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Systematic identification of molecular mediators of interspecies sensing in a community of two frequently coinfecting bacterial pathogens [1]

['Tiffany M. Zarrella', 'Laboratory Of Molecular Biology', 'Center For Cancer Research', 'National Cancer Institute', 'National Institutes Of Health', 'Bethesda', 'Maryland', 'United States Of America', 'Postdoctoral Research Associate Training Program', 'National Institute Of General Medical Sciences']

Date: 2022-07

Bacteria typically exist in dynamic, multispecies communities where polymicrobial interactions influence fitness. Elucidating the molecular mechanisms underlying these interactions is critical for understanding and modulating bacterial behavior in natural environments. While bacterial responses to foreign species are frequently characterized at the molecular and phenotypic level, the exogenous molecules that elicit these responses are understudied. Here, we outline a systematic strategy based on transcriptomics combined with genetic and biochemical screens of promoter-reporters to identify the molecules from one species that are sensed by another. We utilized this method to study interactions between the pathogens Pseudomonas aeruginosa and Staphylococcus aureus that are frequently found in coinfections. We discovered that P. aeruginosa senses diverse staphylococcal exoproducts including the metallophore staphylopine (StP), intermediate metabolites citrate and acetoin, and multiple molecules that modulate its iron starvation response. We observed that StP inhibits biofilm formation and that P. aeruginosa can utilize citrate and acetoin for growth, revealing that these interactions have both antagonistic and beneficial effects. Due to the unbiased nature of our approach, we also identified on a genome scale the genes in S. aureus that affect production of each sensed exoproduct, providing possible targets to modify multispecies community dynamics. Further, a combination of these identified S. aureus products recapitulated a majority of the transcriptional response of P. aeruginosa to S. aureus supernatant, validating our screening strategy. Cystic fibrosis (CF) clinical isolates of both S. aureus and P. aeruginosa also showed varying degrees of induction or responses, respectively, which suggests that these interactions are widespread among pathogenic strains. Our screening approach thus identified multiple S. aureus secreted molecules that are sensed by P. aeruginosa and affect its physiology, demonstrating the efficacy of this approach, and yielding new insight into the molecular basis of interactions between these two species.

Funding: This work was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research. TMZ was supported by a Postdoctoral Research Associate Training (PRAT) Fellowship award 1FI2GM137843-01 from the National Institute of General Medical Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

In this study, we outline a strategy to systematically identify the secreted molecules from one species that are sensed by another, utilizing transcriptomics in conjunction with genetic and biochemical screens of promoter-reporters. Our approach revealed that P. aeruginosa senses at least four distinct signals secreted by S. aureus: the metallophore staphylopine (StP), which induces zinc starvation, the intermediate metabolites citrate and acetoin, which induce their uptake and catabolism, and unidentified molecules that affect iron-related pathways. Through the genome-wide screen, we also delineate the S. aureus genes that alter the production of these sensed molecules. Further, we find that these staphylococcal exoproducts mediate both antagonistic and beneficial effects on P. aeruginosa physiology. StP participates in metal competition and abrogates P. aeruginosa biofilm formation, while the intermediate metabolites can serve as sole carbon sources, and the iron-chelating secreted factors deliver iron to P. aeruginosa. Several clinical isolates of S. aureus and P. aeruginosa show induction and responses, respectively, of varying intensity, and other bacterial species can also induce some of the same pathways in P. aeruginosa, indicating the generality of these phenomena. Finally, we show that these S. aureus secreted molecules explain a major part of the P. aeruginosa response to S. aureus, demonstrating the utility of our approach to identify the secreted factors that underlie interspecies interactions.

Interactions between these two species have been extensively studied (reviewed by [ 28 – 31 ]). P. aeruginosa affects the growth, metabolism, antibiotic resistance, and transcriptional state of S. aureus, mainly via the secretion of multiple antimicrobials [ 6 , 32 – 35 ]. S. aureus induces exploratory motility in P. aeruginosa via unknown exoproducts, and the quorum sensing molecule autoinducer-2 and cell wall precursor N-acetyl glucosamine that can be produced by S. aureus affect the regulation of P. aeruginosa virulence factors [ 36 – 42 ]. However, characterization of the P. aeruginosa transcriptional response to S. aureus and the comprehensive identification of the underlying sensed S. aureus secreted factors and their production pathways have not been fully accomplished.

The goal of this work was to develop a comprehensive approach to define which exoproducts from one species are sensed by another and use this framework to study interspecies sensing in a model two-species system consisting of Pseudomonas aeruginosa and Staphylococcus aureus. Both are opportunistic pathogens that coexist in wound infections, nosocomial pneumonia, as well as lung infections in cystic fibrosis (CF) patients [ 21 – 24 ], where coinfection with both pathogens has been associated with increased disease severity [ 25 – 27 ]. Identifying the molecular determinants of interactions between these two coinfecting pathogens can thus provide novel insights into persistent infections and multispecies communities in general.

One powerful strategy to determine the cues being sensed by bacteria is to infer them from the cellular response. Examples include the SOS response indicating the sensing of DNA damage or the heat shock response revealing the perception of high environmental temperature [ 16 , 17 ]. In multispecies communities, specific bacterial responses have been used to identify the interspecific mediators. These include the up-regulation of oxidative stress pathways in response to secreted redox-active molecules, alleviation of nutritional requirements due to sensing and use of secreted amino acids or other nutrients, and induction of iron starvation pathways by sensed iron competition caused by siderophores secreted by a foreign species [ 18 – 20 ]. However, most such studies of interactions typically focus on a single response and the underlying molecules.

Previous studies on pairwise interspecies interactions have focused on the identification of molecules produced by one species and the effect of these molecules on specific phenotypes of another species, such as growth, biofilm formation, or antibiotic resistance [ 6 – 12 ]. Recent work has described the genome-wide response of one species to another at the molecular level using either transcriptomics, proteomics, or metabolomics in multispecies conditions, revealing that these responses can be complex and affect numerous pathways [ 13 – 15 ], and are therefore likely mediated by multiple sensed secreted factors.

Bacteria frequently exist in multispecies communities in which cooperative and competitive interactions govern community composition and physiological outputs. Interactions among pathogenic bacterial species in multispecies infections can affect disease outcomes and antibiotic susceptibility [ 1 , 2 ]. Elucidating the molecules underlying these interactions is therefore critical to understand and modulate bacterial behavior in communities, and pairwise interaction studies using unbiased genetic and biochemical approaches are powerful tools for such analyses [ 3 – 5 ].

Results

Framework to comprehensively define the molecular mediators of bacterial interspecies sensing We developed a systematic methodology within a two-species system to identify the sensed foreign molecules that underlie the global response of one species to the other (Fig 1A). Our approach first defines the complex response resulting from exposure to a foreign species and identifies the different individual components of the response. Then, these individual responses are used to identify the respective causal foreign molecules via unbiased genetic and biochemical screens. Unlike previous approaches, our strategy does not focus on the effect of individual molecules on a foreign species, or on globally identifying all molecules that are produced by a species, rather each response is used to identify the respective specific foreign molecule(s) that together make up the interspecies interaction. PPT PowerPoint slide

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TIFF original image Download: Fig 1. P. aeruginosa differentially regulates metal deprivation and intermediate metabolite uptake pathways in the presence of S. aureus supernatant. (A) Schematic for identification of molecular mediators of interspecies sensing in a two species system. Global transcriptional response of one species to another species, or its secreted exoproducts, compared to monocultures, is determined by RNA-seq. Analysis of the differentially regulated genes and pathways may be used to identify the signaling molecules that are sensed. Promoter-reporters are constructed from representative up-regulated genes and used to screen for the signaling molecules by 2 complementary methods. In the first method, a mutant library is screened for mutants that disrupt production or export of the signaling molecules and therefore have lower reporter induction. In the second, the supernatant is biochemically fractionated and fractions are screened for induction of the promoter. (B) Venn diagrams of up-regulated and down-regulated genes (Log 2 fold change ≥ 1 or ≤ −1 and p < 0.05 cutoff) in P. aeruginosa after S. aureus supernatant exposure compared to media control after 20 minutes, 1 hour, and 2 hours. (C) GO enrichment of differentially expressed genes in P. aeruginosa after 20 minutes [43–45]. Nonredundant categories for down-regulated and up-regulated biological processes are shown. (D) Scatterplot of mean expression levels of transcripts after S. aureus supernatant exposure compared to media control after 20 minutes. Genes annotated previously in P. aeruginosa strain PAO1 as being regulated by Fur, PvdS (IS box), or Zur are shown [46–48]. (E) Log 2 fold change of select transcripts in metabolite-uptake operons that increase in abundance over time. The data underlying panels B, C, D, and E can be found in Tables B, C, and D, and both Tables A and B, in S1 File, respectively. GO, Gene Ontology; RNA-seq, RNA sequencing. https://doi.org/10.1371/journal.pbio.3001679.g001 In the initial step, global transcriptional analysis is performed on one species exposed to another species or its cell-free supernatant compared to monocultures, to identify which pathways are differentially regulated by interspecies sensing. Next, promoter-reporter constructs are designed using representative genes from these up-regulated classes. These promoter-reporter strains are then employed in two complementary approaches to identify the sensed interspecific molecules. In the first approach, an arrayed transposon mutant library in the species being sensed is screened to determine the mutants that are deficient in inducing reporter expression. The mutants are likely to be involved in the regulation, biosynthesis, or secretion of the cues, and mutant gene function or characterization of the mutant supernatant is therefore used to identify the sensed molecules. In the second, the sensed supernatant is fractionated to identify fractions that contain the active molecules inducing the promoters that can then be further analyzed by additional fractionation and mass spectrometry. This two-pronged unbiased approach has the potential to comprehensively reveal the molecules that lead to complex responses in a foreign species, irrespective of which pathways and mechanisms constitute the response. We applied this scheme to study the S. aureus secreted products that are sensed by P. aeruginosa.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001679

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