of culture extracts showed that production of all six compounds was significantly decreased in ΔpvfC cultures and restored in ΔpvfC + pvf cultures as well as ΔpvfC cultures supplemented with WT extracts (Figures S11 and S12), confirming upregulation of these compounds by PVF autoinducers.

Figure 4.

Figure 4.

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Compounds 1–6 are regulated by pvf. (A) Absorbance at 270 nm of HPLC traces of WT extract (blue) and Δpvf extract (red). (B) Chemical structures of compounds 1–6.

We determined the molecular formula of 1 ([M + H]+ = 241.1335, C15H17N2O+), 2 ([M + H] + = 275.1179, C18H15N2O+), and 3 ([M + H]+ = 255.1492, C16H19N2O+) based on LC-HRMS analysis (Table S3, Figure S13). Compounds 1—3 exhibited similar MS/MS fragmentation patterns and shared a mass fragment with a [M + H]+ of 130.0651 that corresponds to an indole (Figures S14, S15), suggesting that these compounds are related. 1H and (1H, 13C) HMBC NMR analyses confirmed the indole moiety in 1–3 and revealed connectivity to an oxazole in all three molecules, indicating the presence of a common 3-indolyl oxazole core (Tables S4–S6, Figures S16–S30). The 3-indolyl oxazole core is further connected to an isobutyl (1), benzyl (2), or n-pentyl (3) group, consistent with labradorin 1 (1), pimprinaphine (2), and labradorin 2 (3; Figure 4B).35–37

The structures of 4–6 are related based on LC-HRMS and NMR analyses (Figures S13, S31–S45 and Tables S7–S9). The molecular formula was determined for 4 ([M + H]+ = 266.1751, C15H24NO3+), 5 ([M + H]+ = 294.2064, C17H28NO3+), and 6 ([M + H]+ = 320.2220, C19H30NO3+), and similar MS/MS fragmentation patterns of these compounds were observed (Figures S14, S15). (1H, 1H)-COSY and (1H, 13C)-HMBC correlations of 4–6 support structures that contain a common bicyclic pyrrolizidine core with differing fatty acid chains, consistent with pyreudione A (4), pyreudione B (5), and pyreudione D (6; Figure 4B).38 Besides 1—6, we identified several other metabolites upregulated by PVF autoinducers. MS/MS analysis revealed the upregulation of a labradorin (7, [M + H]+ = 227.1179, C14H15N2O+) and a pyreudione (8, [M + H]+ = 292.1907, C17H26NO3+), although insufficient materials were obtained for NMR structural elucidation (Table S3, Figure S15, Data Set 2).

Discussion.

Our work showed that pvf-encoded biosynthetic enzymes produce autoinducing molecules that regulate the protein and small-molecule secretome of P. entomophila. The autoinducers exhibit quorum sensing characteristics: they induce the expression of their own biosynthetic genes and other virulence-factor encoding genes (e.g., mnl) in a cell-density and concentration-dependent manner and regulate the production of diverse proteins and secondary metabolites. To the best of our knowledge, PVF signaling molecules are the first autoinducers discovered in P. entomophilato date.

We demonstrate that PVF autoinducers regulate the production of a wide range of proteins, which exhibit significant biological roles in virulence and bacteria—bacteria and bacteria–host interactions. Identification of these proteins provides a global view of the regulatory effects of PVF autoinducers and yields a molecular understanding of how PVF autoinducers promote virulence. We obtained additional insights regarding PVF’s regulatory mechanism of the known virulence factors, monalysin and the protease AprA, which cause host damage and suppress host defenses.16,20,27 The deletion of pvf did not alter aprA expression,15 although AprA protein abundance decreased compared to WT (RW:D = 74), suggesting that PVF autoinducers regulate AprA at a post-transcriptional level. In addition, we discovered components of the type VI secretion system (T6SS) among the most highly PVF-upregulated proteins. T6SS can directly target the eukaryotic host, causing damage to host cells and modulating the host immune response, and T6SS can also drive interbacterial interactions and competition.21 Deletion of tssJ, an outer membrane component of T6SS, in P. entomophila did not reduce fly infection,16 suggesting that the P. entomophila T6SS instead participates in interbacterial interactions or targets alternative hosts.39,40 Interestingly, P. entomophila lacks the type III secretion system, the predominant injection-based secretion system in many Gram-negative pathogens.41,42 Thus, T6SS likely plays a major role in translocating effector proteins into the host or neighboring bacteria for P. entomophila, and PVF autoinducers may facilitate cross-talk and competition of P. entomophila with other organisms.

While PVF autoinducers upregulate production of many proteins for virulence and competition, they downregulate proteins involved in motility, including those in flagellar assembly and regulation. Indeed, pvfC deletion significantly increased the swarming motility of P. entomophila. Production of surfactants also contributes to motility,32,33 however, deletion of pvf reduced surfactant production, suggesting that increased expression of flagellar components played a larger role in boosting the swarming motility in the ΔpvfC mutant. Motility is essential for initial colonization of the host, but it is also resource-intensive, and the downregulation of motility is thought to help Pseudomonas redistribute resources.43 Thus, PVF autoinducers might downregulate motility after bacteria establish stable colonization in the host. In addition to playing a role in host attachment, flagellar components in P. aeruginosa have been shown to be recognized by the mammalian innate immune system.43,44 Together, downregulation of motility suggests a key role for PVF autoinducers in regulating colonization and evasion of the host immune system.

PVF autoinducers also regulate the expression of receptors and transporters for siderophores, required for acquisition of metal ions that are essential for bacterial survival, virulence, and infection.45 PVF autoinducers upregulate the siderophore receptors BauA and BauB but downregulate two putative TonB-dependent siderophore transporters. BauA shares 55% identity with the TonB-dependent transporter BauA from Acinetobacter baumannii that transports the siderophore preacinetobactin,46 while BauB shares 54% identity with the periplasmic siderophore-binding protein BauB from A. baumannii that transports two different siderophores, acinetobactin and fimsbactin.47 P. entomophila produces pseudomo-nine,45 a structural analog to acinetobactin. Thus, the BauA and BauB homologues from P. entomophila are likely responsible for the uptake of pseudomonine or prepseudomo-nine, but their roles remain to be experimentally confirmed. The siderophores specified by the putative TonB-dependent transporters are unknown. Thus, P. entomophila employs multiple siderophore uptake pathways that may help the bacterium respond to different environmetal cues like many other bacteria.48,49

In addition to regulating the production of nearly 200 secreted and membrane proteins, PVF autoinducers upregulate the production of diverse secondary metabolites. We showed that PVF autoinducers upregulate secondary metabolite production and structurally characterized six (1–6), including 3-indolyl-oxazoles (1–3). Labradorins 1 (1) and 2 (3) were first isolated from Pseudomonas syringae pv. coronafaciens and exhibited anticancer activities.36 Pimprinaphine (2) was first identified from Streptoverticillium olivoreticuli37 and found to exert antiviral activity against the tobacco mosaic virus.50 Interestingly, labradorin 1 and the closely related analog, WS-30581, inhibited Galleria insect hemocytes.35 The insecticidal activity of labradorins suggests that these compounds play a role in Drosophila infection by damaging insect cells.11,15 The biosynthesis of these compounds was recently shown to involve an N-acyltransferase and a non-heme diiron desaturase-like enzyme.51

Metabolites 4—6 are bicyclic pyrrolizidines, pyreudiones A, B, and D, respectively. The pyreudiones were first identified in Pseudomonas f luorescens.38 Their gene clusters were also found in P. entomophila and confirmed by heterologous expression in Pseudomonas putida, however, the pyreudiones were not produced by P. entomophila under the reported conditions.52 We elicited production of pyreudiones in P. entomophila using PVF autoinducers and identified additional PVF-upregulated metabolites, demonstrating the use of quorum sensing systems for natural product discovery; this strategy was also employed in the discovery of bactobolin from Bukholderia thailandensis and azabicyclene from P. aeruginosa.53,54 Further, pyreudione production is necessary and sufficient for preventing amoebal predation in P. f luorescens,38 and pyreudiones exhibit antibacterial activity against Mycobacterium aurin and M. vaccae.38 Thus, pyreudiones may play a role in bacteria-host interactions and interbacterial competition. In sum, by regulating protein and secondary metabolite production, PVF autoinducers promote virulence in a concerted and multifaceted fashion.

While the structures of the PVF autoinducers remain unknown, we may glean insights into their structures from biosynthetic studies of pvf-encoded enzymes and their homologues. The genes pvfC, pvf B, and pvf D are required for full virulence against Drosophila.15 In P. entomophilia, PvfC activates and tethers valine,11,12 while PvfB oxygenates the amine of PvfC-tethered valine, generating a hydroxylamine.13 These transformations are also catalyzed by the homologues of PvfC and PvfB in Burkholderia.13 Thus, PVF autoinducers made by different bacteria may be derived from N-oxygenated valine. Further, more than 200 PvfC homologues, including 78 from P. syringae phytopathogens, are predicted to prefer leucine instead of valine as a substrate,12 suggesting these strains produce leucine-derived, structurally distinct autoinducers. Identification of the structures of PVF autoinducers and their receptors in multiple pathogens is underway to elucidate the quorum-sensing circuits.

In conclusion, we revealed that pvf-encoded enzymes in the model pathogen P. entomophila synthesize PVF autoinducers that regulate proteins and small molecules involved in a wide range of biological processes including virulence, competition, and colonization. The pvf gene cluster is conserved in over 500 bacterial strains of different species, many of which are pathogens or exhibit biocontrol activities.12,13 We expect PVF autoinducers to play a central role for quorum sensing in these bacteria, particularly P. syringae, in which pvf is widespread and quorum sensing is not well understood. Elucidating the remaining steps in the biosynthesis of PVF autoinducers, their structures, and their signal transduction will likely uncover new bacterial cell-to-cell signaling systems, bacteria–host interactions, and virulence factors.

METHODS

Detailed experimental procedures are included in the Supporting Information.

Supplementary Material

Supplemental Dataset 2
Supplemental Dataset 1
Supplemental materials and methods

ACKNOWLEDGMENTS

The authors thank I. Vallet-Gély (I2BC Paris-Saclay) for P. entomophila L48 entA::lacZ and entA::lacZ IM pvfC strains (PSEEN3045-lacZ and PSEEN3045-lacZ IMpvfC, respectively). Proteomic analysis was conducted using the UNC Proteomics Core Facility, which is supported in part by P30 CA016086 Cancer Center Core Support Grant to the UNC Lineberger Comprehensive Cancer Center. This work is supported by the National Institutes of Health (DP2HD094657 to B.L.), the Rita Allen Foundation, and the University of North Carolina at Chapel Hill. A.M.K. acknowledges support from a Burroughs Wellcome Graduate Fellowship.

Footnotes

Notes

The authors declare no competing financial interest.

Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acschembio.0c00901.

Supplemental tables and figures (PDF)

Complete contact information is available at: https://pubs.acs.org/10.1021/acschembio.0c00901

Contributor Information

Ashley M. Kretsch, Department of Chemistry, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States

Gina L. Morgan, Department of Chemistry, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States

Katie A. Acken, Department of Chemistry, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States

Sarah A. Barr, Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States

Bo Li, Department of Chemistry and Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental Dataset 2
NIHMS1700170-supplement-Supplemental_Dataset_2.xlsx (1.1MB, xlsx)
Supplemental Dataset 1
NIHMS1700170-supplement-Supplemental_Dataset_1.xlsx (819.3KB, xlsx)
Supplemental materials and methods
NIHMS1700170-supplement-Supplemental_materials_and_methods.pdf (17.9MB, pdf)

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