3.1. Experimental Design and General Metrics of Omic Data
Noteworthy, along each pre-weaned lamb GIT, microbial communities are expected to vary according to specific and dynamic combinations of dietary patterns, host enzymes, metabolites, and immunological effectors. In turn, the microbial antigenic repertoire and their functional arsenal are expected to maintain the intestinal homeostasis in each GIT. With the aim of achieving knowledge of both taxonomy and functions of the different GIT microbiotas in the pre-weaned lamb and, to this end, to investigate the feasibility of a metaproteogenomic approach, 10 GITs (rumen, reticulum, omasum, abomasum, duodenum, jejunum, ileum, cecum, colon, and rectum) were collected from a 30-day-old Sarda lamb fed mother’s milk and forage. In view of the large number of tracts to be analyzed and of the multi-omic approach selected, we chose to perform this proof-of-principle study on a single animal. All luminal (n = 10) and mucosal (n = 10) samples were divided into two portions, for DNA and protein extraction, respectively.
DNA extracted from luminal and mucosal contents was first directly subjected to amplification of the V4 hypervariable region of the 16S rRNA gene. However, the majority of the DNA samples did not provide evidence of bacterial PCR products when checked on 2% agarose gel (data not shown), possibly due to the lowest microbial genomic contents. In view of this, we decided to perform a full-length 16S rRNA gene amplification, which was able to reach a much higher amplification yield. Then, a nested amplification was devised, comprising V4 amplification on full-length 16S amplicons, which led to obtain a satisfactory amount of PCR products for all samples. Therefore, we decided to employ the nested amplification approach, although being aware of the possible biases that this method can generate with respect to quantitative ratios among microbial species. Amplicons were subsequently subjected to deep sequencing with a MiSeq Illumina sequencer (1,147,915 reads in total; the number of reads obtained per each sample are listed in Table S1
). For the sake of brevity, data concerning the sequencing of nested rRNA gene amplicons (V4 region on full-length 16S) will be hereafter referred to as “16S”. Additionally, a selection of 15 whole metagenomes were sequenced, with the purpose of creating a custom sequence database for metaproteomic analyses.
In parallel, proteins were extracted from luminal and mucosal samples, processed according to the FASP procedure, and finally analyzed by high-resolution LC-MS/MS. Multiple database search strategies were employed to maximize microbial and host protein identification, according to previous reports from ours and other groups [32
]. Specifically concerning the microbial identifications, the first database was composed by the metagenomic sequences (raw and assembled) obtained in this study, while the second was a selection of UniProtKB sequences belonging to the microbial genera identified through 16S metagenomics. A total of 528,572 peptide-spectrum matches (PSMs), of which 412,940 related to the host and 115,632 classified as microbial, were identified. As reported in Table S2
, a low depth of microbial-related information was reached for some GITs, especially those belonging to the small intestine and mucosal samples in general.
Richness and alpha-diversity of taxonomic and functional data concerning the lamb GITs are showed in Figure 1
. As illustrated by the line graphs, the highest values of taxonomic alpha-diversity and richness were observed in the forestomachs, while a remarkable drop was observed in the abomasum and in all the intestinal tracts, with a slight increase of the richness in the large intestine; luminal and mucosal samples showed comparable trends. Concerning the number and complexity of microbial functions (functional families), those detected at the mucosa interface appeared consistent throughout the GITs, while those associated to the digesta were higher in the tracts with higher fermentative activity (i.e., forestomachs and large intestine). The complexity of the microbiota and its functions, therefore, is in keeping with the massive fermentation capability to be reached in the rumen, reticulum and omasum, and with the significant contribution to feed fermentation provided also by microorganisms that colonize the colon, as recently reported [4
]. The harsher conditions of the abomasum and the duodenum, due to the gastric secretion in the former and the bile and pancreatic secretion in the latter, might explain the drop of microbiota richness and alpha diversity in the abomasum and the small intestine. Furthermore, when considering functional data, richness and diversity of host protein functions were higher than those of microbial functions along all GITs, with a remarkable increase in the small intestine where the microbial function exhibited the lowest values.
Beta-diversity was also evaluated through PCA plots (Figure 2
). The plot based on 16S taxonomic data showed a clear separation between forestomachs and the other GITs according to the first component; in addition, small and large intestine clustered separately according to the second component. No clear separation among luminal and mucosal samples could be observed (Figure 2
A). According to metaproteomic data, a global separation was seen between luminal contents of forestomachs and large intestine on one hand, and luminal contents of small intestine and all mucosal contents on the other hand (Figure 2
B). According to these data, similar fibrolytic taxa are expected to be active in the lumen of the forestomachs and the larger intestine. Furthermore, abomasum lumen was clearly different from the other samples based on the second component. Noteworthy, abomasum, duodenum and jejunum showed low diversity at taxonomic level (Figure 2
A). On the other hand, at functional level, abomasum showed high diversity compared to duodenum and jejunum (Figure 2
B), suggesting that the same assortment of microorganism is functionally divergent, possibly as a consequence of the stressful exposure to lowest pH in the former tract. Similar results were found when considering host proteins (Figure 2
3.2. Taxonomic Distribution of the Microbiota along the Gastrointestinal Tract of a Pre-Weaned Lamb
Focusing on taxonomy, important differences were detected along the GITs of the pre-weaned lamb under study. At total of 37 bacterial and archaeal phyla (Dataset S1
) were detected. Firmicutes and Bacteroidetes were the most abundant phyla in all GITs consistently with previous reports from other authors on the adult sheep [12
]. Figure 3
reports the top 10 microbial families and their relative abundances, computed by aggregating OTUs at family level.
At first glance, a strong divergence was observed between the forestomachs and all the other tracts, in accordance with alpha- and beta-diversity. As depicted in Figure 3
A, rumen and reticulum showed high levels of Pirellulaceae (belonging to the phylum Planctomycetes; 22% and 30%, respectively), Ruminococcaceae (phylum Firmicutes; 12% and 14%), F16 (phylum TM7; 13% and 11%), BS11 (phylum Bacteroidetes; 10% and 9%) and Dethiosulfovibrionaceae (phylum Synergistetes; 10% and 5%) in the lumen, plus several other families with a moderate relative abundance (>3%). Planctomycetes, forming the PVC superphylum together with Verrucomicrobia and Chlamydiae [37
], belong to the usual components of the ruminal microbiota of sheep [38
], cattle [39
] and dairy cows [40
]. Specifically, the family Pirellulaceae was found in a greater extent in this work than in the rumen microbiota of mature sheep, cattle and deer [41
]. The role of Planctomycetes in the GIT is still unclear, although anaerobic sulfur reduction and sugar fermentation have been suggested as two possible metabolic processes enabling Planctomycetes to grow in anaerobic environments [42
]. The Bacteroidetes family BS11 is able to ferment hemicellulosic sugar to VFAs, which are considered essential to ruminant energy supply [43
]. The composition of the mucosal microbiota inhabiting the pre-weaned lamb forestomachs appeared to be globally comparable to that found in the lumen, in spite of a drop of Pirellulaceae counterbalanced by higher levels of Veillonellaceae (phylum Firmicutes; 12% in rumen and 15% in reticulum) and Lachnospiraceae (phylum Firmicutes; both 11%). Furthermore, Veillonellaceae (19% in the luminal and 11% in the mucosal community) and Xanthomonadaceae (phylum Proteobacteria; both 18%) were the most abundant families observed in the omasum microbiota, where some families observed in the first two tracts were also detected (such as Ruminococcaceae, Pirellulaceae and F16). Veillonellaceae and Lachnospiraceae have been recently identified as part of the core rumen microbiome in cattle [44
]. The Veillonellaceae family produces propionate as a major fermentation product, while Lachnospiraceae synthesize butyrate that plays an important role in rumen development [8
]. Several members of the Xanthomonadaceae are able to produce xylanases, and therefore to ferment xylan, a major structural component of plant cell wall. This may make them capable to survive in anaerobic environments, as the omasum; furthermore, members of Proteobacteria might play a role in scavenging oxygen diffusing from the capillary system, creating the condition for the establishment of anaerobic communities [6
]. Considering both luminal and mucosal microbial communities, rumen, reticulum and omasum presented a well-assorted microbiota, with several families overtaking the 3% of their relatives abundance. In addition, similar values of Firmicutes/Bacteroidetes (F/B) ratio were observed in the luminal and mucosal content of rumen and reticulum (0.77 vs. 0.95, and 0.94 vs. 1.13), while a higher F/B ratio was detected in the luminal content of omasum compared to the mucosal microbiota (2.46 vs. 0.82) (Dataset S1
). Archaea families were also well represented (around 1% and up to 3% in the omasum) (Dataset S2
The remaining seven GITs showed a completely different structure (Figure 3
B,C), with Lactobacillaceae (phylum Firmicutes) being the most predominant (and almost exclusive) family in the abomasum (94% lumen and 87% mucosa) and gradually decreasing along the intestine, reaching a minimum of 46% in the ileal mucosal microbiota, and remaining around 50% in the large intestine tracts, in line with the diet still mainly based on milk. This also affected the F/B ratio, clearly unbalanced towards Firmicutes. Other Firmicutes families exhibiting a remarkable abundance in the gut were Lachnospiraceae (duodenal mucosa 12%, ileum, 10%, and up to 29% in colonic lumen), Clostridiaceae (13% and 8% in the lumen of ileum and cecum, respectively) and Ruminococcaceae (around 8% in colon and rectum). With the exception of Lactobacillaceae, these families were predominant also in the adult sheep, followed by Veillonellaceae and Mogibacteriaceae; compared to the adult animal, the most striking difference was the very much lower abundance of families belonging to Bacteroidetes and Verrucomicrobia [16
]. Such differences might be explained with a lower complexity of dietary glycans escaping the rumen in the pre-weaning, since this heterogeneous group of molecules is known to shape the composition of the gut microbiota [47
]. Finally, very few Archaea were detected, consistently with a lower methanogenic activity compared to lamb forestomachs and to large intestine in the adult sheep (Datasets S1 and S2
). Indeed, the syntrophic relationships between cellulolytic, H2
-producing bacteria (Ruminococcaceae, Lachnospiraceae, etc.) and H2
-consuming methanogenic archaea, commonly observed in the rumen, might be displaced here by the capability of Lactobacillaceae to inhibit hydrogen fermentation by substrate competition (lactic acid fermentation).
3.3. Functions and Metabolic Pathways of the Pre-Weaned Lamb Gastrointestinal Microbiota
As illustrated in Figure 4
, several enzymes involved in glycolysis and pyruvate metabolism, as well as ribosomal proteins, were detected in the top 10 microbial functions upon metaproteomic analysis (the InterPro database was selected for functional annotation, while assignment to metabolic pathways was based on UniProtKB annotation). Glutamate dehydrogenase overtook the 8% of relative abundance in rumen and reticulum, dropping to much lower percentages in the small (<1%) and large intestine (<2%). Phosphoserine aminotransferase exhibited a similar trend. These findings are in line with the known role of these enzymes in several catabolic and anabolic pathways of amino acid metabolism, consistently with the intense microbial proliferation in these sites.
According to pathway classification (Figure 4
right), proteins involved in starch degradation were almost exclusively detected in the luminal samples from rumen, reticulum, colon and rectum, consistently with the known biogeography of fiber fermentation. The presence of enzymes devoted to starch degradation in the lower GITs is in agreement with the mentioned ingestion of small pieces of corn grain from the mother’s diet in early postnatal phases, even before the first stages of rumen development. Nonetheless, up to 50% of the starch may escape to the small intestine also in mature ruminants [48
], where the hindgut may thus avoid the inefficiencies of rumen fermentation [49
]. This phenomenon has been demonstrated to be highly dependent on the diet and in particular on the presence of corn, sorghum, and legumes [50
We then focused our attention on glycoside hydrolases (GHs), in view of their key role in plant biomass degradation. Peptides belonging to ten different GH families were detected. GH 28, comprising mainly polygalacturonases digesting pectins, representing the high digestible fiber sources for ruminants [52
], was present almost in all GITs, in agreement with the fact that fibrous particles ingested could bypass the forestomach, not only before the rumen development [38
]. Other families were more typical of specific tracts. These include GH 9 (formerly known as cellulose E) that was only found in the ruminal lumen, GH 36 (alpha-galactosidase) higher in the omasum, and GH 2 (beta-galactosidase) present mainly in the lumen of the large intestine (Dataset S3
Next, given the relevant fermentative activity exerted by both rumen and colon, we sought to compare the functional assortment of the microbiota inhabiting the lumen of these two GITs, and to determine if similar/different enzymatic activities are performed by similar/different members of the microbiota in a pre-weaned lamb. As a result, methanogenic enzymes, expressed by Euryarchaeota (specifically Methanobacteriaceae), were only found in the rumen, while several proteins responsible for acetate biosynthesis (including carbon monoxide dehydrogenase and enzymes of the Wood-Ljungdahl pathway, mainly produced by Firmicutes) were exclusively detected in the colon (Datasets S3–5
). Colonization of the rumen by methanogens is more competitive than that of reductive acetogens and starts in the first days of life to sustain a functional rumen by reducing hydrogen concentration [53
Moreover, when considering proteins assigned to Ruminococcaceae, some functions, including pilus retraction protein PilT, aconitase/isopropylmalate dehydratase, enolase and flagellin, were found to be rumen-specific, while others, comprising many ribosomal proteins and enzymes involved in pyruvate and butyrate metabolism, appeared to be colon-specific.
As illustrated previously (Figure 1
), there were considerable differences in the amount of microbial peptides detected based on the specific sample taken into consideration (GIT and/or lumen vs mucosa). This may have led to some biases, especially when comparing relative abundances between samples with much different identification depth. However, glyceraldehyde-3-phosphate dehydrogenase, known to be a housekeeping protein with quite constant abundance in different organisms/tissues, was consistently detected at comparable levels in samples with different in identification depth (e.g., lumen and mucosa of large intestinal tracts).
3.4. Host Protein Functions along the Pre-Weaned Lamb Gastrointestinal System: Focus on Digestion
Metaproteomics allows the identification of both microbial and host proteins in the same sample. Therefore, we exploited this omic approach to elucidate the distribution of lamb proteins along the different GITs. Many structural proteins were identified, especially in the mucosal samples. However, we decided to focus on host digestive enzymes, in view of their importance for animal production and management. As shown in Figure 5
(and detailed in Dataset S6
), intestinal-type alkaline phosphatase, lactase, trypsin, trefoil factor, alpha-amylase, colipase and other enzymes were detected at very high levels in lumen of the various tracts of the large intestine. Previous studies confirm the high activity of these enzymes in duodenum and colon, as well as the association of this catalytic activity to GIT development stages in small ruminants [54
]. It is generally assumed that lactase activity decreases with age whereas all the other hydrolytic enzymes increase reaching stable ranges at ruminal level, 30 days after birth, in dairy calves [55
]. In pre-weaned lambs, fed only milk, a quite stable enzymatic hydrolytic activity in different traits of the small intestine was also observed from birth to 56 days [56
On the other hand, in this study, gastrokine, pepsin and somatostatin were detected in the omasum and, at even higher amounts, in the abomasum. Fatty acid-binding protein was instead typical of duodenum, whereas bile salt-activated lipase was found mostly in abomasum and duodenum.