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The chemical topology of a bacterial swarm

  • Neha Garg
    Correspondence
    To whom correspondence should be addressed: School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332
    Affiliations
    School of Chemistry and Biochemistry, Atlanta, Georgia 30332

    Emory–Children’s Cystic Fibrosis Center, Atlanta, Georgia 30322
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  • Marvin Whiteley
    Affiliations
    School of Biological Sciences, Georgia Institute of Technology, Atlanta, Georgia 30332

    Emory–Children’s Cystic Fibrosis Center, Atlanta, Georgia 30322
    Search for articles by this author
Open AccessPublished:June 15, 2018DOI:https://doi.org/10.1074/jbc.H118.003178
      Microbes respond to antibiotics by initiating a suite of defense mechanisms, including the production of small-molecule effectors. However, it is not well-known how these defenses vary according to the particular effector or antibiotic and bacterial state, due in part to the challenges of monitoring small molecules in complex environments. A new study uses state-of-the-art imaging techniques to track the location of secreted small molecules produced by a bacterial swarm in response to different antibiotics, providing unexpected insights into the spatial heterogeneity of bacterial stress responses.

      Introduction

      Bacteria occupy a wide array of diverse niches, where they may encounter numerous external stresses. As a result, they have evolved a variety of mechanisms to counter distinct stresses, often by coordinating community-wide responses that may protect at least a subset of the bacterial population. These mechanisms can be mediated by secreted small molecules; compounds discovered thus far have been shown to play roles in the cell-to-cell communication known as quorum-sensing, as well as virulence, aggregation and biofilm formation, antibiotic tolerance, the stress response, and interspecies interactions. Our understanding of the roles of specific small-molecule effectors in mediating these behavioral responses has been established mostly by population level studies. However, a more detailed understanding of where, when, and why these molecules are produced has been difficult to unravel. A new study by Morales-Soto et al. (
      • Morales-Soto N.
      • Dunham S.J.B.
      • Baig N.F.
      • Ellis J.F.
      • Madukoma C.S.
      • Bohn P.W.
      • Sweedler J.V.
      • Shrout J.D.
      Spatially dependent alkyl quinolone signaling responses to antibiotics in Pseudomonas aeruginosa swarms.
      ) sheds light on this topic, using advanced analytical strategies to directly image the alkyl quinolones produced by swarming Pseudomonas aeruginosa in response to two different antibiotics. Their results redefine the role of one well-known quorum–sensing molecule and demonstrate the geographical precision with which bacteria can control their communities.
      P. aeruginosa is an opportunistic pathogen that has the ability to become tolerant as well as resistant to several antibiotics. It has also become an important model system for studying biofilm formation and quorum-sensing. It secretes more than 50 different small molecules, referred to as 2-alkyl-4(1H)-quinolones, or alkyl quinolones (
      • Lépine F.
      • Milot S.
      • Déziel E.
      • He J.
      • Rahme L.G.
      Electrospray/mass spectrometric identification and analysis of 4-hydroxy-2-alkylquinolines (HAQs) produced by Pseudomonas aeruginosa.
      ). Two of these molecules—2-heptyl-3-hydroxyquinolone (also termed Pseudomonas quinolone signal, PQS)
      The abbreviations used are: PQS
      Pseudomonas quinolone signal
      HQNO
      2-heptyl-4-hydroxyquinoline N-oxide.
      and 2-heptyl-4-hydroxyquinoline N-oxide (HQNO)—are well-studied with the former serving as a quorum-sensing signal (
      • Pesci E.C.
      • Milbank J.B.
      • Pearson J.P.
      • McKnight S.
      • Kende A.S.
      • Greenberg E.P.
      • Iglewski B.H.
      Quinolone signaling in the cell-to-cell communication system of Pseudomonas aeruginosa.
      ), whereas the latter possesses antimicrobial activity (
      • Szamosvari D.
      • Böttcher T.
      An unsaturated quinolone N-oxide of Pseudomonas aeruginosa modulates growth and virulence of Staphylococcus aureus.
      ), in addition to various other biological roles. Previous work, including our own, has shown spatial variability in the production of these molecules and others at the macro scale within lungs of humans and pigs infected with P. aeruginosa (
      • Garg N.
      • Wang M.
      • Hyde E.
      • da Silva R.R.
      • Melnik A.V.
      • Protsyuk I.
      • Bouslimani A.
      • Lim Y.W.
      • Wong R.
      • Humphrey G.
      • Ackermann G.
      • Spivey T.
      • Brouha S.S.
      • Bandeira N.
      • Lin G.Y.
      • Rohwer F.
      • Conrad D.J.
      • Alexandrov T.
      • Knight R.
      • Dorrestein P.C.
      Three-dimensional microbiome and metabolome cartography of a diseased human lung.
      ,
      • Davies S.K.
      • Fearn S.
      • Allsopp L.P.
      • Harrison F.
      • Ware E.
      • Diggle S.P.
      • Filloux A.
      • McPhail D.S.
      • Bundy J.G.
      Visualizing antimicrobials in bacterial biofilms: Three-dimensional biochemical imaging using TOF-SIMS.
      • Jorth P.
      • Staudinger B.J.
      • Wu X.
      • Hisert K.B.
      • Hayden H.
      • Garudathri J.
      • Harding C.L.
      • Radey M.C.
      • Rezayat A.
      • Bautista G.
      • Berrington W.R.
      • Goddard A.F.
      • Zheng C.
      • Angermeyer A.
      • Brittnacher M.J.
      • Kitzman J.
      • Shendure J.
      • Fligner C.L.
      • Mittler J.
      • Aitken M.L.
      • Manoil C.
      • Bruce J.E.
      • Yahr T.L.
      • Singh P.K.
      Regional isolation drives bacterial diversification within cystic fibrosis Lungs.
      ). However, the spatial distribution of individual alkyl quinolones at the cellular level is unknown. Furthermore, the interplay between environmental cues and bacterial responses remains poorly understood during the “swarming” state, a term for bacterial translocation across a solid (or semisolid) substrate that has been suggested to be a pre-biofilm state. Swarming bacteria have been shown to be more resistant to some antimicrobials; thus, it may have importance in vivo. In addition, the swarming phenotype may be important for finding favorable conditions in vivo prior to biofilm formation. Understanding how bacteria spread on surfaces and how these processes are affected during stress response is crucial to creating effective treatment strategies.
      Morales-Soto et al. began their study by confirming that both the ribosome-binding tobramycin and the cell-wall biosynthesis–disrupting carbenicillin inhibit swarming of P. aeruginosa, resulting in different cellular morphologies. The authors then used confocal Raman microscopy, which is able to distinguish PQS and HQNO families of compounds, to monitor the chemical outcome of antibiotic treatment. In contrast to untreated swarms, in which PQS-like molecules were localized in the center of the swarms, these compounds were found at the edge of the swarm after addition of a higher dose of tobramycin (Fig. 1). HQNO-like compounds, which were uniformly distributed in untreated swarms, were also found at the swarm periphery upon challenge with the same dose of tobramycin, but at the opposite side of the swarm. Surprisingly, PQS-like compounds could not be detected after carbenicillin treatment. Tests of bacterial strains deficient in the PQS or HQNO biosynthetic pathways confirmed a unique relationship between PQS and tobramycin though, paradoxically, swarms of cells deficient in PQS production were actually less susceptible to tobramycin.
      Figure thumbnail gr1
      Figure 1Cartoon representation of variation in distribution of alkyl quinolones on exposure to tobramycin displaying the significant differences. A, distribution of PQS and HQNO to unexposed swarm. B, distribution of PQS and HQNO to swarm exposed to tobramycin.
      To gain further insights, the authors turned to MS-based imaging. This technique can theoretically distinguish individual compounds, but traditionally has relied on the detection of MS parent ions; as the PQS and HQNO families of alkyl quinolones have identical molecular mass, it is difficult to distinguish between them using only MS data. Instead, Morales-Soto et al. used MS/MS-based (product ion) MS imaging, which allowed them to monitor four specific compounds (PQS, HQNO, and their longer-chain analogs). This method confirmed the presence of the compounds at the swarm periphery upon tobramycin treatment as well as the disparate distribution of PQS near the tobramycin and HQNO away from it.
      These data show for the first time that the PQS and HQNO family alkyl quinolones are present on different spatial scales and have distinct biological responses to environmental stress. Importantly, it was previously thought that PQS was a general signal induced by stress, but its presence in swarms exposed to tobramycin but not carbenicillin and its specific localization in the tobramycin-treated swarms suggests it is instead a short-range signal that is only induced by certain stresses. Indeed, we have described a general lack of PQS detection in P. aeruginosa–dominant sputum (
      • Quinn R.A.
      • Phelan V.V.
      • Whiteson K.L.
      • Garg N.
      • Bailey B.A.
      • Lim Y.W.
      • Conrad D.J.
      • Dorrestein P.C.
      • Rohwer F.L.
      Microbial, host and xenobiotic diversity in the cystic fibrosis sputum metabolome.
      ) and lung tissue (
      • Garg N.
      • Wang M.
      • Hyde E.
      • da Silva R.R.
      • Melnik A.V.
      • Protsyuk I.
      • Bouslimani A.
      • Lim Y.W.
      • Wong R.
      • Humphrey G.
      • Ackermann G.
      • Spivey T.
      • Brouha S.S.
      • Bandeira N.
      • Lin G.Y.
      • Rohwer F.
      • Conrad D.J.
      • Alexandrov T.
      • Knight R.
      • Dorrestein P.C.
      Three-dimensional microbiome and metabolome cartography of a diseased human lung.
      ) of patients afflicted with cystic fibrosis, whereas other alkyl quinolones were detected. Thus, alkyl quinolone signaling varies with the stage of biofilm formation and in response to different environmental stresses. The higher localization of PQS and not HQNO at the edge of swarm further suggests that PQS and HQNO send or respond to distinct messages to/from cells depending on the stress and that PQS, but not HQNO, provides protection against tobramycin to pre-biofilm swarms. Morales-Soto et al. (
      • Morales-Soto N.
      • Dunham S.J.B.
      • Baig N.F.
      • Ellis J.F.
      • Madukoma C.S.
      • Bohn P.W.
      • Sweedler J.V.
      • Shrout J.D.
      Spatially dependent alkyl quinolone signaling responses to antibiotics in Pseudomonas aeruginosa swarms.
      ) propose that PQS may serve to modulate cell permeability, resulting in lysis and release of DNA, which could sequester the positively charged tobramycin.
      The work from Morales-Soto et al. (
      • Morales-Soto N.
      • Dunham S.J.B.
      • Baig N.F.
      • Ellis J.F.
      • Madukoma C.S.
      • Bohn P.W.
      • Sweedler J.V.
      • Shrout J.D.
      Spatially dependent alkyl quinolone signaling responses to antibiotics in Pseudomonas aeruginosa swarms.
      ) highlights several new directions to explore. First, it will be useful to understand the molecular basis for decreased susceptibility to tobramycin in the PQS-deficient strain, as this may provide new insights into the mechanism of action of this compound. Second, the authors noted differences in PQS and HQNO spatial profiles at varying antibiotic dosages. Are such conditions present in a clinical infection? Previous work, including our own, has demonstrated regional diversification in antibiotic resistance, antibiotic penetration, and the properties of P. aeruginosa in patient lungs and biofilms (
      • Garg N.
      • Wang M.
      • Hyde E.
      • da Silva R.R.
      • Melnik A.V.
      • Protsyuk I.
      • Bouslimani A.
      • Lim Y.W.
      • Wong R.
      • Humphrey G.
      • Ackermann G.
      • Spivey T.
      • Brouha S.S.
      • Bandeira N.
      • Lin G.Y.
      • Rohwer F.
      • Conrad D.J.
      • Alexandrov T.
      • Knight R.
      • Dorrestein P.C.
      Three-dimensional microbiome and metabolome cartography of a diseased human lung.
      ,
      • Davies S.K.
      • Fearn S.
      • Allsopp L.P.
      • Harrison F.
      • Ware E.
      • Diggle S.P.
      • Filloux A.
      • McPhail D.S.
      • Bundy J.G.
      Visualizing antimicrobials in bacterial biofilms: Three-dimensional biochemical imaging using TOF-SIMS.
      • Jorth P.
      • Staudinger B.J.
      • Wu X.
      • Hisert K.B.
      • Hayden H.
      • Garudathri J.
      • Harding C.L.
      • Radey M.C.
      • Rezayat A.
      • Bautista G.
      • Berrington W.R.
      • Goddard A.F.
      • Zheng C.
      • Angermeyer A.
      • Brittnacher M.J.
      • Kitzman J.
      • Shendure J.
      • Fligner C.L.
      • Mittler J.
      • Aitken M.L.
      • Manoil C.
      • Bruce J.E.
      • Yahr T.L.
      • Singh P.K.
      Regional isolation drives bacterial diversification within cystic fibrosis Lungs.
      ). Thus, microbial communities are exposed and respond to differential antibiotic stresses, even within the same organ. But, previous to the paper by Morales-Soto et al. (
      • Morales-Soto N.
      • Dunham S.J.B.
      • Baig N.F.
      • Ellis J.F.
      • Madukoma C.S.
      • Bohn P.W.
      • Sweedler J.V.
      • Shrout J.D.
      Spatially dependent alkyl quinolone signaling responses to antibiotics in Pseudomonas aeruginosa swarms.
      ), a direct correlation between antibiotic exposure and spatial differences in specific alkyl quinolone levels was not known. In this regard, Morales-Soto et al. narrowed our gap in the understanding of individual functions of alkyl quinolones and how they contribute to antibiotic tolerance. Future investigations into the role of alkyl quinolone signaling in Pseudomonas clinical isolates from patients with prior exposure to antibiotics will reveal how these pathways evolve in complex infection environments such as cystic fibrosis. Such studies are necessary to explore the efficacy and timing of administration and development of new therapeutics.

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      Linked Article

      • Spatially dependent alkyl quinolone signaling responses to antibiotics in Pseudomonas aeruginosa swarms
        Journal of Biological ChemistryVol. 293Issue 24
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          There is a general lack of understanding about how communities of bacteria respond to exogenous toxins such as antibiotics. Most of our understanding of community-level stress responses comes from the study of stationary biofilm communities. Although several community behaviors and production of specific biomolecules affecting biofilm development and associated behavior have been described for Pseudomonas aeruginosa and other bacteria, we have little appreciation for the production and dispersal of secreted metabolites within the 2D and 3D spaces they occupy as they colonize, spread, and grow on surfaces.
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