Read more……>click Here< We used anthropometric data collected from members of a birth cohort study (14) of 100 children living in Mirpur thana in Dhaka, Bangladesh, to define whether they were healthy or undernourished (table S1). Those with height-for-age z scores (HAZ) greater than or equal to −2 were classified as “healthy,” whereas those with scores less than or equal to −3 were deemed severely stunted. At 18 months, 30 and 25 children satisfied these criteria for healthy and severely stunted, respectively, whereas at 24 months, 27 and 20 children received these designations; the remaining children were classified as moderately stunted (HAZ between −2 and −3). A PCR-based screen for ETBF targeting all three fragilysin gene subtypes (14) was performed using DNA isolated from fecal samples that had been collected from these children at 18 and 24 months of age. The results revealed that ETBF was variably present between individuals and within a given individual over time, with a total of 25% of 18-month-old and 14% of 24-month-old children having a positive test (table S1). In this small cohort, ETBF carriage was not significantly correlated with indices of linear or ponderal growth [HAZ, weight-for-age z score (WAZ), and weight-for-height z score (WHZ) measured at 12 and 24 months of age (P = 0.8 and P = 0.4, P = 0.7 and P = 0.2, and P = 0.5 and P = 0.2, respectively; two-tailed Student’s t test)]. We combined anthropometric and PCR data to select fecal samples collected at 24 months from two children: (i) a healthy individual (child ID 7114 in table S1) with a HAZ score of −0.71, a WAZ score of −1.49, and a WHZ score of −1.62 who was ETBF-negative at the two time points tested, and (ii) a severely stunted and moderately underweight individual (child ID 7004) with a HAZ score of −3.02, a WAZ score of −2.51, and a WHZ score of −1.34 who was ETBF-positive at both time points. Of the 35 individuals with a positive ETBF test at either time point, only this stunted/underweight child was positive at both 18 and 24 months of age. Fecal samples obtained from members of this singleton birth cohort were screened for parasites using microscopic methods (5); neither of the two donors tested positive (see Materials and Methods for details).
To define the effects of diet and these two childrens’ gut microbiota on host biology, we generated three representative versions (embodiments) of the diets consumed by the population represented by the donors. To do so, we determined the relative daily caloric contributions of various selected ingredient types, based on a study by Arsenault and coworkers (16). Selection of specific food items as representative of each ingredient type was based on consumption incidence surveys tabulated by Islam et al. (17), and the results were incorporated into a database consisting of 54 food ingredients. We filtered this database to remove items consumed by <20% of households and categorized each of the remaining 39 items (see Materials and Methods for additional details). From the resulting diet ingredient matrix, we randomly sampled (without replacement) one item each from cereals, pulse vegetables, roots/tubers, leafy vegetables, fruits, and fish, plus three nonleafy vegetables, to populate three separate diet lists. Using the U.S. Department of Agriculture National Nutrient Database for Standard References (18), we determined the caloric information for each ingredient and subsequently calculated proportions required to match the predetermined contributions of each ingredient type. Food items were cooked in a manner intended to simulate Bangladeshi practices, and the resulting three embodiments of a Bangladeshi diet were sterilized by irradiation. This approach allowed us to generate several representative Bangladeshi diets that were not dominated by the idiosyncrasies of a single individual’s diet or by our own biases. The composition and results of nutritional analysis of the three diet embodiments are described in table S2 (A and B). The nutritional requirements of mice and children are compared in table S2C.
The results of a 12-year survey of demographic variations in the nutritional status of 16,278 Bangladeshi children found no significant sex differences in WHZ, WAZ, or HAZ (19). Therefore, in these and subsequent experiments, we eliminated gender as an experimental variable and only studied male mice. We gavaged separate groups of 8- to 9-week-old germfree C57BL/6 mice with the intact uncultured fecal microbiota samples obtained from the healthy or stunted/underweight Bangladeshi donors (two independent experiments; n = 4 singly caged mice per donor microbiota per experiment; see fig. S1A for study design). Fecal microbiota transplantation occurred 2 days after mice had been switched from an irradiated, nutritionally complete, low-fat/high-plant polysaccharide (LF/HPP) mouse chow that they had received since weaning to the first of the three embodiments of the Bangladeshi diet. Animals were subsequently fed, ad libitum, embodiment 1 for 1 week, followed by embodiment 2 for 1 week, and finally embodiment 3 for 1 week, with frequent sampling of their fecal microbiota during the course of each diet. Sequencing PCR amplicons generated from variable region 4 (V4) of bacterial 16S ribosomal RNA (rRNA) genes present in the donor’s fecal sample and in fecal samples collected over time from recipient gnotobiotic mice (table S3) provided an in vivo assay of colonization efficiency for each human donor sample. 16S rRNA sequencing reads were grouped into operational taxonomic units (OTUs) on the basis of a threshold of ≥97% nucleotide sequence identity (97% ID). The results revealed that at the conclusion of the experiment, 65.8 ± 2.5% (mean ± SEM) of OTUs in the stunted/underweight donor’s fecal microbiota sample and 68.4 ± 8.8% (mean ± SEM) of the OTUs in the healthy donor’s microbiota were detectable in recipient mice (that is, each OTU had a relative abundance of ≥0.1% in ≥1% of fecal samples obtained from the animals).
Although gnotobiotic animals colonized with the healthy donor’s intact uncultured fecal microbiota maintained weight, recipients of the severely stunted/underweight donor’s intact uncultured fecal microbiota exhibited progressive and significant weight loss (P < 0.005, paired two-tailed Student’s t test, comparison of final versus initial weights between the two treatment groups; Fig. 1A). In contrast to mice colonized with the healthy donor’s microbiota, those that received the stunted donor’s microbiota exhibited statistically significant weight loss at 10 days postgavage (dpg), during consumption of diet embodiment 2. Weight loss in this group worsened progressively, reaching 31 ± 6% (mean ± SEM) of original starting weight by 21 dpg (P < 0.001, two-tailed Student’s t test, comparison of final weights; Fig. 1A); in a linear mixed-effects model, both dpg and the interaction between microbiota and dpg were significant factors affecting weight throughout the experiment (P < 1 × 10−7 for each). Food consumption was not different between the two treatment groups as their weight phenotypes diverged. The relative abundance of B. fragilis, defined by V4-16S rRNA analysis of fecal samples obtained at the time of killing, was significantly greater in mice colonized with the stunted/underweight donor’s microbiota than in mice colonized with the healthy donor’s microbiota (P = 1.9 × 10−6, two-tailed Student’s t test; Fig. 1B).
(A) Germfree male C57BL/6 mice (8 to 9 weeks old) (n = 8 per treatment group) gavaged with intact uncultured fecal microbiota from Bangladeshi donors were fed a sequence of three embodiments of a representative Bangladeshi diet consumed by members of the donor population. See fig. S1A for experimental design. Mean weights (±SEM) as a function of dpg are shown as percentages of weights immediately before fecal microbiota transplantation. (B) Efficiency of capture of bacterial OTUs present in the donor’s intact uncultured fecal samples in gnotobiotic mice. Mean relative abundances (±SEM) of 97% ID OTUs representing ≥1% of the total fecal microbial communities in recipient animals. Results are based on V4-16S rRNA data sets and summarized at the species level (or genus when species could not be determined). OTUs present at lower abundances are not shown and account for the proportion not represented in each stacked barplot. (C) Transplantation of culture collections (dashed lines) generated from the fecal microbiota of the healthy or stunted/underweight donors recapitulated the discordant weight phenotype seen with the corresponding intact uncultured microbiota (solid lines) (n = 6 mice per treatment group, mean weights ± SEM plotted). *P < 0.05 (paired two-tailed Student’s t test and linear mixed-effects model, as above). (D) The weight-loss phenotype observed in recipients of the stunted/underweight donor’s culture collection is not significantly different between the three Bangladeshi diet embodiments tested (P > 0.05; two-tailed Student’s t test). Mean weights (±SEM) are plotted as a function of dpg (n = 6 mice per culture collection per diet embodiment). Significant weight differences were seen between mice colonized with the healthy donor’s compared to the stunted/underweight donor’s culture collection in the context of all three embodiments of the Bangladeshi diet. *P < 0.05 (paired two-tailed Student’s t test and linear mixed effects model). (E) Intergenerational transmission of discordant weight phenotypes. See fig. S1C for experimental design. Mean weights (±SEM) of offspring of female gnotobiotic mice colonized with the indicated donors’ microbiota (n = 3 to 4 mice per treatment group) are plotted as a function of age. Animals were switched from a nutrient sufficient LF/HPP mouse chow to embodiments of the Bangladeshi diets beginning on postnatal day 56. *P < 0.05 (tested by both paired two-tailed Student’s t test comparing weights at killing and linear mixed effects model assessing interaction of weight, dpg, and microbiota through the experiment). The efficiency of intergenerational transmission of 97% ID OTUs was 96 ± 1.8% and 88 ± 2.3% (mean ± SEM) for the healthy and stunted/underweight donor’s microbiota, respectively (defined at the time of killing).
We next cultured bacterial strains from the healthy and stunted/underweight donors’ fecal samples (20, 21). Each collection of cultured strains was clonally arrayed in multiwell plates so each well contained a monoculture of a given bacterial isolate (20). Each culture collection consisted of organisms that had coexisted in the donor’s gut and thus were the products of the donor’s history of environmental exposures to various microbial reservoirs (including those of family members and various enteropathogens endemic to the Mirpur thana), as well as the selective pressures and evolutionary events placed on and operating within their microbiota (for example, immune, antibiotic, dietary, and horizontal gene transfer). Individual isolates in the clonally arrayed culture collection were grouped into “strains” if they shared an overall level of nucleotide sequence identity of >96% across their assembled draft genomes (21). On the basis of this criterion and the results of sequencing amplicons generated from the isolates’ 16S rRNA genes, we determined that the healthy and stunted donors’ culture collections contained 53 and 37 strains, respectively. Only one strain was shared between the two culture collections: Bifidobacterium breve hVEW9 [see table S4 for a list of all isolates in the culture collection derived from the stunted/underweight child and (21) for details of the healthy donor’s culture collection]. The two B. fragilis strains present in the healthy donor’s culture collection (hVEW46 and hVEW47) lacked a BfPAI and were therefore classified as NTBF. The stunted donor’s collection contained a single B. fragilis strain (mVEW4) with a bft-3 allele. ETBF strains of this type are globally distributed but most common in Southeast Asia (22) (see table S5 for a comparison of the functions encoded by genes in the genomes of these ETBF and NTBF strains and the reference B. fragilis type strain ATCC 25285).
To ascertain whether the contrasting weight phenotypes conferred by the two intact uncultured fecal microbiota samples could be transmitted by the strains captured in their derivative culture collections, we colonized 8-week-old adult male germfree C57BL/6 mice with all members of either of these two culture collections (n = 6 singly caged mice per collection; all mice receiving a given culture collection were maintained in a single gnotobiotic isolator). As a reference control for this experiment, and to compare results between this and the previous experiment, we colonized mice with the corresponding intact uncultured fecal microbiota samples, housing these mice in separate isolators from those used for the culture collection transplants. All mice were fed three embodiments of the Bangladeshi diet (1 week per diet) in the same order described for the previous experiment. As with the intact uncultured microbiota, the corresponding culture collections transmitted discordant weight phenotypes to recipient animals (P < 0.002, two-tailed Student’s t test, comparison of final weights; Fig. 1C). Moreover, the weight phenotypes (change in body weight over time as a percentage of initial weight before gavage) observed with each intact uncultured fecal microbiota and the corresponding derivative culture collection were not significantly different (P > 0.05 for both microbiota donors, two-tailed Student’s t test; Fig. 1C). The difference in weight phenotypes first became statistically significant between the two groups of mice midway through consumption of diet embodiment 2, continued to increase with diet embodiment 3 (Fig. 1C), and again were not attributable to differences in food consumption.
Effect of diet.
To test whether the weight loss phenotype was sensitive or robust to diet embodiment type, we gavaged the two clonally arrayed bacterial culture collections into separate groups of 8-week-old adult male germfree C57BL/6 mice who were monotonously fed Bangladeshi diet embodiment 1, 2, or 3 for 3 weeks (n = 6 singly caged recipient mice per culture collection per diet embodiment; fig. S1B). The discordant weight phenotype observed previously was preserved irrespective of the Bangladeshi diet embodiment consumed (P < 0.01, two-tailed Student’s t test, comparison of final weights of mice regardless of diet embodiment consumed; n = 18 mice per culture collection; Fig. 1D). Moreover, no significant differences in weights were noted between groups of mice colonized with the same culture collection but fed different diet embodiments (P > 0.05 for embodiments 1 versus 2, 1 versus 3, and 2 versus 3; two-tailed Student’s t test, comparison of final weights; Fig. 1D).
Transmission of strains was assessed by short-read shotgun sequencing of DNA isolated from fecal samples collected at the end of the experiment. This method, known as community profiling by sequencing (COPRO-Seq) (21), maps reads onto the draft genome assemblies of community members. At the depth of sequencing used [354,352 ± 23,216 (mean ± SEM), 50-nucleotide (nt) unidirectional reads/fecal DNA sample], we could reliably detect strains whose relative abundance is ≥0.1%. COPRO-Seq demonstrated that transplantation of the culture collections was efficient and reproducible, with 98.1 ± 0.6% and 94.5 ± 1.6% (mean ± SEM) of strains in the collections derived from the healthy and stunted donors, respectively, appearing in recipient animals. The relative abundance of ETBF in the fecal microbiota of mice containing the stunted/underweight donor’s culture collection was significantly greater than the cumulative relative abundance of the two NTBF strains in recipients of the healthy culture collection irrespective of the diet embodiment consumed (75.5 ± 4.1% versus 17.0 ± 4.4%; P = 2.8 × 10−9, two-tailed Student’s t test; Fig. 2A). The relative abundances of the ETBF strain in recipients of the stunted/underweight donor’s culture collection, the two NTBF strains in the healthy donor’s collection, and all other Bacteroides species did not differ significantly between diet embodiments [P > 0.2 for all Bacteroides, one-way analysis of variance (ANOVA); Fig. 2B].
(A) Gut microbial community composition, defined by COPRO-Seq, in mice colonized with either of the two unmanipulated culture collections or the derived manipulated versions. Mean values for relative abundances ± SEM are plotted using aggregate data generated from fecal samples collected from mice colonized with a given community. Taxa present at abundances lower than 1% are not represented in the stacked barplots. (B) The proportional representation of Bacteroides taxa in unmanipulated culture collections installed in gnotobiotic mice does not differ significantly as a function of the diet embodiments animals were fed. Means ± SEM for data generated from feces are shown (n = 5 to 6 per group; one-way ANOVA). (C) Schematic illustrating the different groups of gnotobiotic mice generated by manipulating the presence/absence of ETBF and NTBF within the stunted/underweight or healthy donors’ culture collections and the questions addressed by the indicated comparisons. (D) Removal of ETBF prevents weight loss in mice colonized with the stunted/underweight donor’s culture collection. In contrast, addition of ETBF with the simultaneous removal of NTBF does not significantly affect weight in mice colonized with the culture collection derived from the healthy child (n = 5 to 6 mice per treatment group). Means ± SEM are plotted. *P < 0.05 (paired two-tailed Student’s t test and linear mixed effects model as above). (E) Addition of NTBF to the stunted/underweight donor’s culture collection ameliorates ETBF-associated weight loss in gnotobiotic mice fed embodiment 2 of a representative Bangladeshi diet (n = 6 mice per treatment group). Means ± SEM are plotted. *P < 0.05 (paired two-tailed Student’s t test and linear mixed effects model as above).
Intergenerational transmission of weight phenotypes.
To assess whether this weight loss phenotype was transmissible across generations of mice, two C57BL/6 male mice from the transplant experiment, one containing the stunted/underweight donor’s culture collection and the other containing the healthy donor’s collection, were switched to and subsequently maintained on an irradiated nutritionally enhanced mouse breeder chow from 21 to 48 dpg, at which time they were each cohoused with two germfree 6-week-old female mice that had received breeder chow since weaning. Seven days after cohousing, each male mouse was withdrawn from each mating trio, and the female mice were subsequently maintained on breeder chow throughout their pregnancy and as their pups completed the suckling period (fig. S1C). Male pups (n = 3 to 4 per litter) were then weaned onto an irradiated, nutritionally sufficient, LF/HPP chow, until they were 9 weeks old, at which time they were switched to the Bangladeshi diets (10 days per diet; same order of sequential presentation of the embodiments as before). Mice born to mothers colonized with either of these arrayed culture collections experienced identical weight gain profiles while consuming the LF/HPP diet (P = 0.9, two-tailed Student’s t test; table S6). However, once they were transitioned to the sequence of three Bangladeshi diet embodiments (consumed from postnatal days 56 to 86), mice born to mothers harboring a stunted/underweight donor’s microbial community exhibited significantly greater weight loss (P = 0.03, two-tailed Student’s t test comparing weights at killing). The total relative abundance of the two NTBF strains in fecal samples obtained from recipients of the healthy donor’s culture collection was 4.2 ± 0.7% at the conclusion of the LF/HPP diet period and 4.6 ± 0.9% at the conclusion of the Bangladeshi diet embodiment sequence, whereas the relative abundance of ETBF at these two time points was 34.3 ± 4.2% and 50.0 ± 0.7%, respectively, in mice colonized with the stunted/underweight donor’s culture collection.
An independent intergenerational transfer experiment was performed, in this case using the donors’ intact uncultured fecal microbiota. The efficiency of ETBF and NTBF transmission from mothers to pups was 100%. As with the culture collections, there was diet-dependent transmission of the discordant weight loss phenotype (Fig. 1E; compare with Fig. 1A).
To establish whether ETBF is necessary and sufficient to cause marked weight loss in multiple community contexts, we performed a series of manipulations that involved removing the ETBF strain from the stunted/underweight donor’s culture collection and adding it to the healthy donor’s culture collection, with or without subtraction of its two NTBF strains (Fig. 2C). These manipulations allowed us to characterize (i) the role of community context in determining ETBF pathogenicity, (ii) the community/host responses to ETBF, (iii) the ability of NTBF to modulate ETBF effects, and (iv) the effects of ETBF on NTBF. Recipient C57BL/6 male mice in each of the different treatment groups were 8 to 9 weeks old at the time of colonization; all were placed on diet embodiment 2 for 2 days before gavage and subsequently maintained on this diet for 14 days until they were killed (n = 5 singly caged animals per treatment group, maintained in separate gnotobiotic isolators). Fecal samples were collected at the time points described in fig. S1D.
Weight phenotypes.
Removal of the ETBF strain from the stunted/underweight donor’s culture collection prevented the transmissible weight loss phenotype (Fig. 2D; P = 5.9 × 10−8, two-tailed Student’s t test, comparison of weights at killing). However, addition of the ETBF strain to the healthy donor’s culture collection did not produce significant weight loss, regardless of whether the NTBF strains were present or absent (P = 0.3 and P = 0.2, respectively, two-tailed Student’s t test, comparison of weights at killing; Fig. 2D). On the basis of these findings, we concluded that whether ETBF produces weight loss (cachexia) is dependent on microbial community context.
COPRO-Seq analysis of the fecal microbiota of recipients of the unmanipulated ETBF(−) NTBF(+) healthy donor’s culture collection revealed that it contained the two NTBF strains [total relative abundance of 14.5 ± 3.0% (mean ± SEM), with B. fragilis hVEW46 and B. fragilis hVEW47 comprising 1.1 and 13.5%, respectively], two other Bacteroides (B. thetaiotaomicron and B. caccae), plus Bifidobacterium breve and Enterococcus. The relative abundance of B. fragilis was not significantly different between mice harboring the transplanted unmanipulated healthy donor’s culture collection and its two manipulated ETBF(+) NTBF(−) and ETBF(+) NTBF(+) versions (P > 0.5, two-tailed Student’s t test; Fig. 2A). (The term “unmanipulated” indicates that all bacterial isolates that comprise a culture collection were pooled before transplantation, whereas “manipulated” refers to the inclusion and/or exclusion of B. fragilis strains as part of the gavaged consortium.) The fecal microbiota of recipients of the unmanipulated stunted donor’s culture collection was dominated by ETBF (relative abundance, 62.3 ± 4.0%). Removal of ETBF led to significant increases in the relative abundances of B. breve, another Bifidobacterium strain, Enterococcus lactis, and Enterococcus gallinarum (P < 0.02, two-tailed Student’s t test; Fig. 2A).
To determine whether NTBF alone is sufficient to protect mice from ETBF’s cachectic effects, we colonized three groups of C57BL/6 male gnotobiotic mice, each with a different version of the stunted donor’s culture collection: the unmanipulated culture collection containing ETBF alone or one of two manipulated versions, one with NTBF alone, and the other with both ETBF and NTBF strains. Mice were placed on diet embodiment 2 for 2 days before gavage and maintained on this diet for 2 weeks until killed (n = 6 animals per treatment group, all singly caged; one treatment group per gnotobiotic isolator; fig. S1D). We observed a significant difference in weight phenotypes between mice colonized with the unmanipulated undernourished donor’s ETBF(+) NTBF(−) culture collection compared to the manipulated ETBF(−) NTBF(+) version (P = 0.01, one-tailed Student’s t test; Fig. 2E). Addition of NTBF [yielding the ETBF(+) NTBF(+) community] markedly ameliorated the weight loss phenotype (P = 0.0004 for weights at killing compared to mice with the unmanipulated community, one-tailed Student’s t test; Fig. 2E). Follow-up COPRO-Seq analysis revealed that the relative abundances of ETBF at the conclusion of the experiment were 38.9 ± 3.9% and 39.0 ± 3.5% when animals were colonized with and without NTBF, respectively. Thus, NTBF does not appear to mediate its effects by reducing the fractional representation of ETBF in the community. However, ETBF appears to reduce the relative abundance of NTBF, which constituted 41.8 ± 3.2% of the total community when ETBF was absent but only 19.2 ± 2.6% when ETBF was present (P = 0.04, one-tailed Student’s t test).
The effects of intraspecific interactions on microbial gene expression.
We performed microbial RNA sequencing (RNA-seq) of cecal contents harvested at killing to characterize the transcriptomes of members of the unmanipulated and manipulated versions of the healthy and stunted communities. Our goal was to assess (i) the effects of intraspecific competition (NTBF on ETBF and vice versa) in the healthy and stunted community contexts, (ii) the effects of the cultured stunted/underweight versus healthy donor community on ETBF, and (iii) the effects of cocolonization with ETBF on other bacterial members (including other Bacteroides). ETBF genes with significant differential expression attributable to the presence or absence of NTBF, in both healthy and stunted community contexts, are listed in table S8 (B and F). Conversely, NTBF genes with significant differential expression attributable to the presence or absence of ETBF, in both healthy and stunted community contexts, are highlighted in table S8 (C and E).
Fragipain is a cysteine protease that activates fragilysin by removing its autoinhibitory prodomain. In mouse models of colitis, host proteases can also serve this function, but fragipain is required for sepsis to occur (23, 24). In the presence of NTBF, ETBF expression of fragilysin (bft-3) in the cecal metatranscriptome of mice harboring the manipulated ETBF(+) NTBF(+) healthy donor’s community was significantly decreased compared to the manipulated version of the community where ETBF, but not NTBF, was present (39-fold, based on normalized transcript counts; P = 0.002, one-tailed Student’s t test). Fragipain expression was also significantly reduced (14.2-fold; P = 0.0005, one-tailed Student’s t test) (table S8B). In the context of the stunted community, the reduction in bft-3 expression associated with introducing NTBF was considerably more modest (5.9-fold; P = 0.09, one-tailed Student’s t test), whereas fragipain expression was not significantly different between the two treatment groups (P > 0.5, one-tailed Student’s t test; table S8).
When we abrogated fragilysin (bft-3) expression through insertional mutagenesis (fig. S2), the mutant Δbft-3 strain grew robustly in vitro. However, when germfree mice were gavaged with a manipulated version of the stunted donor’s culture collection containing this isogenic strain with a disrupted bft-3 locus substituted for the wild-type ETBF strain, we observed no detectable colonization of the mutant (n = 5 mice fed diet embodiment 2 for 14 days); the number of COPRO-Seq reads mapping to the mutant Δbft-3 strain was no greater than background, and a PCR assay that used B. fragilis–specific bft primers was negative. However, these results led us to conclude that this locus functions as an important colonization factor for this particular ETBF strain in this community context. However, these experiments did not allow us to directly address the hypothesis that attenuation of bft-3 expression produced by inclusion of NTBF in the stunted community contributed to the observed mitigation of weight loss.
Looking beyond the effects of intraspecific interactions on btf-3 expression, we compared the cecal metatranscriptomes of gnotobiotic mice colonized with the unmanipulated NTBF(+) ETBF(−) healthy donor’s culture collection versus mice harboring the two manipulated versions where ETBF was added, with or without removal of the two NTBF strains. The results revealed that ETBF in the absence of NTBF produced significant alterations in the expression of a number of transcripts related to various features of stress responses in several community members [Enterococcus faecalis, E. gallinarum, B. breve, and two members of Enterobacteriaceae; differentially expressed genes identified using the Robinson and Smyth exact negative binomial test (25), with Bonferroni correction for multiple hypotheses] (Fig. 3). Both rpoS, which is a key general stress response sigma factor that positively controls expression of genes involved in transport of carbon sources and iron acquisition, and recD, which is involved in DNA repair, exhibited significant increases in their expression in the setting of ETBF without NTBF (P < 0.05). Several genes involved in the acquisition and metabolism of iron were either up-regulated in the presence of ETBF (for example, ferric aerobactin receptor, ferric uptake regulation protein, and aerobactin synthase) or repressed (for example, an Enterobacteriaceae strain hVEW34 homolog of the Escherichia coli BasSR system component BasS, which is normally induced under high-iron conditions) (26). ETBF’s effect on expression of these latter genes was mitigated when NTBF was present (Fig. 3), highlighting the importance of iron in intraspecific and interspecific interactions in the healthy donor’s consortium of transplanted cultured bacterial strains. In contrast, the presence or absence of ETBF or NTBF did not evoke significant changes in the expression of these or other genes involved in iron metabolism in the context of the stunted/underweight donor’s community. Numerous genes related to prophage and mobile DNA element biology were also expressed at significantly higher levels by healthy community members when ETBF was present in the absence of NTBF (P < 0.05; Fig. 3). Prophage activation occurs in response to stress. Some studies have postulated that phage induction can “shuffle” community structure to favor an increased proportion of pathobionts (27).
Adult mice were colonized with the indicated unmanipulated and manipulated versions of the healthy donor’s culture collection. All treatment groups were monotonously fed diet embodiment 2. Cecal contents were collected at the time of killing 14 days after initial colonization, and gene expression in the community was analyzed by microbial RNA-Seq. Each column represents data from an individual mouse. Each row represents the levels of a given transcript, normalized across that row. Addition of ETBF to and removal of NTBF from the healthy donor’s culture collection (middle set of columns) produced an increase in expression of the indicated genes in strains whose identity is denoted by the color code on the left, compared to their expression in the unmanipulated ETBF(−) NTBF(+) version (left set of columns) or the manipulated version where the NTBF strains were retained when ETBF was added (right set of columns). UniProt-based annotations are shown on the right. ATP, adenosine triphosphate; HTH, helix-turn-helix.
Studies in gnotobiotic mice have shown that signaling by members of the human gut microbiota involving the quorum sensing molecule, autoinducer-2 (AI-2), can alter virulence factor expression in enteropathogens (28) and have linked AI-2 signaling to modulation of the levels of Bacteroidetes in the gut (29). LuxQ is involved in the detection of AI-2. In the context of the healthy community, expression of three of the four luxQ homologs in the ETBF genome was decreased when NTBF was present [log2(fold change) of −2.8, −4.4, and −9.5, P < 0.005, exact negative binomial test; table S8B]. Comparing the mice colonized with the unmanipulated ETBF(−) NTBF(+) and manipulated ETBF(+) NTBF(+) versions of the healthy donor’s culture collection revealed differential regulation of five other luxQ transcripts encoded by Bacteroides members (three in B. thetaiotaomicron hVEW3, and two in B. caccae hVEW51; table S8D). In the context of the stunted donor’s community, the presence of ETBF had no significant effects on lux gene expression in NTBF or any other community members, nor did the presence of NTBF have any effect on lux expression in ETBF (table S8, E and F). Together, these results illustrate the importance of community context in determining the transcriptional effects of intraspecific (and interspecific) interactions involving ETBF.
Metabolism.
The metabolic effects of manipulating the representation of ETBF and NTBF in the healthy and stunted donor’s communities were studied by targeted mass spectrometry (MS) of tissue samples obtained from mice in the fed state (table S7). Quantifying amino acids, organic acids, acylcarnitines, and acyl-CoAs in livers obtained from animals colonized with either of the two unmanipulated culture collections disclosed that compared to mice harboring the healthy donor’s ETBF(−) NTBF(+) culture collection, those colonized with the stunted donor’s ETBF(+) NTBF(−) culture collection had higher concentrations of propionyl-CoA and isovaleryl-CoA [by-products of oxidation of branched-chain and other amino acids; P < 0.05, false discovery rate (FDR)–adjusted two-tailed Student’s t test; Fig. 4A], and lower concentrations of acetyl-CoA (P = 0.07) and its cognate metabolite acetyl carnitine (that is, C2 acylcarnitine; P = 0.001; Fig. 4B). Mirroring these trends, cecal contents harvested at the time of killing from mice harboring the stunted donor’s unmanipulated culture collection contained higher concentrations of branched-chain amino acids (P = 0.066 for isoleucine/leucine and P = 0.1 for valine) and lower concentrations of acetyl-carnitine (P = 0.067). However, these trends were not observed in skeletal muscle.
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Urinary tract infections (UTIs) are primarily caused by uropathogenic Escherichia coli (UPEC), and 1 in 40 women experience chronic UTIs during their lifetime. The antibiotic courses required to treat infections promote antibiotic resistance, and current vaccine options offer limited protection. We have pioneered a strategy using small iron-chelating compounds called siderophores as vaccine antigens. These siderophores are not produced by commensal bacteria and are required for UTI. The siderophore vaccines reported here are easy to formulate and reduce bacterial burdens in a murine model of UTI. This report highlights the untapped resource of bacteria-specific small molecules as potential vaccine antigens and provides a proof of principle for incorporating these compounds into multicomponent vaccines for the prevention of bacterial infections.
Uropathogenic Escherichia coli (UPEC) is the primary cause of uncomplicated urinary tract infections (UTIs). Whereas most infections are isolated cases, 1 in 40 women experience recurrent UTIs. The rise in antibiotic resistance has complicated the management of chronic UTIs and necessitates new preventative strategies. Currently, no UTI vaccines are approved for use in the United States, and the development of a highly effective vaccine remains elusive. Here, we have pursued a strategy for eliciting protective immunity by vaccinating with small molecules required for pathogenesis, rather than proteins or peptides. Small iron-chelating molecules called siderophores were selected as antigens to vaccinate against UTI for this vaccine strategy. These pathogen-associated stealth siderophores evade host immune defenses and enhance bacterial virulence. Previous animal studies revealed that vaccination with siderophore receptor proteins protects against UTI. The poor solubility of these integral outer-membrane proteins in aqueous solutions limits their practical utility. Because their cognate siderophores are water soluble, we hypothesized that these bacterial-derived small molecules are prime vaccine candidates. To test this hypothesis, we immunized mice with siderophores conjugated to an immunogenic carrier protein. The siderophore–protein conjugates elicited an adaptive immune response that targeted bacterial stealth siderophores and protected against UTI. Our study has identified additional antigens suitable for a multicomponent UTI vaccine and highlights the potential use of bacterial-derived small molecules as antigens in vaccine therapies.
Both the physical and financial burdens of urinary tract infections (UTIs) are staggering. Half of all women experience a symptomatic UTI in their lifetime (1). And of those women, almost half suffer a reoccurrence within the next year (1). In the United States, where the annual societal cost of UTI is likely underestimated at $3.5 billion (2), 4 million women have UTIs continuously (3). Uropathogenic Escherichia coli (UPEC) is a subclass of extraintestinal pathogenic E. coli (ExPEC) and is the etiological agent for 80% of all uncomplicated UTIs (1). In 2006, there were 11 million physician visits, over 1.7 million emergency room visits, and 479,000 hospitalizations of both men and women in the United States for UTI (2, 4). Altogether, these estimates place UTIs first among kidney and urologic diseases in terms of total cost.
UTIs occur when bacteria, most commonly UPEC (5), contaminate the periurethral area and traverse the urethra to colonize the bladder and its underlying epithelium, causing cystitis (6, 7). If left untreated, UPEC may ascend the ureters and establish a secondary infection in the kidney parenchyma, causing pyelonephritis. At this juncture, UPEC can elicit serious complications, including renal scarring, septicemia, and death.
UTIs are routinely treated with antibiotic therapy, including trimethroprim–sulfamethoxazole (TMP–SMX) and ciprofloxacin. Women experiencing at least two UTIs per year are frequently given antibiotics prophylactically (8). Not surprisingly, the rates of resistance to these antibiotics in UPEC strains have steadily risen over the past few decades. In the United States, Canada, and elsewhere, ∼10–25% of uncomplicated UTI isolates are resistant to TMP–SMX (9⇓–11). This trend is forcing physicians to reach for more expensive and sometimes less effective drugs to treat UTIs (10, 12, 13). Even more troubling is the rise in multidrug resistance among UPEC strains, as a recent international study found that over 10% of E. coli cystitis isolates are resistant to at least three different classes of antimicrobial agents (14). These trends challenge the prescription choices of physicians to address shifting microbial susceptibilities (15).
To compound the danger of antibiotic resistance, there are no currently licensed vaccines in the United States to combat recurrent UTIs in women. In Europe, two vaccines against UTIs called SolcoUrovac and Uro-Vaxom are licensed for use in women with recurrent UTIs (16). SolcoUrovac is a vaginal suppository containing 10 heat-killed UPEC strains that provides relatively poor protection in the absence of frequent administration (17). Uro-Vaxom is an oral capsule containing a lyophilized mixture of membrane proteins from 18 UPEC isolates that is expected to be taken daily. Although this vaccine offers protection against UPEC, its success is limited due to toxicity and poor adherence to the daily regimen (18). Due to these drawbacks, no vaccines are licensed for use in the United States (19). Given the paucity of effective vaccines, the increasing rate of UPEC antibiotic resistance, the decline in novel antibiotic scaffolds, and the need to reduce healthcare expenditures, new therapeutic strategies to manage UTIs must be explored.
Previous work using unbiased genomic and proteomic screens identified bacterial targets that are expressed in vivo by UPEC during UTIs in women, reside on the surface of the bacterium, are immunogenic, and carry out a critical function for survival of E. coli in the host (20). Six bacterial iron acquisition system proteins met all criteria. These findings are supported by a rich history of genomic, transcriptomic, and proteomic studies that have also identified iron acquisition systems as prime anti-UTI targets (21⇓⇓⇓⇓⇓–27).
Iron is an essential cofactor in many biological processes, including DNA synthesis, electron transfer, and central metabolism (28). Iron acquisition is generally required for bacterial growth during infection (28, 29). One facet of innate immunity, coined “nutritional immunity,” restricts bacterial infections by limiting access to critical metal cofactors (28, 30). The mammalian host limits intracellular and freely circulating iron by sequestering iron in proteins such as lactoferrin, transferrin, ferritin, and hemoglobin (31). Notably, the primary site of UPEC infection, the bladder, has lower iron levels than serum (32). Thus, it is not surprising that over 14 gene clusters implicated in iron acquisition have been identified as important virulence factors in UPEC strains (33⇓⇓⇓–37); these gene clusters encode up to four siderophore biosynthesis and uptake systems as well as receptors for the acquisition of heme, ferric citrate, and ferrous iron. Of the many classes of siderophores, UPEC strains typically encode at least three of the following siderophores: yersiniabactin (Ybt), aerobactin (Aer), enterobactin (Ent), and the glucosylated Ent, salmochelin (Glc-Ent) (38, 39).
Bacterial iron acquisition is a natural target of the host immune system. For example, serum albumin and lipocalin-2 bind and inactivate Ent (40, 41). To evade host immunity, pathogenic E. coli strains typically encode a combination of Ybt, Aer, and Glc-Ent stealth siderophores, which are not recognized by host defenses (39). By evading host defenses to secure nutrient iron, Ybt, Aer, and Glc-Ent serve as urovirulence factors (33, 36). Notably, Ybt and Aer are more prevalent among pathogenic E. coli strains than commensal isolates (39). Moreover, previous studies that systematically assessed the use of surface-exposed iron receptors as potential vaccine antigens found that two of the stealth siderophore receptors, those that recognize Ybt and Aer, protect against UTI (20, 42, 43). The hydrophobic nature of these outer-membrane receptors, however, makes these antigens insoluble in water, complicating the purification and formulation of the vaccines. Here, we examine the potential use of the small-molecule siderophores Ybt and Aer as protective vaccine antigens that could bolster the efficacy of other immunoprotective strategies.
By virtue of the confirmed importance of Ybt and Aer in uropathogenesis (33⇓⇓⇓–37), the proven efficacy of their receptors in experimental vaccines (20, 42), their increased prevalence among pathogenic E. coli (39), and amenable biochemical features, we hypothesized that Ybt and Aer could represent valid vaccine candidates. Because the siderophores are small (<564 Da) and unlikely to be immunogenic, Ybt and Aer were conjugated to cationized BSA (cBSA), an immunogenic carrier protein that has aminoethyl-capped carboxylic acids (44⇓–46). The positive surface charge of cBSA increases vaccine binding to immune cells, and the aminoethyl modifications improve the coupling reaction by both eliminating carboxylic acids and providing additional primary amines on the carrier protein (44⇓–46). Both Ybt and Aer have carboxylic acid moieties; thus, standard amide coupling conditions were used to prepare the cBSA–siderophore conjugates using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as the coupling reagent (SI Appendix, Fig. S1 A and B). cBSA incubated with EDC in the absence of siderophore was used as a negative control.
Liquid chromatography and tandem mass spectrometry on trypsin-digested cBSA–siderophore conjugates confirmed linkage of the siderophores to cBSA. For the cBSA–Aer conjugate, peptide fragment ions supporting the linkage of Aer to Lys266, aminoethyl-Asp474, and aminoethyl-Glu267 were detected (SI Appendix, Fig. S2 A–C and Tables S1–S3). For cBSA–Ybt, peptide ions supporting the linkage of Ybt to aminoethyl-Asp272 and Lys437 were detected (SI Appendix, Fig. S2D and Tables S4 and S5). Altogether, these data confirm at least two conjugation sites on cBSA for each siderophore.
To assess the efficacy of vaccination with the stealth siderophore conjugates, mice were immunized intranasally with 10 μg of vaccine conjugate prepared in 20 μL of PBS (SI Appendix, Fig. S1C). Three vaccine groups were examined along with a cBSA control, including cBSA–Ybt, cBSA–Aer, or a 1:1 mixture of cBSA–Ybt:cBSA–Aer, which was composed of 5 μg of each conjugate. Previous reports have shown that intranasal vaccination provides the most consistent protection in murine UTI vaccine studies (20, 42, 47). Therefore, mice were boosted intranasally with 20 μL of PBS containing 2.5 μg of vaccine conjugate 7 and 14 d postimmunization. UPEC strain HM69, a strain recently isolated from a patient with uncomplicated cystitis, was selected for challenge because it encodes Ent, Ybt, and Aer (38). On day 21 postimmunization, mice were transurethrally inoculated with 108 colony-forming units (cfus) of HM69, and after 48 h, the bacterial burdens in the urinary tract were quantified.
Vaccination with cBSA–Ybt reduced bacterial burden by 12-fold in the urine (P = 0.04) and 10-fold in the kidneys (P = 0.01), whereas cBSA–Aer reduced bacterial burden by 19-fold (P = 0.02) in the urine (Fig. 1 A–C). Coimmunization with 1:1 cBSA–Ybt:Aer also decreased bacterial burdens in the urine by 14-fold (P = 0.3) and, most dramatically, reduced bacterial burden in the kidneys by 126-fold (P = 0.002) (Fig. 1 A–C). Altogether these data demonstrate that the siderophore–protein conjugates significantly reduce the bacterial burden in experimental UTI, particularly dissemination to the kidneys. At the time of sacrifice, a subset of the kidneys and bladders from infected mice were fixed in neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Five-micrometer histologic sections were randomized and blindly scored for neutrophilic inflammation by a board-certified veterinary pathologist. Scores were semiquantitative and ranged from 0 (no inflammation) to 3 (severe inflammation) (SI Appendix, Fig. S3). Vaccination with cBSA–Ybt, cBSA–Aer, and 1:1 cBSA–Ybt:cBSA–Aer significantly reduced pyelonephritis and overall inflammation scores (Fig. 1 D–F).
Vaccination with siderophores reduces bacterial burdens and inflammation in the urine and kidneys during UTI. CBA/J mice were vaccinated with cBSA, cBSA–Ybt, cBSA–Aer, or 1:1 cBSA–Ybt:cBSA–Aer. At 21 d after the first immunization, mice were transurethrally inoculated with 108 cfus of wild-type HM69. After 48 h, cfus in the (A) urine, (B) bladder, and (C) kidneys were enumerated by serial dilution and plating on LB agar. Limit of detection was 102 cfu/mL of urine or cfu/g of organs. Bladders and kidneys were fixed, embedded in paraffin, sectioned, and H&E stained. Tissue sections were randomized and blindly scored for inflammation in the (E) bladders and (F) kidneys. The pathology scores in E and F were combined to quantify overall inflammation in D. Significance was calculated using a one-tailed Mann–Whitney test. In all panels, bars represent the median; in A–C, n ≥ 38; D–F, n ≥ 8.
To ascertain whether the vaccine specifically targets Ybt and Aer, three isogenic mutants were generated; E. coli HM69 ybtS::cam (ybtS) and iucA::kan (iucA) have the Ybt and Aer biosynthetic machinery disrupted, respectively. In addition, both biosynthetic operons were disrupted in a ybtS–iucA double mutant. We hypothesized that if the vaccines specifically target Ybt or Aer during infection, then infection with each siderophore biosynthesis mutant would result in loss of protection by the corresponding vaccine. Mice were vaccinated with either cBSA or cBSA–Ybt (SI Appendix, Fig. S1C) and then transurethrally challenged with ybtS. After 48 h, there were no significant differences in the bacterial burdens of ybtS between cBSA- and cBSA–Ybt-vaccinated mice (Fig. 2A and SI Appendix, Fig. S4A). Similar experiments were conducted by vaccinating mice with cBSA–Aer and then challenging with iucA, as well as by covaccinating with 1:1 cBSA–Ybt:cBSA–Aer and then challenging with ybtS–iucA (Fig. 2 B and C and SI Appendix, Fig. S4 B and C). In all instances, no significant differences were identified between the vaccinated groups and the cBSA control mice (Fig. 2 and SI Appendix, Fig. S4), indicating that the vaccines elicit an immune response specifically targeting Ybt and Aer during UTI.
Protection provided by siderophore vaccines requires the biosynthesis of bacterial stealth siderophores. CBA/J mice were vaccinated according to the protocol but transurethrally inoculated with the corresponding siderophore biosynthesis mutant. (A) cBSA–Ybt-vaccinated mice were inoculated with HM69 ybtS, (B) cBSA–Aer-vaccinated mice were inoculated with HM69 iucA, and (C) 1:1 cBSA–Ybt:cBSA–Aer-vaccinated mice were infected with HM69 ybtS–iucA. For each experiment, cBSA was used as the negative control (dark gray bars). In all instances, mice were transurethrally inoculated with 108 cfus of the indicated strain. After 48 h, cfus in the urine and kidneys were enumerated by serial dilution and plating on LB agar. Statistical analyses using a one-tailed Mann–Whitney test identified no significant differences between negative control and siderophore-vaccinated groups (0.2336 < P < 0.4761). Limit of detection was 102 cfu/mL of urine or cfu/g of kidneys; bars represent the median; and n ≥ 14.
The unexpected differences in kidney colonization for iucA and ybtS–iucA mutants (Fig. 2 B and C) compared with the cBSA–Aer- and 1:1 cBSA–Ybt:cBSA–Aer-vaccinated and infected with WT HM69 (Fig. 1C) could be due to disparities between the effects of genetically disrupting intracellular siderophore biosynthesis and immunologically targeting extracellular siderophore activity. In the case of iucA (Fig. 2B) and cBSA–Aer (Fig. 1C), it is possible that the immune response does not inactivate Aer-mediated iron acquisition as well as genetically disrupting Aer biosynthesis. Whereas in the case of ybtS–iucA (Fig. 2C) and 1:1 cBSA–Ybt:cBSA–Aer (Fig. 1C), ybtS–iucA may colonize the kidneys better than WT in 1:1 cBSA–Ybt:cBSA–Aer-vaccinated mice because of increased virulence or Ent production in ybtS–iucA. Studies have shown that disrupting siderophore biosynthesis impacts central metabolism, which could affect the elaboration of other virulence factors and ultimately pathogenesis (48). Alternatively, elevated Ent levels have been detected in bacterial strains with mutations in the Aer and Ybt pathways, which could improve pathogenesis (49, 50). Elevated Ent levels in ybtS–iucA may also explain the surprising increase in kidney colonization of cBSA-vaccinated mice infected with ybtS–iucA compared with iucA (Fig. 2 B and C), as the iucA and ybtS–iucA mutants have been rigorously confirmed to be genetically correct and found to grow similarly in vitro (SI Appendix, Fig. S5). Despite these confounding factors presented by the use of isogenic siderophore biosynthesis mutants, the data presented in Fig. 2 reveal that the bacteria must encode the targeted siderophore to be susceptible to the corresponding cBSA–siderophore vaccine. This emphasizes that the protection elicited by vaccination with Ybt and Aer is target-specific, suggestive of an adaptive immune response.
Because the molecular masses of Ybt and Aer are relatively small (481 Da and 564 Da, respectively), we hypothesized that Ybt and Aer are unable to stimulate an immune response alone and are acting as haptens. To test the immunogenicity of Ybt and Aer, mice were vaccinated with Ybt, Aer, or a 1:1 mixture of Ybt:Aer. Based on the theoretical maximum amount of siderophores administered in the cBSA–conjugate vaccine, mice were immunized with 0.75 μg of siderophore and boosted with 0.19 μg of siderophore on days 7 and 14 postimmunization (SI Appendix, Fig. S1C). On day 21 postimmunization, mice were challenged with 108 cfu of HM69 and bacterial burdens were quantified 48 h later. Without a carrier protein, immunization with Ybt and Aer no longer protected against UTI (Fig. 3 A and B and SI Appendix, Fig. S6A), revealing that Ybt and Aer require cBSA to elicit a protective immune response.
Research Update Nov. 14, 2016
A long-term NIDDK study reports that keeping blood glucose (sugar) as close to normal as possible for an average of 6.5 years early in the course of type 1 diabetes reduces cardiovascular (heart) disease for up to 30 years. The landmark Diabetes Control and Complications Trial (DCCT) began in 1983. The DCCT randomly assigned half its participants to an intensive blood glucose management regimen designed to keep blood glucose levels as close to normal as safely possible, and half to the less-intensive conventional treatment at the time. When DCCT ended in 1993, it was clear that intensive management had significantly reduced eye, nerve, and kidney complications, but at that time the participants were too young to determine their rates of cardiovascular disease. All DCCT participants were taught the intensive management regimen and invited to join the Epidemiology of Diabetes Interventions and Complications (EDIC) study. EDIC continued to monitor DCCT/EDIC participants’ health, and overall blood glucose management has since been similar in both DCCT treatment groups.
To study the long-term effects of the different treatments tested in the DCCT, researchers examined differences in cardiovascular problems, which can take many years to develop, between the former intensive and conventional treatment groups. After an impressive average 30-year follow-up, DCCT/EDIC researchers found that those who practiced intensive blood glucose management during the DCCT still had significantly reduced cardiovascular disease compared to those who did not, despite having similar blood glucose management for 20 years after the DCCT ended. Compared to the former conventional treatment group, the former intensive management group had a 30 percent reduced incidence of cardiovascular disease and 32 percent fewer major cardiovascular events (such as non-fatal heart attack, stroke, or death from cardiovascular disease), after 30 years of follow-up. These results were similar for both men and women who participated in the studies. However, the beneficial effects of intensively managing blood glucose during the DCCT appeared to be wearing off over time. Previously, after 20 years of follow-up, DCCT/EDIC researchers reported that the former intensive treatment group had a 42 percent reduced risk of cardiovascular disease compared to the former conventional treatment group. After 30 years of follow-up, that number had fallen to 30 percent. Even with this reduction in protection, these new data show that a finite period of near-normal blood glucose management early in the course of type 1 diabetes can have substantial beneficial effects on cardiovascular health for up to 30 years. Overall, this finding adds to DCCT/EDIC’s decades of evidence demonstrating how people with type 1 diabetes can dramatically reduce their risk for complications later in life by practicing early, intensive blood glucose management.
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