Integration host factor regulates colonization factors in the bee gut symbiont Frischella perrara

  1. Konstantin Schmidt
  2. Gonçalo Santos-Matos
  3. Stefan Leopold-Messer
  4. Yassine El Chazli
  5. Olivier Emery
  6. Théodora Steiner
  7. Joern Piel
  8. Philipp Engel

  • Curated by eLife

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    Evaluation Summary:

    The gut of honeybees is colonized with stable community of bacterial symbionts. The molecular mechanisms involved in this stable associations are poorly understood. Here Schmidt et. al. focused on the identification of bacterial factors required for the colonization of the bee gut symbiont Frischella perrara. They identified a major regulator involved in controlling the expression of multiple colonization factors in F. perrara which include factors that are either required for the induction of a distinct scab phenotype upon gut colonization, or are involved in promoting colonization through adhesin or the production of secondary metabolites. The work is of potential interest for researchers in the field of host-microbe interactions and symbiosis, and for the study of bee health. The valuable integration of different techniques used in this study and rigor of the experiments lead to solid data and justified conclusions.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

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Abstract

Bacteria colonize specific niches in the animal gut. However, the genetic basis of these associations is often unclear. The proteobacterium Frischella perrara is a widely distributed gut symbiont of honey bees. It colonizes a specific niche in the hindgut and causes a characteristic melanization response. Genetic determinants required for the establishment of this association, or its relevance for the host, are unknown. Here, we independently isolated three point mutations in genes encoding the DNA-binding protein integration host factor (IHF) in F. perrara . These mutants abolished the production of an aryl polyene metabolite causing the yellow colony morphotype of F. perrara . Inoculation of microbiota-free bees with one of the mutants drastically decreased gut colonization of F. perrara . Using RNAseq, we found that IHF affects the expression of potential colonization factors, including genes for adhesion (type 4 pili), interbacterial competition (type 6 secretion systems), and secondary metabolite production (colibactin and aryl polyene biosynthesis). Gene deletions of these components revealed different colonization defects depending on the presence of other bee gut bacteria. Interestingly, one of the T6SS mutants did not induce the scab phenotype anymore despite colonizing at high levels, suggesting an unexpected role in bacteria-host interaction. IHF is conserved across many bacteria and may also regulate host colonization in other animal symbionts.

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  1. Version published to 10.7554/elife.76182 on eLife
  2. Version published to 10.1101/2021.12.06.471457v3 on bioRxiv
  3. eLife

    Evaluation Summary:

    The gut of honeybees is colonized with stable community of bacterial symbionts. The molecular mechanisms involved in this stable associations are poorly understood. Here Schmidt et. al. focused on the identification of bacterial factors required for the colonization of the bee gut symbiont Frischella perrara. They identified a major regulator involved in controlling the expression of multiple colonization factors in F. perrara which include factors that are either required for the induction of a distinct scab phenotype upon gut colonization, or are involved in promoting colonization through adhesin or the production of secondary metabolites. The work is of potential interest for researchers in the field of host-microbe interactions and symbiosis, and for the study of bee health. The valuable integration of different techniques used in this study and rigor of the experiments lead to solid data and justified conclusions.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    The authors are using the honeybee and its gut microbial symbionts to understand the factors that enable the stable association between the host and its symbiont. They are also interested in understanding the factors involved in niche specificity, namely the mechanisms that are required for bacteria to colonize specific physical niches in the gut.

    Here they focus on the bee gut symbiont Frischella perrara. A gammaproteobacterial that colonizes a restricted region of the gut (midgut and pylorus/ileum). One unique characteristic of this bacterial symbiont is that upon colonization of the gut it induces the scab phenotype, characterized by the accumulation of a brown material deposited on the host epithelium, which might be related to the host immune response to this symbiont.

    The authors isolated spontaneous mutations in a regulator called IHF, and studied the phenotype of these mutations in vivo, using gnotobiotic model with bees mono-colonized with F. perrara. They determined the genes differentially expressed in a ihf mutant in comparison with WT, both in vitro and in vivo. With these transcriptomics experiments they observed that mutants in IHF affects expression of a large number of genes. Among the genes with low expression in the ihf mutant, were genes involved in production of colibactin, pili formation, production of the yellow pigment aryl polyene, and components of type VI secretion system (T6SS). They constructed deletion mutants in genes involved in these processes and show that the deletion mutants had distinct defects in gut colonization. Mutants affecting pili formation (presumably involved in adhesion to the host epithelium) or the production of the aryl polyenes were defective in colonization. Interestingly, mutants in the T6SS had only a mild colonization defect but were in impaired in inducing the scab phenotype. T6SS are usually thought to be important in interbacterial warfare, the results with the T6SS shown here with gnotobiotic bees mono-colonized with F. perrara provides evidence for the role of this system in a direct interaction with the host.

    In conclusion, they showed that IHF is major regulator in F. perrara, involved in the regulation of the expression of colonization factors with distinct roles in colonization. It remains to be shown if the regulation of IHF is direct or indirect, but it is clear that this regulator is a major hub controlling many different factors required for colonization in F. perrara. There are homologues of this regulator in other bacteria, so it is possible it plays a role in controlling colonization factors in other symbionts.
    One interesting point that was not addressed here was if this regulator, or some of the factors that it controls will play a role in spatial occupancy during gut colonization. Given that this bacterium colonizes a particular section of the gut it will be interesting to see if any of the factors regulated by IHF play a role in distinct niche colonization.

    Overall, the experiment included here are technically sound and the conclusions are well supported by the data. The paper is well-written and clear.

  5. eLife

    Reviewer #2 (Public Review):

    This is a very interesting, well conducted and conclusive research paper. On the positive side, beyond the scientific results of the work, this reviewer emphasizes the development of a protocol for RNA extraction and sequencing in vivo as well as a directed mutagenesis protocol for this peculiar symbiont which constitute great technical assets for future research in the bee microbiota field. On the less positive side, this reviewer feels there is a lack of characterization of the molecular phenotype induced by ihfA* on host biology, maybe as a start at the transcriptional level. Also, it would be interesting to study the impact of the deletion mutant on the bacterial physiology (cell shape, growth...) to help rationalize how the mutant is compromised in its ability to colonize the bee gut. In addition, given the ecological context of the bee gut, competition assays between the mutants and wt strains of F.perrara in bi-associated animals or within the a more complex gut microbiota would help to further understand the impact of the mutations on the fitness of the bacterial strain in the bee gut.

  6. eLife

    Reviewer #3 (Public Review):

    The manuscript by Schmidt and coauthors explores bacterial molecular factors influencing the colonization of the gut bacterial symbiont Frischella perrara in the honeybee gut, as well as the formation of the so called "scab phenotype". The authors elucidate a major role of the DNA-binding protein integration host factor (IHF) in regulating the expression of several colonization factors, which have been shown to be involved in other symbiotic systems. The use of a combination of techniques leads to the conclusion that specific pili genes, two Type VI secretion systems and the biosynthesis gene clusters for two secondary metabolites have particular effects on colonization success and/or the emergence of the scab phenotype.

    The thorough and extensive work presented provides well-supported conclusions on the role of specific genes for colonization in one strain of F. perrara. In particular, the valuable integration of infection bioassays, gene expression analyses, analytical chemistry and genetic manipulation result in a solid and wide-ranging set of results. Also, the text is well written, and the experimental procedures and presentation of the results are properly designed and detailed.

    It is generally interesting that they identify genes already discovered in other bacterial symbioses, as this reinforces and deepens on their relevance for establishment of bacteria in an animal host. Despite these links, a clearer case could be made on the relevance of the findings in connection to natural or non-experimental conditions in honeybees. It is not immediately clear to the reader why the scab phenotype might be interesting, how frequently it is found (at least not from this paper) and if it is associated to honeybee performance or health. Additionally, there is no information or discussion on whether the F. perrara strain used is representative for the system, whether other F. perrara strains are often found and have the same "wt" genotype for the IHF gene. It also seems relevant to think about the possible impact of other microbial associates colonizing the gut, since most conclusions are drawn from assays in gnotobiotic bees.

    In summary, I find that the experiments and conclusions of this work are well supported and properly presented. Placing the motivation for the work and the interpretations in a more general context would be useful to better convey their relevance.

  7. Version published to 10.1101/2021.12.06.471457v2 on bioRxiv
  8. Version published to 10.1101/2021.12.06.471457v1 on bioRxiv