Caloric restriction induces rapid and deep changes in the faecal microbiota metaproteome

We recently reported that significant compositional changes arise in the faecal microbiota of young growing rats after short-term administration of a CR diet15. Here, we sought to verify if those structural changes in the GM are associated with modification in its functional and metabolic profile, through the application of a shotgun metaproteomic approach. To this end, we collected faecal samples from rats undergoing 3, 5 and 8 weeks of CR, as well as from control AL rats at the same time points, and characterised the metaproteomic profile of their GM. A total of 142,942 mass spectra could be matched to 878 different protein functions, belonging to over 250 different microbial genera (complete metrics, identification and annotation data can be found in Supplementary Dataset S1).

First, in order to investigate compositional changes in the GM, we focused on taxonomic data based on the abundance of the proteins expressed by each microbial member. Genus abundance data were initially subjected to a Principal Component Analysis (PCA), revealing that a separation between CR- and AL-fed rats can be observed since 3 weeks of treatment, becoming even clearer up to 8 weeks (p < 0.001 PERMANOVA between groups considering all weeks), as shown in Fig. 1, top. We then performed a differential abundance analysis (edgeR comparison of all AL samples vs all CR samples, followed by Benjamini-Hochberg correction) to identify which genera were mainly responsible for the segregation between groups. Heatmap in Fig. 1 illustrates 24 genera with significantly differential distribution between the GMs of AL- and CR-fed rats. Interestingly, the abundance of 8 Bacteroidetes genera, including Prevotella and Bacteroides, resulted higher in CR-fed rats compared to AL controls, while 11 Firmicutes genera, including Clostridium, Eubacterium and Oscillibacter, were more abundant in AL compared to CR-fed rats. The only Firmicutes genus to be enriched in CR-fed rats was Lactobacillus, with remarkable significance values (FDR = 9 × 10−20) and in line with our previous 16S rDNA gene sequencing results15.

Figure 1 Changes in GM taxonomic composition at genus level, based on metaproteomic results obtained upon caloric restriction treatment on young rats. Top, PCA plot based on microbial genera relative abundance data. Each dot indicates a sample (different time points, expressed in weeks, are illustrated with different shapes), while dotted ellipses indicate 95% confidence level. AL, ad libitum; CR, caloric restriction. Bottom, heatmap illustrating microbial genera with significantly differential abundance between AL and CR groups (edgeR analysis followed by Benjamini-Hochberg correction). Columns represent samples, while rows represent genera. The color gradient is based on the standardized abundance (z-score). Only genera with abundance >0.25% are shown, and ordered first according to the group in which they are significantly more abundant, and then based on the phylum to which they belong (B, Bacteroidetes; F, Firmicutes; G, Glomeromycota; P, Proteobacteria; S, Spirochaetes). Full size image

Our main interest was directed to elucidate which functional traits could discriminate the activity of the GM of CR- and AL-fed animals. Again, the PCA plot (Fig. 2, top) clearly shows as CR and AL groups can be discriminated based on functional abundance data (p < 0.001 PERMANOVA between groups considering all weeks). Differential abundance analysis (edgeR comparison of all AL samples vs all CR samples, followed by Benjamini-Hochberg correction) allowed us to identify 167 functions that vary significantly between the two groups (the 62 functions exceeding 0.25% of relative abundance in at least one group are listed in the heatmap of Fig. 2). The CR treatment was found to induce a deep rearrangement of the microbial metaproteome, involving both catalytic and structural/antigenic functions belonging to several different COG categories (with “carbohydrate metabolism and transport” being the most represented). In particular, several enzymes responsible for carbohydrate degradation and acetate/butyrate biosynthesis were significantly downregulated after CR, while the expression of various ribosomal, outer membrane, DNA-binding and stress-related proteins appeared to be induced by the CR treatment. In most cases, the differential trend started after 3 weeks of treatment and reached similar, top values at 5 and 8 weeks.

Figure 2 Changes in metaproteome functional expression observed upon caloric restriction treatment on young rats. Top, PCA plot based on relative abundance of microbial protein functions. Each dot indicates a sample (different time points are illustrated with different shapes), while dotted ellipses indicate 95% confidence level. AL, ad libitum; CR, caloric restriction. Bottom, heatmap illustrating microbial functions with significantly differential abundance between AL and CR groups (edgeR analysis followed by Benjamini-Hochberg correction). Columns represent samples, while rows represent functions. The color gradient is based on the standardized abundance (z-score). Only functions with abundance >0.25% are shown, and ordered first according to the group in which they are significantly more abundant, and then based on the COG category to which they belong (C, Energy production and conversion; E, Amino acid transport and metabolism; G, Carbohydrate transport and metabolism; I, Lipid metabolism; J, Translation, ribosomal structure and biogenesis; K, Transcription; L, Replication, recombination and repair; M, Cell wall/membrane/envelop biogenesis; N, Cell motility; O, Posttranslational modification, protein turnover, chaperones; T, Signal transduction mechanisms). Full size image

Caloric restriction-induced metaproteomic changes in the faecal microbiota are kept in the adulthood but start to be reversed after 1 week of ad libitum diet

We recently demonstrated that compositional changes early produced by CR in the rat faecal microbiota mainly involve Lactobacillus spp. and are maintained up to 9 months of dietary intervention15. Here, we aimed to investigate, under a structural and functional perspective: (i) if these changes can be still observed in older rats; (ii) if reverting from long-term CR diet to short-term AL diet causes a shift of the faecal microbiota composition and activity. To this purpose, we collected faecal samples from rats treated with CR for 1.5 years, as well as from control AL rats at the same time point; then, CR-fed rats were switched back to the AL diet, and their faecal samples were collected after a week (CR → AL). DNA and proteins were extracted from faecal samples, with the aim of carrying out both 16S rRNA gene sequencing and metaproteome analysis. 16S rRNA gene sequencing provided 1,429,463 reads, assigned to 217 different microbial genera (detailed information is provided in Supplementary Dataset S2). When considering metaproteomic analysis, a total of 126,913 mass spectra could be matched to 1,000 different protein functions, belonging to 241 different microbial genera (complete metrics, identification and annotation data can be found in Supplementary Dataset S1).

Figure 3 shows taxonomic results from 16S rDNA (A) and metaproteomic (B) analyses. Long-term CR was found to induce structural changes that are kept up to 1.5 years of dietary treatment, as clearly illustrated by PCA plots (top) based on relative genus abundances. Even more interestingly, samples collected from rats after only one week of “diet reversion” (CR → AL) clustered in between AL and CR groups, indicating a perturbation of the GM composition in these aged animals due to an increase in the administered quantity of the very same feed. Comparing the three groups analysed, according to 16S rDNA and metaproteomic data, a number of microbial genera showed a significantly differential abundance (again, edgeR comparison of all AL samples vs all CR samples, followed by Benjamini-Hochberg correction), as depicted in the heatmaps of Fig. 3. On the whole, genus abundances were quite heterogeneously distributed among groups and among phyla according to 16S rDNA data; conversely, a clearer trend could be observed based on metaproteomic profiles, with CR → AL abundances placing almost always in the middle between AL and CR, and ‒ consistently with the results obtained on younger rats ‒ all Bacteroidetes differential genera being enriched in CR-treated rats. A consensus could be found between 16S rDNA and metaproteomic results when considering Lactobacillus and Ruminococcus spp., which were found to be more represented in CR microbiota compared to AL controls according to both approaches. The quite low consensus between 16S rDNA and metaproteomic differential results may be mainly due to the use of two different taxonomic databases (Greengenes and NCBI/UniProt, respectively).

Figure 3 Changes in taxonomic composition at genus level based on 16S rDNA gene sequencing (A) and metaproteomics (B) results obtained on adult rats (1.5 years of treatment). AL, ad libitum; CR, caloric restriction; CR → AL, 1-week reversion from caloric restriction to ad libitum. (A) Top, PCA plot based on 16S rDNA gene sequencing relative abundance data at the genus level. Each dot indicates a sample, while dotted ellipses indicate 95% confidence level. Bottom, heatmap illustrating microbial genera with significantly differential abundance between groups upon edgeR analysis followed by Benjamini-Hochberg correction. Heatmap columns represent samples, while rows represent genera. Asterisks in supplementary columns 1, 2 and 3 indicate genera with significantly differential abundance upon AL vs CR, AL vs CR → AL, and CR vs CR → AL comparisons, respectively. The asterisk colour refers to the group in which the genus was found as more abundant. The color gradient is based on the standardized abundance (z-score). Only genera with abundance >0.1% are shown, and ordered according to the phylum to which they belong (B, Bacteroidetes; F, Firmicutes; P, Proteobacteria; V, Verrucomicrobia). (B) Same as in (A), but concerning metaproteomic data instead of 16S rDNA gene sequencing data. Further phylum abbreviations: A, Actinobacteria; Ge, Gemmatimonadetes; Gl, Glomeromycota; S, Spirochaetes; T, Tenericutes. Full size image

The metaproteome dataset was further investigated to identify differences in the expression of protein functions. Once assessed that functional data led to a PCA-based group clustering consistent to that observed for taxonomic data (Fig. 4, top), we focused our attention on differential functions. A total of 113 functions were found with significantly differential abundance among the three sample groups (as a result of edgeR comparison of all AL samples vs all CR samples, followed by Benjamini-Hochberg correction); among them, 73 (65%) had also been consistently found as differential in the young rat experiment. Heatmap in Fig. 4 lists 45 differential functions, exceeding 0.25% of relative abundance in at least one group, belonging to many different COG categories (“carbohydrate metabolism and transport” was again the most represented). Consistent with the data obtained in the young rat experiment, enzymes responsible for acetate/butyrate biosynthesis (carbon monoxide dehydrogenase/acetyl-CoA synthase, formate–tetrahydrofolate ligase and phosphate acetyltransferase for acetogenesis; acetyl-CoA acetyltransferase, butyryl-CoA dehydrogenase and short-chain-enoyl-CoA hydratase for butyrogenesis) were significantly downregulated after CR, while the expression of those involved in propionogenesis (methylmalonyl-CoA mutase and propionyl-CoA carboxylase) appeared to be induced by the CR treatment. Several catalytic functions participating to pentose metabolism (including L-rhamnose and xylose isomerases and altronate oxidoreductase) were also revealed to be consistently more abundant in CR samples. In addition, in almost all cases of differential protein abundance between AL and CR rats, levels in CR → AL rats were in between those measured in CR and AL rats, confirming that a single week of “diet reversion” is sufficient to induce changes in the GM functional profile.

Figure 4 Changes in metaproteome functional expression observed upon caloric restriction treatment on adult rats (1.5 years of treatment). AL, ad libitum; CR, caloric restriction; CR → AL, 1-week reversion from caloric restriction to ad libitum. Top, PCA plot based on relative abundance of microbial protein functions. Each dot indicates a sample, while dotted ellipses indicate 95% confidence level. Bottom, heatmap illustrating microbial functions with significantly differential abundance between groups upon edgeR analysis followed by Benjamini-Hochberg correction. Heatmap columns represent samples, while rows represent functions. Asterisks in supplementary columns 1, 2 and 3 indicate functions with significantly differential abundance upon AL vs CR, AL vs CR → AL, and CR vs CR → AL comparisons, respectively. The asterisk colour refers to the group in which the function was found as more abundant. The color gradient is based on the standardized abundance (z-score). Only functions with abundance >0.25% are shown, and ordered first according to the group in which they are significantly more abundant, and then based on the COG category to which they belong (C, Energy production and conversion; E, Amino acid transport and metabolism; G, Carbohydrate transport and metabolism; I, Lipid metabolism; J, Translation, ribosomal structure and biogenesis; M, Cell wall/membrane/envelop biogenesis; O, Posttranslational modification, protein turnover, chaperones; T, Signal transduction mechanisms). Full size image

Genus-specific functional analysis reveals peculiar functional shifts related to caloric restriction

To gain insight into the contribution of specific microbial genera to the functional activity of the GM in CR- and AL-fed rats, we combined functional and taxonomic (genus level) annotations and investigated the abundance profile of the main proteins expressed by some of the most represented members of the faecal microbiota, including Lactobacillus, Clostridium (see Fig. 5), Bacteroides, Prevotella, Oscillibacter and Ruminococcus (see Supplementary Fig. S1).

Figure 5 Functional expression profile of Lactobacillus (top) and Clostridium (bottom) metaproteomes. Relative abundance values concerning the young rat experiment (left, up to 8 weeks of treatment) and adult rat experiment (right, 1.5 years of treatment) are shown. AL, ad libitum; CR, caloric restriction; CR → AL, 1-week reversion from caloric restriction to ad libitum. Heatmap columns represent samples, while rows represent functions. The color gradient is based on the standardized abundance (z-score). Only functions with abundance >0.1% (Lactobacillus) and >0.05% (Clostridium) are shown (ribosomal proteins were excluded), and ordered according to the COG category to which they belong (C, Energy production and conversion; E, Amino acid transport and metabolism; G, Carbohydrate transport and metabolism; I, Lipid metabolism; J, Translation, ribosomal structure and biogenesis; M, Cell wall/membrane/envelop biogenesis; N, Cell motility; O, Posttranslational modification, protein turnover, chaperones; Q, Secondary structure). Full size image

Lactobacillus was found to be significantly enriched in CR samples at all the time points analysed in this study, consistently with previous reports15. Therefore, we were particularly interested in understanding which functions and metabolic activities related to Lactobacillus spp. are actually increased in the CR microbiota. Among Lactobacillus-specific proteins significantly more abundant in the CR metaproteome, we found many enzymes involved in hexose (including glycolytic enzymes, but also alpha-galactosidases comprised in the glycoside hydrolase family 36), pentose (belonging to pentose phosphate and pentose-glucuronate interconversion pathways) and pyruvate (to lactate and formate) metabolism, but also in oxalate and urea degradation. Proteins responsible for transport and phosphorylation of carbohydrates were also observed as higher in CR-fed rats. Intriguingly, a single Lactobacillus protein function followed the opposite trend (significantly higher in AL-fed rats), i.e. the S-layer protein.

Clostridium spp. were instead reduced after CR treatment, with a consequent and significant decrease in expression of various clostridial functions, including several enzymes mainly involved in glycolysis, but also in pyruvate and butyrate metabolism. Furthermore, only flagellin, a clostridial protein with relevant antigenic properties, was strikingly observed as higher in CR-fed rats.

When looking at the two main Bacteroidetes genera, namely Bacteroides and Prevotella, we found several proteins consistently more abundant in the CR rat faecal microbiota, mainly involved in membrane transport, metabolism and protein folding. Bacteroides enzymes responsible for pentose catabolism and Prevotella proteins belonging to the TonB-dependent transport system were typically increased in the CR metaproteome.

We also examined the metaproteome of two Firmicutes members with different behaviour, Oscillibacter and Ruminococcus. Oscillibacter proteins, similarly to most Firmicutes, were depleted in the CR metaproteome, including several enzymes participating to butyrate, pyruvate and acetate metabolic pathways. On the contrary, we observed an enrichment of Ruminococcus after long-term CR, mainly related to the overexpression of cellulases (glycoside hydrolase family 9).

Caloric restriction promotes expression of propionogenic enzymes and limits abundance of butyrogenic and acetogenic enzymes

We noticed that several enzymes implicated in short-chain fatty acid (SCFA) biosynthesis exhibited differential expression between CR and AL faecal microbiota. To go deeper in the characterisation of these relevant metabolic pathways, we inspected the identified functions in search for all catalytic activities related to butyrate, propionate and acetate metabolism, according to the orthologous genes listed in the corresponding KEGG pathways. First, we calculated the global relative abundance of all enzymes contributing to butyrogenesis, propionogenesis and acetogenesis in the different study groups (listed in Supplementary Fig. S2), and found ‒ consistently both in young and adult rats ‒ that propionogenic functions were significantly higher in the CR metaproteome, whereas a decrease in proteins participating to butyrate and acetate biosynthesis was observed in CR-fed rats (Fig. 6). Interestingly, adult rats fed AL for 1 week after 1.5 years of CR treatment showed a rapid restoration of the levels of acetogenic enzymes typical of an AL diet, while changes in butyrogenic and propionogenic enzyme abundances appeared to be slower.

Figure 6 Scatter plots showing the relative abundance of enzymes involved in short-chain fatty acid biosynthesis. AL, ad libitum; CR, caloric restriction; CR → AL, 1-week reversion from caloric restriction to ad libitum. Each dot indicates a sample; different time points are illustrated with different shapes (same as in Fig. 1). The young rat experiment results are shown on the left of each scatter plot, while the adult rat experiment results are shown on the right. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 10−5. Full size image

To investigate the taxonomic contribution to these metabolic pathway, a map illustrating the expression profiles of the SCFA biosynthetic enzymes identified in this study, including the related taxonomic classification, was generated (Supplementary Fig. S2). A high level of cross feeding appears to occur for butyrogenesis, as each pathway step was found to be due to different members of Clostridia (including Butyricicoccus, Butyrivibrio, Clostridium, Eubacterium, Faecalibacterium, Oscillibacter and Roseburia), while the central, typical reactions of propionate biosynthesis (catalysed by methylmalonyl-CoA mutase and propionyl-CoA carboxylase) could be attributed mainly to Bacteroides and Prevotella spp. Of note, abundance data revealed that the expression of the acetogenic carbon monoxide dehydrogenase (assigned mostly to Blautia) was rapidly and strongly induced in formerly CR-fed rats after just a week of AL feeding, restoring abundance levels comparable to those reached in the long-term AL-fed group.

Caloric restriction modifies abundance of some relevant host proteins detectable in stool

In addition to the microbial portion of a stool sample (microbiota), metaproteomics allows the characterisation of the host proteins that are released in the gut lumen and embodied within the faecal material. We therefore compared the host proteins abundance profile in AL- and CR-fed rats, both after short-term (3–8 weeks) and long-term (1.5 years) dietary treatment (Supplementary Tables S1 and 2). Of interest, enzymes involved in lipid degradation, including bile salt-activated lipase, neutral ceramidase and inactive pancreatic lipase related protein 1, were detected as more abundant in the CR faecal proteome in young and older rats. Further, host proteins involved in mucus production and mucosal anti-inflammatory response were higher in the gut of AL-fed rats (i.e., murinoglobulin-1, calcium activated chloride channel regulator 1 and mucin-2), together with indicators of cell death burden (cytochrome c and actin). Vice versa, a number of keratins, whose alteration in colon is reported during inflammatory stress18, were more abundant in CR-treated rats, including keratin 1, a protein that is key in maintaining epithelial barrier and in the control of intestinal mucosa permeability.