Mutational Analysis of the Quorum-Sensing Receptor LasR Reveals Interactions that Govern Activation and Inhibition by Nonlactone Ligands

SummaryGram-negative bacteria use N-acyl L-homoserine lactone (AHL) quorum-sensing (QS) signals to regulate the expression of myriad phenotypes. Non-native AHL analogs can strongly attenuate QS receptor activity and thereby QS signaling; however, we currently lack a molecular understanding of the mechanisms by which most of these compounds elicit their agonistic or antagonistic profiles. In this study, we investigated the origins of striking activity profile switches (i.e., receptor activator to inhibitor, and vice versa) observed upon alteration of the lactone head group in certain AHL analogs. Reporter gene assays of mutant versions of the Pseudomonas aeruginosa QS receptor LasR revealed that interactions between the ligands and Trp60, Tyr56, and Ser129 govern whether these ligands behave as LasR activators or inhibitors. Using this knowledge, we propose a model for the modulation of LasR by AHL analogs—encompassing a subtly different interaction with the binding pocket to a global change in LasR conformation

Genetic mapping of microbial and host traits reveals production of immunomodulatory lipids by Akkermansia muciniphila in the murine gut.

The molecular bases of how host genetic variation impacts the gut microbiome remain largely unknown. Here we used a genetically diverse mouse population and applied systems genetics strategies to identify interactions between host and microbe phenotypes including microbial functions, using faecal metagenomics, small intestinal transcripts and caecal lipids that influence microbe-host dynamics. Quantitative trait locus (QTL) mapping identified murine genomic regions associated with variations in bacterial taxa; bacterial functions including motility, sporulation and lipopolysaccharide production and levels of bacterial- and host-derived lipids. We found overlapping QTL for the abundance of Akkermansia muciniphila and caecal levels of ornithine lipids. Follow-up in vitro and in vivo studies revealed that A. muciniphila is a major source of these lipids in the gut, provided evidence that ornithine lipids have immunomodulatory effects and identified intestinal transcripts co-regulated with these traits including Atf3, which encodes for a transcription factor that plays vital roles in modulating metabolism and immunity. Collectively, these results suggest that ornithine lipids are potentially important for A. muciniphila-host interactions and support the role of host genetics as a determinant of responses to gut microbes

Competition Studies Confirm Two Major Barriers That Can Preclude the Spread of Resistance to Quorum-Sensing Inhibitors in Bacteria

The growing threat of antibiotic resistance necessitates the development of novel antimicrobial therapies. Antivirulence agents that target group-beneficial traits in microorganisms (i.e., phenotypes that help the cells surrounding the producer cell instead of selfishly benefiting only the producer cell) represent a new antimicrobial approach that may be robust against the spread of resistant mutants. One prominent group-beneficial antivirulence target in bacteria is quorum sensing (QS). While scientists are producing new QS inhibitors (QSIs) at an increasing pace for use as research tools and potential therapeutic leads, substantial work remains in empirically demonstrating a robustness against resistance. Herein we report the results of <i>in vitro</i> competition studies in <i>Pseudomonas aeruginosa</i> that explicitly confirm that <i>two separate barriers</i> can impede the spread of resistance to QSIs: (1) insufficient native QS signal levels prevent rare QSI-resistant bacteria from expressing their QS regulon, and (2) group-beneficial QS-regulated phenotypes produced by resistant bacteria are susceptible to cheating by QSI-sensitive neighbors, even when grown on a solid substrate with limited mixing to mimic infected tissue. These results underscore the promise of QSIs and other antivirulence molecules that target group beneficial traits as resistance-robust antimicrobial treatments and provide support for their further development

Chemical factors induce aggregative multicellularity in a close unicellular relative of animals

Regulated cellular aggregation is an essential process for development and healing in many animal tissues. In some animals and a few distantly related unicellular species, cellular aggregation is regulated by diffusible chemical cues. However, it is unclear whether regulated cellular aggregation was part of the life cycles of the first multicellular animals and/or their unicellular ancestors. To fill this gap, we investigated the triggers of cellular aggregation in one of animals’ closest unicellular living relatives—the filasterean Capsaspora owczarzaki. We discovered that Capsaspora aggregation is induced by chemical cues, as observed in some of the earliest branching animals and other unicellular species. Specifically, we found that calcium ions and lipids present in lipoproteins function together to induce aggregation of viable Capsaspora cells. We also found that this multicellular stage is reversible as depletion of the cues triggers disaggregation, which can be overcome upon reinduction. Our finding demonstrates that chemically regulated aggregation is important across diverse members of the holozoan clade. Therefore, this phenotype was plausibly integral to the life cycles of the unicellular ancestors of animals.We thank the Light Microscopy Center at the Indiana University for support in image acquisition and analysis (funding provided by the NIH grant NIH1S10OD024988-01) and the Advanced Light Microscopy Unit of the Center for Genomic Regulation for support on image acquisition. We also thank the Indiana University Nanoscale Characterization Facility, Electron Microscopy Center, and Laboratory for Biological Mass Spectrometry for use of their instruments. We want to special thank Claudio Scazzocchio for fruit-ful discussions and Sebastián R. Najle for feedback and support in early work on Capsaspora aggregation. We acknowledge Omaya Dudin for feedback and support in image acquisition and analysis, Michelle M. Leger for feedback on the manuscript, and Koryu Kin for support in immunocytochemistry experi-ments, image acquisition and analysis, and stimulating discussions. We also thank many people from the lab for insights and support that helped advance this project. This work was supported by a NIH grant (R35GM138376) to J.P.G. and grants BFU2017-90114-P from Ministerio de Economía y Competitividad, Agencia Estatal de Investigación (AEI), and Fondo Europeo de Desarrollo Regional and PID2020-120609GB-I00 by Ministerio de Ciencia e Innovación/AEI/10.13039/501100011033 and “European Regional Development Fund: A way of making Europe” to I.R.-T. N.R.-R. was supported by a “Formación del Profesorado Universitario (FPU13/01840)” predoctoral fellowship from Ministerio de Educación, Cultura y Deporte and R.Q.K. was supported by a NIH training grant (T32GM131994

Mating in the Closest Living Relatives of Animals Is Induced by a Bacterial Chondroitinase

We serendipitously discovered that the marine bacterium Vibrio fischeri induces sexual reproduction in one of the closest living relatives of animals, the choanoflagellate Salpingoeca rosetta. Although bacteria influence everything from nutrition and metabolism to cell biology and development in eukaryotes, bacterial regulation of eukaryotic mating was unexpected. Here, we show that a single V.&nbsp;fischeri protein, the previously uncharacterized EroS, fully recapitulates the aphrodisiac-like activity of live V.&nbsp;fischeri. EroS is a chondroitin lyase; although its substrate, chondroitin sulfate, was previously thought to be an animal synapomorphy, we demonstrate that S.&nbsp;rosetta produces chondroitin sulfate and thus extend the ancestry of this important glycosaminoglycan to the premetazoan era. Finally, we show that V.&nbsp;fischeri, purified EroS, and other bacterial chondroitin lyases induce S.&nbsp;rosetta mating at environmentally relevant concentrations, suggesting that bacteria likely regulate choanoflagellate mating in nature

Interbacterial Biofilm Competition through a Suite of Secreted Metabolites

Polymicrobial biofilms are ubiquitous, and the complex interspecies interactions within them are cryptic. We discovered the chemical foundation of antagonistic interactions in a model dual-species biofilm in which Pseudomonas aeruginosa inhibits the biofilm formation of Agrobacterium tumefaciens. Three known siderophores produced by P. aeruginosa (pyoverdine, pyochelin, and dihydroaeruginoic acid) were each capable of inhibiting biofilm formation. Surprisingly, a mutant that was incapable of producing these siderophores still secreted an antibiofilm metabolite. We discovered that this inhibitor was N5-formyl-N5-hydroxy-l-ornithine (fOHOrn)a precursor in pyoverdine biosynthesis. Unlike the siderophores, this inhibitor did not appear to function via extracellular metal sequestration. In addition to this discovery, the compensatory overproduction of a new biofilm inhibitor illustrates the risk of pleiotropy in genetic knockout experiments. In total, this work lends new insight into the chemical nature of dual-species biofilm regulation and reveals a new naturally produced inhibitor of A. tumefaciens biofilm formation

Chemical Interrogation of LuxR-type Quorum Sensing Receptors Reveals New Insights into Receptor Selectivity and the Potential for Interspecies Bacterial Signaling

Cell–cell signaling between bacteria, including quorum-sensing (QS) communication systems, may play a role in the establishment and maintenance of polymicrobial communities. To better understand and model these interactions, we must uncover the degree to which neighboring species recognize each another’s signals. In the current study, we tested the likelihood of whether the QS systems of two opportunistic pathogens (<i>Acinetobacter baumannii</i> and <i>Pseudomonas aeruginosa</i>) that frequently arise in polymicrobial infections would be affected by the QS signals of neighboring species. Through the synthesis and screening of a library of native and non-native <i>N-</i>acyl l-homoserine lactones (AHLs), we found that the AbaR LuxR-type receptor protein of <i>A. baumannii</i> is highly selective for its native AHL signal. However, a homologous LuxR-type receptor in <i>P. aeruginosa</i>, LasR, is far more promiscuously activated by AHLs relative to AbaR, suggesting that LasR-regulated QS could be more susceptible to activation by neighboring species. To explain the observed difference in signal selectivity between AbaR and LasR, we developed a model based on (i) the activity profiles of these proteins and (ii) previously reported structural data and activity profiles for related LuxR-type receptors. This model may facilitate the study of signal selectivities for hundreds of LuxR-type QS receptors from bacteria, many of which grow in polymicrobial communities and may sense each other’s signals. In addition, we discovered a set of AHLs that could be used to selectively activate LasR and selectively inhibit AbaR in polymicrobial experiments