DSF-family quorum sensing signal-mediated intraspecies, interspecies, and inter-kingdom communication

DSFs are cis-2-unsaturated fatty acids containing fatty acid carbon chains of various lengths and cis double-bond configurations. DSF-producing bacteria are widely distributed in nature.Based on their genomic origins, DSF-mediated intraspecies communication pathways are generally affiliated to three different groups, respectively represented by Xanthomonas campestris pv. campestris (Xcc), Burkholderia cenocepacia (Bcc), and Pseudomonas aeruginosa.DSF-mediated interspecies communication occurs between DSF-producing bacteria and B. cenocepacia, Stenotrophomonas maltophilia, Bacillus, Francisella novicida, Salmonella, and Bdellovibrio bacteriovorus.DSF-mediated inter-kingdom communication occurs between DSF-producing bacteria and Candida albicans.DSF signaling occurs in plant development and defense, Xylella fastidiosa colonization of insects, and the zebrafish inflammatory response. While most bacteria are unicellular microbes they communicate with each other and with their environments to adapt their behaviors. Quorum sensing (QS) is one of the best-studied cell–cell communication modes. QS signaling is not restricted to bacterial cell-to-cell communication – it also allows communication between bacteria and their eukaryotic hosts. The diffusible signal factor (DSF) family represents an intriguing type of QS signal with multiple roles found in diverse Gram-negative bacteria. Over the last decade, extensive progress has been made in understanding DSF-mediated communication among bacteria, fungi, insects, plants, and zebrafish. This review provides an update on these new developments with the aim of building a more comprehensive picture of DSF-mediated intraspecies, interspecies, and inter-kingdom communication. While most bacteria are unicellular microbes they communicate with each other and with their environments to adapt their behaviors. Quorum sensing (QS) is one of the best-studied cell–cell communication modes. QS signaling is not restricted to bacterial cell-to-cell communication – it also allows communication between bacteria and their eukaryotic hosts. The diffusible signal factor (DSF) family represents an intriguing type of QS signal with multiple roles found in diverse Gram-negative bacteria. Over the last decade, extensive progress has been made in understanding DSF-mediated communication among bacteria, fungi, insects, plants, and zebrafish. This review provides an update on these new developments with the aim of building a more comprehensive picture of DSF-mediated intraspecies, interspecies, and inter-kingdom communication. Microbes are ubiquitous and populate plants and animals, as well as soil, water, and air. They form diverse communities in a multitude of environments. It is becoming increasingly apparent that microbes are not isolated in specific communities but rather interact and communicate with each other and with their surroundings [1.Thompson L.R. et al.The Earth microbiome project consortium. A communal catalogue reveals Earth’s multiscale microbial diversity.Nature. 2017; 551: 457-463Crossref PubMed Scopus (1204) Google Scholar]. One of the key challenges in microbiology is to dissect their diversity, metabolic activities, and interactions within their community and between communities and associated environments. Quorum sensing (QS) (see Glossary), a process whereby bacteria monitor their population density and regulate gene expression using chemical signals, is the best-studied type of bacterial communication [2.Leoni L. et al.Leoni L. Rampioni G. Quorum Sensing Methods and Protocols. Humana Press, 2017: 1-4Google Scholar]. QS is widespread in eubacteria, archaea, and fungi. Research in recent decades has identified a rich inventory of QS signals and QS-regulated beneficial or competitive interactions [2.Leoni L. et al.Leoni L. Rampioni G. Quorum Sensing Methods and Protocols. Humana Press, 2017: 1-4Google Scholar]. Recent evidence shows that QS signaling is not limited to bacterial cell–cell communication but also allows communication between bacteria and their eukaryotic hosts [3.Hughes D. et al.Inter-kingdom signaling: communication between bacteria and their hosts.Nat. Rev. Microbiol. 2008; 6: 111-120Crossref PubMed Scopus (550) Google Scholar, 4.Hartmann A. et al.Importance of N-acyl-homoserine lactone-based quorum sensing and quorum quenching in pathogen control and plant growth promotion.Pathogens. 2021; 10: 1561Crossref PubMed Scopus (8) Google Scholar, 5.Fan Q. et al.Structure and signal regulation mechanism of interspecies and interkingdom quorum sensing system receptors.J. Agric. Food Chem. 2022; 70: 429-445Crossref PubMed Scopus (7) Google Scholar]. Understanding the mechanisms and outcomes of QS signal-mediated communication has important implications for appreciating host–pathogen interactions and, ultimately, may provide new targets and strategies for antimicrobial therapies that block or interfere with these communication networks. The diffusible signal factor (DSF) family represents an intriguing type of QS signal found in diverse Gram-negative bacteria. Over the last decade, extensive progress has been made in understanding DSF-mediated intraspecies communication and interspecies communication [6.Deng Y. et al.Listening to a new language: DSF-based quorum sensing in Gram-negative bacteria.Chem. Rev. 2011; 111: 160-173Crossref PubMed Scopus (191) Google Scholar,7.Zhou L. et al.The DSF family of quorum sensing signals: diversity, biosynthesis, and turnover in Xanthomonas.Trends Microbiol. 2017; 25: 293-303Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar]. Recently, inter-kingdom communication between DSF-producing bacteria and insects, plants, and zebrafish has been reported [8.Tran T.M. et al.The bacterial quorum sensing signal DSF hijacks Arabidopsis thaliana sterol biosynthesis to suppress plant innate immunity.Life Sci. Alliance. 2020; 3e202000720Crossref Google Scholar, 9.Ma Z. et al.Formin nanoclustering-mediated actin assembly during plant flagellin and DSF signaling.Cell Rep. 2021; 34108884Abstract Full Text Full Text PDF Scopus (15) Google Scholar, 10.Zhu H. et al.Bacterial quorum sensing signal DSF inhibits LPS-induced inflammations by suppressing Toll-like receptor signaling and preventing lysosome mediated apoptosis in zebrafish.Int. J. Mol. Sci. 2022; 23: 7110Crossref PubMed Scopus (1) Google Scholar]. This review provides an update on these new developments with the aim of building a more comprehensive picture of DSF-mediated intraspecies, interspecies, and inter-kingdom communication. More detailed descriptions of the chemical structures, biological functions, biosynthesis, and signaling pathways of the DSF family signals can be found in previous reviews [6.Deng Y. et al.Listening to a new language: DSF-based quorum sensing in Gram-negative bacteria.Chem. Rev. 2011; 111: 160-173Crossref PubMed Scopus (191) Google Scholar,7.Zhou L. et al.The DSF family of quorum sensing signals: diversity, biosynthesis, and turnover in Xanthomonas.Trends Microbiol. 2017; 25: 293-303Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar,11.He Y.W. et al.Quorum sensing and virulence regulation in Xanthomonas campestris.FEMS Microbiol. Rev. 2008; 32: 842-857Crossref PubMed Scopus (200) Google Scholar, 12.Dow J.M. Diffusible signal factor-dependent quorum sensing in pathogenic bacteria and its exploitation for disease control.J. Appl. Microbiol. 2016; 122: 2-11Crossref PubMed Scopus (32) Google Scholar, 13.Wang M. et al.The cis-2-dodecenoic acid (BDSF) quorum sensing system in Burkholderia cenocepacia.Appl. Environ. Microbiol. 2022; 88e0234221Crossref Scopus (4) Google Scholar]. For convenience, DSF will be used as a collective term for the DSF-family signals unless otherwise specified. The first discovered DSF was the cis-11-methyl-dodecenoic acid (XcDSF) in the phytopathogen Xanthomonas campestris pv. campestris (Xcc). Subsequently, several analogs, cis-2-dodecenoic acid (BDSF), cis, cis-11-methyldodeca-2,5-dienoic acid (CDSF), cis-10-methyl-2-dodecenoic acid (IDSF), 13-methyltetradecanoic acid (LeDSF), cis-2-tetradecenoic acid (XfDSF1), cis-2-hexadecenoic acid (XfDSF2), and cis-2-decenoic acid (PDSF), have been identified. Except for LeDSF, all DSFs are cis-2-unsaturated fatty acids containing fatty acid carbon chains of various lengths and cis double-bond configurations (Figure 1). Until now, DSF members have been detected in a range of Gram-negative bacterial species belonging to the γ-proteobacteria and β-proteobacteria groups, including Xanthomonas spp., Xylella fastidiosa, Stenotrophomonas maltophilia, Lysobacter enzymogenes and L. brunescens, Leptospirillum ferri philum and L. ferrooxidans, Burkholderia cenocepacia, Cronobacter turicensis, and Pseudomonas aeruginosa [7.Zhou L. et al.The DSF family of quorum sensing signals: diversity, biosynthesis, and turnover in Xanthomonas.Trends Microbiol. 2017; 25: 293-303Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar,14.An S.Q. et al.Diffusible signal factor signaling regulates multiple functions in the opportunistic pathogen Stenotrophomonas maltophilia.BMC Res. Notes. 2018; 11: 569Crossref PubMed Scopus (16) Google Scholar, 15.Li L. et al.Diffusible signal factor (DSF)-mediated quorum sensing modulates expression of diverse traits in Xanthomonas citri and responses of citrus plants to promote disease.BMC Genomics. 2019; 20: 55Crossref PubMed Scopus (24) Google Scholar, 16.Feng T. et al.Interspecies and intraspecies signals synergistically regulate Lysobacter enzymogenes twitching motility.Appl. Environ. Microbiol. 2019; 85e01742Crossref Scopus (12) Google Scholar, 17.Ling J. et al.LbDSF, the Lysobacter brunescens quorum-sensing system diffusible signaling factor, regulates anti- Xanthomonas XSAC biosynthesis, colony morphology, and surface motility.Front. Microbiol. 2019; 10: 1230Crossref PubMed Scopus (7) Google Scholar, 18.Alcaraz E. et al.The rpf/DSF signalling system of Stenotrophomonas maltophilia positively regulates biofilm formation, production of virulence-associated factors and β-lactamase induction.FEMS Microbiol. Lett. 2019; 366fnz069Crossref PubMed Scopus (18) Google Scholar, 19.Yero D. et al.Genetic variants of the DSF quorum sensing system in Stenotrophomonas maltophilia influence virulence and resistance phenotypes among genotypically diverse clinical isolates.Front. Microbiol. 2020; 11: 1160Crossref PubMed Scopus (11) Google Scholar, 20.Bellenberg S. et al.Diffusible signal factor signaling controls bioleaching activity and niche protection in the acidophilic, mineral-oxidizing Leptospirilli.Sci. Rep. 2021; 11: 16275Crossref PubMed Scopus (5) Google Scholar, 21.Singh P. et al.The diffusible signal factor synthase, RpfF, in Xanthomonas oryzae pv. oryzae is required for the maintenance of membrane integrity and virulence.Mol. Plant Pathol. 2022; 23: 118-132Crossref PubMed Scopus (4) Google Scholar]. In silico analysis suggests that Methylobacillus flagellates, Thiobacillus denitrificans, and bacteria in the genera Frateuria, Luteibacter, Pseudoxanthomonas, and Rhodanobacter also produce DSF. By definition, intraspecies communication occurs among individuals of the same species. QS signal-mediated intraspecies communication helps bacteria to sense their population size or degree of confinement, allowing them to function as a multicellular organism to execute a broad range of functions [7.Zhou L. et al.The DSF family of quorum sensing signals: diversity, biosynthesis, and turnover in Xanthomonas.Trends Microbiol. 2017; 25: 293-303Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar]. Based on their genomic origins, DSF-mediated intraspecies communication pathways are generally affiliated with three different groups, respectively represented by Xcc, B. cenocepacia, and P. aeruginosa. This DSF-based communication system is distinguished by the presence of a ‘regulation of pathogenicity factors’ (rpf) cluster encoding key signaling components such as DSF synthase RpfF and DSF perception system RpfC/RpfG (Figure 1). This system has been functionally verified in the phytopathogens Xanthomonas spp., X. fastidiosa, S. maltophilia, and strains of environmental relevance such as Lysobacter spp. and Leptospirillum spp. [14.An S.Q. et al.Diffusible signal factor signaling regulates multiple functions in the opportunistic pathogen Stenotrophomonas maltophilia.BMC Res. Notes. 2018; 11: 569Crossref PubMed Scopus (16) Google Scholar,16.Feng T. et al.Interspecies and intraspecies signals synergistically regulate Lysobacter enzymogenes twitching motility.Appl. Environ. Microbiol. 2019; 85e01742Crossref Scopus (12) Google Scholar, 17.Ling J. et al.LbDSF, the Lysobacter brunescens quorum-sensing system diffusible signaling factor, regulates anti- Xanthomonas XSAC biosynthesis, colony morphology, and surface motility.Front. Microbiol. 2019; 10: 1230Crossref PubMed Scopus (7) Google Scholar, 18.Alcaraz E. et al.The rpf/DSF signalling system of Stenotrophomonas maltophilia positively regulates biofilm formation, production of virulence-associated factors and β-lactamase induction.FEMS Microbiol. Lett. 2019; 366fnz069Crossref PubMed Scopus (18) Google Scholar, 19.Yero D. et al.Genetic variants of the DSF quorum sensing system in Stenotrophomonas maltophilia influence virulence and resistance phenotypes among genotypically diverse clinical isolates.Front. Microbiol. 2020; 11: 1160Crossref PubMed Scopus (11) Google Scholar, 20.Bellenberg S. et al.Diffusible signal factor signaling controls bioleaching activity and niche protection in the acidophilic, mineral-oxidizing Leptospirilli.Sci. Rep. 2021; 11: 16275Crossref PubMed Scopus (5) Google Scholar, 21.Singh P. et al.The diffusible signal factor synthase, RpfF, in Xanthomonas oryzae pv. oryzae is required for the maintenance of membrane integrity and virulence.Mol. Plant Pathol. 2022; 23: 118-132Crossref PubMed Scopus (4) Google Scholar]. Bacteria using this communication system produce DSF through a classic fatty acid synthesis pathway and a final bifunctional enzyme, RpfF, having both dehydratase and thioesterase activities [22.Zhou L. et al.The multiple DSF-family QS signals are synthesized from carbohydrate and branched-chain amino acids via the FAS elongation cycle.Sci. Rep. 2015; 5: 13294Crossref PubMed Scopus (55) Google Scholar]. However, DSF biosynthesis is strictly regulated during bacterial growth. The whole QS-dependent communication comprises three stages: the pre-QS, QS, and the post-QS, in Xcc [7.Zhou L. et al.The DSF family of quorum sensing signals: diversity, biosynthesis, and turnover in Xanthomonas.Trends Microbiol. 2017; 25: 293-303Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar]. At the pre-QS stage, cell density-dependent DSF biosynthesis is autoinduced via a protein–protein interaction mechanism. The DSF sensor RpfC forms a complex with RpfF through its receiver domain to control DSF biosynthesis [23.He Y.W. et al.Dual signaling functions of the hybrid sensor kinase RpfC of Xanthomonas campestris involve either phosphorelay or receiver domain–protein interaction.J. Biol. Chem. 2006; 281: 33414-33421Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar]. At the post-QS stage, a RpfB-dependent QS signal turnover system is induced and DSF levels return to those of the pre-QS stage [24.Zhou L. et al.Identification and characterization of naturally occurring DSF-family quorum sensing signal turnover system in the phytopathogen Xanthomonas.Environ. Microbiol. 2015; 17: 4646-4658Crossref PubMed Scopus (41) Google Scholar] (Figure 2A ). DSF is perceived and transduced by a two-component system comprising the sensor kinase RpfC and the response regulator RpfG. In Xcc, the activated HD-GYP domain of RpfG functions as a phosphodiesterase to degrade the second messenger cyclic-di-GMP, a ligand of the global regulator cyclic AMP receptor-like protein (Clp), which, in turn, directly or indirectly regulates the production of virulence factors [25.He Y.W. et al.Xanthomonas campestris cell-cell communication involves a putative nucleotide receptor protein Clp and a hierarchical signalling network.Mol. Microbiol. 2007; 64: 281-292Crossref PubMed Scopus (157) Google Scholar,26.Tao F. et al.The cyclic nucleotide monophosphate domain of Xanthomonas campestris global regulator Clp defines a new class of cyclic di-GMP effectors.J. Bacteriol. 2010; 192: 1020-1029Crossref PubMed Scopus (148) Google Scholar]. Activated RpfG was also shown to interact with two GGDEF domain-containing proteins to control Xcc motility [27.Ryan R.P. et al.Cell-cell signal-dependent dynamic interactions between HD-GYP and GGDEF domain proteins mediate virulence in Xanthomonas campestris.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 5989-5994Crossref PubMed Scopus (117) Google Scholar]. In addition, Clp also negatively regulates Xcc aggregate gene cluster ABC (xagABC)-dependent biofilm formation [28.Tao F. et al.Quorum sensing modulation of a putative glycosyltransferase gene cluster essential for Xanthomonas campestris biofilm formation.Environ. Microbiol. 2010; 12: 3159-3170Crossref PubMed Scopus (60) Google Scholar] (Figure 2B). Interestingly, atypical RpfF/RpfC-regulated gene expression and phenotypes were also reported in Xanthomonas oryzae pv. oryzae (Xoo) strain BXO43 and X. fastidiosa [29.Rai R. et al.Cell-cell signaling promotes ferric iron uptake in Xanthomonas oryzae pv. oryzicola that contribute to its virulence and growth inside rice.Mol. Microbiol. 2015; 96: 708-727Crossref PubMed Scopus (20) Google Scholar,30.Ionescu M. et al.Promiscuous diffusible signal factor production and responsiveness of the Xylella fastidiosa rpf System.mBio. 2016; 7e01054Crossref PubMed Scopus (31) Google Scholar]. These findings suggest that the category I DSF signaling might be more complex than what the current models suggest. In Xanthomonas plant pathogens, DSF signaling is generally associated with the regulation of biofilm, multidrug resistance, membrane integrity, and production of virulence factors [15.Li L. et al.Diffusible signal factor (DSF)-mediated quorum sensing modulates expression of diverse traits in Xanthomonas citri and responses of citrus plants to promote disease.BMC Genomics. 2019; 20: 55Crossref PubMed Scopus (24) Google Scholar,21.Singh P. et al.The diffusible signal factor synthase, RpfF, in Xanthomonas oryzae pv. oryzae is required for the maintenance of membrane integrity and virulence.Mol. Plant Pathol. 2022; 23: 118-132Crossref PubMed Scopus (4) Google Scholar,31.He Y.W. et al.Genome scale analysis of diffusible signal factor regulon in Xanthomonas campestris pv. campestris: identification of novel cell-cell communication-dependent genes and functions.Mol. Microbiol. 2006; 59: 610-622Crossref PubMed Scopus (150) Google Scholar,32.Barel V. et al.Virulence and in planta movement of Xanthomonas hortorum pv. pelargonii are affected by the diffusible signal factor (DSF)-dependent quorum sensing system.Mol. Plant Pathol. 2015; 16: 710-723Crossref PubMed Scopus (13) Google Scholar]. In the soil bacterium Lysobacter, LeDSF regulates the production of antibiotics and pigment, and twitching motility [16.Feng T. et al.Interspecies and intraspecies signals synergistically regulate Lysobacter enzymogenes twitching motility.Appl. Environ. Microbiol. 2019; 85e01742Crossref Scopus (12) Google Scholar,17.Ling J. et al.LbDSF, the Lysobacter brunescens quorum-sensing system diffusible signaling factor, regulates anti- Xanthomonas XSAC biosynthesis, colony morphology, and surface motility.Front. Microbiol. 2019; 10: 1230Crossref PubMed Scopus (7) Google Scholar]. DSF signaling in the acidophilic mineral-oxidizing Leptospirilli spp. controls bioleaching activity and niche protection [20.Bellenberg S. et al.Diffusible signal factor signaling controls bioleaching activity and niche protection in the acidophilic, mineral-oxidizing Leptospirilli.Sci. Rep. 2021; 11: 16275Crossref PubMed Scopus (5) Google Scholar]. This communication system is characterized by the presence of the rpfF homolog rpfFBC and rpfR, encoding a BDSF sensor protein (Figure 1). It was reported in all strains of the Burkholderia cepacia complex (Bcc) and the Cronobacter genus [33.Deng Y. et al.Structural and functional characterization of diffusible signal factor family quorum-sensing signals produced by members of the Burkholderia cepacia complex.Appl. Environ. Microbiol. 2010; 76: 4675-4683Crossref PubMed Scopus (95) Google Scholar,34.Suppiger A. et al.Evidence for the widespread production of DSF family signal molecules by members of the genus Burkholderia by the aid of novel biosensors.Environ. Microbiol. Rep. 2016; 8: 38-44Crossref PubMed Scopus (16) Google Scholar]. BDSF is the major signal used for category II intraspecies communication. BDSF biosynthesis is positively regulated by a two-component system, RqpS/RqpR, through modulating rpfFBc transcription in B. cenocepacia [35.Cui C. et al.A novel two-component system modulates quorum sensing and pathogenicity in Burkholderia cenocepacia.Mol. Microbiol. 2018; 108: 32-44Crossref PubMed Scopus (26) Google Scholar]. BDSF signaling in B. cenocepacia has been recently reviewed [13.Wang M. et al.The cis-2-dodecenoic acid (BDSF) quorum sensing system in Burkholderia cenocepacia.Appl. Environ. Microbiol. 2022; 88e0234221Crossref Scopus (4) Google Scholar]. RpfR is a BDSF sensor protein containing a Per/Arnt/Sim (PAS), a GGDEF, and an EAL domain. BDSF binds to the PAS domain of RpfR, causing a conformation change. This change plays a critical role in regulating the phosphodiesterase activity of RpfR, thereby influencing intracellular cyclic-di-GMP levels [36.Waldron E.J. et al.Structural basis of DSF recognition by its receptor RpfR and its regulatory interaction with the DSF synthase RpfF.PLoS Biol. 2019; 17e3000123Crossref PubMed Scopus (20) Google Scholar]. RpfR is also a cyclic di-GMP sensor able to bind both BDSF and cyclic di-GMP [34.Suppiger A. et al.Evidence for the widespread production of DSF family signal molecules by members of the genus Burkholderia by the aid of novel biosensors.Environ. Microbiol. Rep. 2016; 8: 38-44Crossref PubMed Scopus (16) Google Scholar]. A global regulator, GtrR, interacts with RpfR to regulate the transcription of cepI, the acyl homoserine lactone (AHL)-type QS signal synthesis gene in B. cenocepacia [37.Yang C. et al.Burkholderia cenocepacia integrates cis-2-dodecenoic acid and cyclic dimeric guanosine monophosphate signals to control virulence.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 13006-13011Crossref PubMed Scopus (43) Google Scholar]. These findings link BDSF signaling with the AHL-dependent QS system, underlining the complexity of DSF-mediated intraspecies signaling. This category is represented by the drug-resistant bacterium P. aeruginosa. It produces the DSF-type molecule PDSF. PDSF biosynthesis was attributed to dspI (PA0745), a putative rpfF homolog located within the cluster PA0743–PA0747 in PAO1 [38.Amari D.T. et al.The putative enoyl-coenzyme A hydratase DspI is required for production of the Pseudomonas aeruginosa biofilm dispersion autoinducer cis-2-decenoic acid.J. Bacteriol. 2013; 195: 4600-4610Crossref PubMed Scopus (46) Google Scholar]. However, another gene cluster, PA4978–PA4983, was also suggested to be involved in PDSF synthesis and perception [39.Rahmani-Badi A. et al.Dissection of the cis-2-decenoic acid signaling network in Pseudomonas aeruginosa using microarray technique.Front. Microbiol. 2015; 6: 383PubMed Google Scholar]. PDSF was first shown to induce biofilm dispersion in the P. aeruginosa strains PAO1 and PA14 [38.Amari D.T. et al.The putative enoyl-coenzyme A hydratase DspI is required for production of the Pseudomonas aeruginosa biofilm dispersion autoinducer cis-2-decenoic acid.J. Bacteriol. 2013; 195: 4600-4610Crossref PubMed Scopus (46) Google Scholar]. Microarray analysis revealed that PDSF is involved in biofilm dispersion, motility, virulence, and persistence in PAO1 [39.Rahmani-Badi A. et al.Dissection of the cis-2-decenoic acid signaling network in Pseudomonas aeruginosa using microarray technique.Front. Microbiol. 2015; 6: 383PubMed Google Scholar]. In PA14, PDSF-deficiency provoked a remarkable reduction in pyoverdine production, swarming motility, biofilm dispersion, and virulence [40.Liu L. et al.Structural and functional studies on Pseudomonas aeruginosa DspI: implications for its role in DSF biosynthesis.Sci. Rep. 2018; 8: 3928Crossref PubMed Scopus (19) Google Scholar]. The perception and signaling transduction of PDSF remains to be elucidated. Given that PDSF-regulated functions mostly overlap with that of cyclic di-GMP signaling, it is likely that PDSF signaling is also mediated by the second messenger cyclic di-GMP in P. aeruginosa. Microbes never live alone in natural ecosystems but rather interact physically and trophically with each other. They detect not only self-produced signals but also signals synthesized by their neighbors [41.Coquant G. et al.(2020) Impact of N-acyl-homoserine lactones, quorum sensing molecules, on gut immunity.Front. Immunol. 1827; 2020: 11Google Scholar]. Given the prodigious diversity of DSF-producing bacteria in nature, it is not surprising that an enormous repertoire of DSF-mediated interspecies communication occurs. P. aeruginosa, B. cenocepacia, and S. maltophilia are all multidrug-resistant pathogens. Chronic colonization and infection by P. aeruginosa occur in 80% of cystic fibrosis (CF) patients, who are often coinfected by B. cenocepacia, S. maltophilia, as well as the fungus Aspergillus sp. [42.Hughes D.A. et al.Clinical characteristics of Pseudomonas and Aspergillus co-infected cystic fibrosis patients: a UK registry study.J. Cyst. Fibros. 2022; 21: 129-135Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar]. As described previously, these three bacteria each produce the DSF molecules, PDSF, BDSF, and XcDSF, respectively, that mediate, in order, category III, II, and I intraspecies communication (Figure 1). In S. maltophilia–P. aeruginosa cocultures, DSF produced by S. maltophilia influences the architecture of the P. aeruginosa biofilm. The response of P. aeruginosa to S. maltophilia DSF requires the sensor kinase PA1396 [43.Ryan R.P. et al.Interspecies signalling via the Stenotrophomonas maltophilia diffusible signal factor influences biofilm formation and polymyxin tolerance in Pseudomonas aeruginosa.Mol. Microbiol. 2008; 68: 75-86Crossref PubMed Scopus (179) Google Scholar]. Mutation of PA1396 or exogenous addition of XcDSF to P. aeruginosa results in increased bacterial stress tolerance and polymyxin resistance, suggesting that DSF from S. maltophilia can be sensed by P. aeruginosa PA1396 [43.Ryan R.P. et al.Interspecies signalling via the Stenotrophomonas maltophilia diffusible signal factor influences biofilm formation and polymyxin tolerance in Pseudomonas aeruginosa.Mol. Microbiol. 2008; 68: 75-86Crossref PubMed Scopus (179) Google Scholar]. Further, XcDSF and BDSF were both detected at physiologically relevant concentrations in sputum samples from CF patients infected with Bcc and S. maltophilia [44.Twomey K.B. et al.Bacterial cis-2-unsaturated fatty acids found in the cystic fibrosis airway modulate virulence and persistence of Pseudomonas aeruginosa.ISME J. 2012; 6: 939-950Crossref PubMed Scopus (78) Google Scholar]. The presence of DSF was correlated with polymicrobial infections involving Burkholderia and/or Stenotrophomonas species together with P. aeruginosa. When P. aeruginosa PAO1 was inoculated intratracheally together with DSF into CF transmembrane conductance regulator (CFTR) knockout mice, P. aeruginosa persistence was observed in the lung [44.Twomey K.B. et al.Bacterial cis-2-unsaturated fatty acids found in the cystic fibrosis airway modulate virulence and persistence of Pseudomonas aeruginosa.ISME J. 2012; 6: 939-950Crossref PubMed Scopus (78) Google Scholar]. Furthermore, P. aeruginosa could form biofilms on human CF-derived lung epithelial cells in an in vitro culture system. Addition of DSF to the culture system significantly enhanced polymyxin tolerance in P. aeruginosa biofilms. DSF-mediated interspecies communication between B. cenocepacia and P. aeruginosa was also identified [45.Deng Y. et al.Cis-2-dodecenoic acid signal modulates virulence of Pseudomonas aeruginosa through interference with quorum sensing systems and T3SS.BMC Microbiol. 2013; 13: 231Crossref PubMed Scopus (41) Google Scholar]. Exogenous addition of BDSF decreases the transcriptional level of the QS regulator genes lasR, rhlR, and pqsR in P. aeruginosa, leading to a reduced production of AHL and Pseudomonas quinolone signal (PQS) [45.Deng Y. et al.Cis-2-dodecenoic acid signal modulates virulence of Pseudomonas aeruginosa through interference with quorum sensing systems and T3SS.BMC Microbiol. 2013; 13: 231Crossref PubMed Scopus (41) Google Scholar]. In addition, BDSF inhibits the expression of type III secretion system (T3SS) genes in P. aeruginosa at micromolar concentrations and reduces P. aeruginosa virulence in both HeLa cell and zebrafish infection models. However, BDSF signaling in P. aeruginosa could not be attributed to the activity of the reported sensor PA1396, or that of the long-chain fatty acid sensor PsrA. Instead, BDSF appeared to inhibit the T3SS and QS systems through two alternative independent signaling pathways [45.Deng Y. et al.Cis-2-dodecenoic acid signal modulates virulence of Pseudomonas aeruginosa through interference with quorum sensing systems and T3SS.BMC Microbiol. 2013; 13: 231Crossref PubMed Scopus (41) Google Scholar]. Taken together, complex DSF-mediated interspecies communication between P. aeruginosa, B. cenocepacia, and S. maltophilia clearly occurs. This interspecies communication may operate in CF lungs a