Genomic changes associated with the evolutionary transition of an insect gut symbiont into a blood-borne pathogen.

The genus Bartonella comprises facultative intracellular bacteria with a unique lifestyle. After transmission by blood-sucking arthropods they colonize the erythrocytes of mammalian hosts causing acute and chronic infectious diseases. Although the pathogen-host interaction is well understood, little is known about the evolutionary origin of the infection strategy manifested by Bartonella species. Here we analyzed six genomes of Bartonella apis, a honey bee gut symbiont that to date represents the closest relative of pathogenic Bartonella species. Comparative genomics revealed that B. apis encodes a large set of vertically inherited genes for amino acid and cofactor biosynthesis and nitrogen metabolism. Most pathogenic bartonellae have lost these ancestral functions, but acquired specific virulence factors and expanded a vertically inherited gene family for harvesting cofactors from the blood. However, the deeply rooted pathogen Bartonella tamiae has retained many of the ancestral genome characteristics reflecting an evolutionary intermediate state toward a host-restricted intraerythrocytic lifestyle. Our findings suggest that the ancestor of the pathogen Bartonella was a gut symbiont of insects and that the adaptation to blood-feeding insects facilitated colonization of the mammalian bloodstream. This study highlights the importance of comparative genomics among pathogens and non-pathogenic relatives to understand disease emergence within an evolutionary-ecological framework

The impact of location-awareness on the perception of information services

It is presently unclear how much individual community members contribute to the overall metabolic output of a gut microbiota. To address this question, we used the honey bee, which harbors a relatively simple and remarkably conserved gut microbiota with striking parallels to the mammalian system and importance for bee health. Using untargeted metabolomics, we profiled metabolic changes in gnotobiotic bees that were colonized with the complete microbiota reconstituted from cultured strains. We then determined the contribution of individual community members in mono-colonized bees and recapitulated our findings using in vitro cultures. Our results show that the honey bee gut microbiota utilizes a wide range of pollen-derived substrates, including flavonoids and outer pollen wall components, suggesting a key role for degradation of recalcitrant secondary plant metabolites and pollen digestion. In turn, multiple species were responsible for the accumulation of organic acids and aromatic compound degradation intermediates. Moreover, a specific gut symbiont, Bifidobacterium asteroides, stimulated the production of host hormones known to impact bee development. While we found evidence for cross-feeding interactions, approximately 80% of the identified metabolic changes were also observed in mono-colonized bees, with Lactobacilli being responsible for the largest share of the metabolic output. These results show that, despite prolonged evolutionary associations, honey bee gut bacteria can independently establish and metabolize a wide range of compounds in the gut. Our study reveals diverse bacterial functions that are likely to contribute to bee health and provide fundamental insights into how metabolic activities are partitioned within gut communities.ISSN:1544-9173ISSN:1545-788

16S amplicon sequencing and qPCR data

This archive contains 1) raw Illumina MiSeq reads (300bp, PE, Reagent Kit v3) for all bacterial 16S V3/V4 amplicons used in our study, 2) metadata for each sample/individual, formatted as a QIIME 1.9 mapping file, 3) preprocessed data and .biom tables used to generate our results using phyloseq 1.22.3, and 4) raw data from qPCR analyses used to generate our results. We used two databases, greengenes 13_8 and SILVA NR Small Subunit v128 to assign taxonomy in our study; the filenames of preprocessed data include the database used to generate each file

Bacterial communities within Phengaris (Maculinea) alcon caterpillars are shifted following transition from solitary living to social parasitism of Myrmica ant colonies

Bacterial symbionts are known to facilitate a wide range of physiological processes and ecological interactions for their hosts. In spite of this, caterpillars with highly diverse life histories appear to lack resident microbiota. Gut physiology, endogenous digestive enzymes, and limited social interactions may contribute to this pattern, but the consequences of shifts in social activity and diet on caterpillar microbiota are largely unknown. Phengaris alcon caterpillars undergo particularly dramatic social and dietary shifts when they parasitize Myrmica ant colonies, rapidly transitioning from solitary herbivory to ant tending (i.e., receiving protein‐rich regurgitations through trophallaxis). This unique life history provides a model for studying interactions between social living, diet, and caterpillar microbiota. Here, we characterized and compared bacterial communities within P. alcon caterpillars before and after their association with ants, using 16S rRNA amplicon sequencing and quantitative PCR. After being adopted by ants, bacterial communities within P. alcon caterpillars shifted substantially, with a significant increase in alpha diversity and greater consistency in bacterial community composition in terms of beta dissimilarity. We also characterized the bacterial communities within their host ants (Myrmica schencki), food plant (Gentiana cruciata), and soil from ant nest chambers. These data indicated that the aforementioned patterns were influenced by bacteria derived from caterpillars’ surrounding environments, rather than through transfers from ants. Thus, while bacterial communities are substantially reorganized over the life cycle of P. alcon caterpillars, it appears that they do not rely on transfers of bacteria from host ants to complete their development.ISSN:2045-775

Summary of the metabolic activities of the bee gut microbiota identified in this study.

<p>(A) Schematic representation of the bee gut depicting the crop, midgut, and hindgut. The hindgut is divided into the ileum and the rectum, where the highest bacterial densities are found. Bacteria in the ileum are shown in magenta and orange (mostly Proteobacteria), and those in the rectum are shown in green and blue (mostly <i>Lactobacilli</i> and <i>Bifidobacteria</i>). Pollen grains are shown in yellow. (B) Pollen is likely predigested in the midgut, where bacterial levels are relatively low [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003467#pbio.2003467.ref045" target="_blank">45</a>]. Here, the host absorbs accessible pollen-derived compounds such as simple sugars (glucose or fructose) and amino acids [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003467#pbio.2003467.ref046" target="_blank">46</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003467#pbio.2003467.ref047" target="_blank">47</a>]. Nondigested pollen compounds enter the hindgut, where bacterial density is higher. We found nucleosides, various carboxylic acids (e.g., citrate, malate, and fumarate), and aromatic compounds (such as quinate) from pollen to be utilized by bee gut bacteria. In the posterior part of the hindgut (rectum), three community members (Firm-5, Firm-4, and <i>B</i>. <i>asteroides</i>) metabolize major components of the outer pollen wall, including flavonoids, phenolamides, and ω-hydroxy acids. The metabolic activities of the microbiota lead to the accumulation of fermentation products and intermediates of aromatic compound degradation. Some of the bacterial products may be utilized by other community members, as exemplified by the cross-feeding between <i>G</i>. <i>apicola</i> and <i>S</i>. <i>alvi</i>, or absorbed by the host. In addition, the gut symbiont <i>B</i>. <i>asteroides</i> seems to increase the production of several host metabolites (juvenile hormone derivatives and prostaglandins) that have key functions in immunity and physiology.</p

Bacterial colonization levels in the guts of microbiota-depleted (MD), colonized (CL), and hive bees.

<p>(A) Total bacterial loads in the gut of 10-d-old MD bees (<i>n</i> = 21), CL bees (<i>n</i> = 18), and hive bees (<i>n</i> = 16) were assessed by quantitative PCR (qPCR) with universal bacterial 16S rRNA primers. (B) The bacterial loads of the seven predominant community members used for experimental colonizations were assessed by qPCR with species-specific 16S rRNA primers for the same bees as shown in panel A. Black lines show median values. Samples with <10⁵ bacterial cells per gut are shown below the red line, which we consider the threshold of detection. Primer characteristics are summarized in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003467#pbio.2003467.s022" target="_blank">S2 Table</a>. n.s., not significant; *<i>P</i> < 0.05; **<i>P</i> < 0.01; and ***<i>P</i> < 0.001 (Wilcoxon Rank Sum test, Benjamini and Hochberg adjusted [BH adj.]). The numerical data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003467#pbio.2003467.s001" target="_blank">S1 Data</a>.</p

Overview of metabolite changes explained by different community members of the bee gut microbiota.

<p>(A) Bar graphs show the fraction of the metabolic changes explained by mono-colonizations and hive bees for substrates (240 ions) and products (132 ions). The category “Total” indicates the total number of ions explained by mono-colonizations, thus excluding hive bees. Heatmap representation of enrichment <i>P</i> values (one-sided Fisher’s exact test <i>P</i> < 0.05) are provided for compound categories enriched in one or several mono-colonizations. (B–E) Z-score transformed ion intensities of selected substrate and product ions are shown for all treatment groups. (B) Four glycosylated flavonoid substrates. (C) Two substrates from the outer pollen wall. (D) Two products corresponding to host-derived metabolites. (E) Succinate, one of the major fermentation products. Groups depicted in color highlight treatment groups displaying a significant difference compared to MD bees in the same direction as the CL versus MD difference (one-way analysis of variance [ANOVA], Tukey honest significant difference [HSD] post hoc test at 99% confidence, <i>P</i> ≤ 0.05). Plots for all 372 ions are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003467#pbio.2003467.s008" target="_blank">S8 Data</a>. Ba, <i>B</i>. <i>apis</i> mono-colonized; Bi, <i>B</i>. <i>asteroides</i> mono-colonized; CL, colonized with the reconstituted microbiota; F4, Firm-4 mono-colonized; F5, Firm-5 mono-colonized; Fp, <i>F</i>. <i>perrara</i> mono-colonized; Ga, <i>G</i>. <i>apicola</i> mono-colonized; Hive, hive bees; MD, microbiota-depleted; Sa, <i>S</i>. <i>alvi</i> mono-colonized. The numerical results of the full enrichment analysis, bar graphs, and mono-colonization plots are provided in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003467#pbio.2003467.s003" target="_blank">S3 Data</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003467#pbio.2003467.s001" target="_blank">S1 Data</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003467#pbio.2003467.s008" target="_blank">S8 Data</a>, respectively.</p

Metabolite changes between microbiota-depleted (MD) and colonized (CL) bees.

<p>An Orthogonal Projection of Least Squares-Differentiation Analysis (OPLS-DA) based S-plot of metabolite changes shows the ions responsible for CL and MD separation. The inset shows OPLS-DA separation between CL and MD along the component that was used for correlating ion intensities. Experiment 2 data (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003467#pbio.2003467.s002" target="_blank">S2A Data</a>) was used for this plot, and annotated ions that were not robustly significantly different between CL and MD in both experiments are plotted in grey. Ions with a first annotation belonging to an enriched category (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003467#pbio.2003467.s003" target="_blank">S3A Data</a>) are plotted in color, except for the category “amino acids and derivatives”, which did not meet the significance threshold for enrichment but was deemed relevant. The “purine nucleosides and analogues” and “pyrimidine nucleosides and analogues” categories were combined into “nucleosides and analogs” for coloring only. The boxed areas show the <i>m/z</i> [M-H⁺]^- of the ion and the first annotation name of the most discriminatory ions, sorted by covariance. Asterisks indicate ions with ambiguous annotations. The numerical data can be found in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003467#pbio.2003467.s001" target="_blank">S1 Data</a>. Conjug., conjugates; Deriv., derivatives; FC, fold change; int., intensity.</p

Cross-feeding between <i>G</i>. <i>apicola</i> and <i>S</i>. <i>alvi</i>.

<p>(A) Evidence for cross-feeding of pyruvate in the honey bee gut. Z-score transformed ion intensities revealed that the ion annotated as pyruvate accumulated in bees mono-colonized with <i>G</i>. <i>apicola</i> but was depleted in hive bees, CL bees, and bees mono-colonized with <i>S</i>. <i>alvi</i> and Firm-5. (B) Growth improvement of <i>S</i>. <i>alvi</i> in <i>G</i>. <i>apicola-</i>conditioned medium. <i>S</i>. <i>alvi</i> was grown in pollen-conditioned medium in the presence (black line) or absence (dashed line) of <i>G</i>. <i>apicola</i> culture supernatant (50%, v/v). Growth was determined based on OD₆₀₀ at time points 0 h, 16 h, 36 h, and 72 h. n.s., not significant; * <i>P</i> < 0.05 (Welch’s <i>t</i> test, Benjamini and Hochberg adjusted [BH adj.]). (C) Six potentially cross-fed ions that accumulated during in vitro growth of <i>G</i>. <i>apicola</i> (left subpanel) and were consumed by <i>S</i>. <i>alvi</i> when it was grown in the presence <i>G</i>. <i>apicola</i> culture supernatant (right subpanel). Data from panels B and C come from the same experiment. Smoothed lines are added for interpretation purposes only in panel C and are dashed in the left subpanel because they are drawn through two points only. Error bars represent the standard deviation based on three replicate cultures. Chemical structures of the first annotation of each ion are shown. Asterisks indicate ions with ambiguous annotations. The numerical data of panel A can be retrieved from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003467#pbio.2003467.s008" target="_blank">S8 Data</a>. All other values are available in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2003467#pbio.2003467.s001" target="_blank">S1 Data</a>.</p