Global Profiling of Carbohydrate Active Enzymes in Human Gut Microbiome.

MotivationCarbohydrate Active enzyme (CAZyme) families, encoded by human gut microflora, play a crucial role in breakdown of complex dietary carbohydrates into components that can be absorbed by our intestinal epithelium. Since nutritional wellbeing of an individual is dependent on the nutrient harvesting capability of the gut microbiome, it is important to understand how CAZyme repertoire in the gut is influenced by factors like age, geography and food habits.ResultsThis study reports a comprehensive in-silico analysis of CAZyme profiles in the gut microbiomes of 448 individuals belonging to different geographies, using similarity searches of the corresponding gut metagenomic contigs against the carbohydrate active enzymes database. The study identifies a core group of 89 CAZyme families that are present across 85% of the gut microbiomes. The study detects several geography/age-specific trends in gut CAZyme repertoires of the individuals. Notably, a group of CAZymes having a positive correlation with BMI has been identified. Further this group of BMI-associated CAZymes is observed to be specifically abundant in the Firmicutes phyla. One of the major findings from this study is identification of three distinct groups of individuals, referred to as 'CAZotypes', having similar CAZyme profiles. Distinct taxonomic drivers for these CAZotypes as well as the probable dietary basis for such trends have also been elucidated. The results of this study provide a global view of CAZyme profiles across individuals of various geographies and age-groups. These results reiterate the need of a more precise understanding of the role of carbohydrate active enzymes in human nutrition

List of CAZyme families having positive correlation with BMI along with their broad carbohydrate substrate classifications.

<p>List of CAZyme families having positive correlation with BMI along with their broad carbohydrate substrate classifications.</p

Differential CAZyme profiles across age and geographies.

<p>Partial Least Square Discriminant Analysis (PLS-DA) score plots showing global profiles of CAZymes across <b>(a)</b> Age and, <b>(b)</b> Geography. While there is a clear distinction in the CAZyme profiles of children/infants as compared to adult individuals, the differences among the adult individuals of various geographies are relatively subtle. However these differences are with respect to the overall CAZyme profiles.</p

Variation of the abundance and diversity of CAZymes across geographies.

<p>Variation of the abundance of CAZymes across <b>(a)</b> Adult individuals belonging to various geographies and, <b>(b)</b> Children/infant individuals belonging to four geographies. Variation of the diversity of CAZymes across <b>(c)</b> Adult individuals belonging to various geographies and, <b>(d)</b> Children/infant individuals belonging to four geographies. For the adult individuals, while there is little variation in the diversity of CAZymes across countries, there are country-specific trends in the abundances of various CAZyme families. High CAZyme/MB content in individuals from rural Venezuela and Malawi may be attributable to their carbohydrate rich diet. It also shows that each adult individual, in general, have a consistent diversity across countries. However, in children and infants, in whom the gut flora is developing and unstable, diversity of CAZymes fluctuates greatly.</p

Variation of contribution index (expressed as GINI coefficient) of CAZyme families in the human gut with age.

<p>The GINI coefficient, an indicator of functional rarefaction, varies as a logarithmic function with age. Adult individuals have a lower GINI coefficient, indicating more equal distribution of CAZyme families, but with progressively younger age, fewer CAZyme families contribute to a greater proportion of CAZymes, thereby increasing the GINI coefficient. Also, high GINI coefficients, close to a maximum of 1, indicate frequency distribution of CAZyme families is highly non-uniform.</p

Variation of CAZyme abundances with BMI.

<p><b>(a)</b> Correlation of BMI with summed abundances of all gut-associated CAZymes <b>(b)</b> Correlation of BMI with summed abundances of the 10 CAZyme families showing significant positive correlation with BMI. The overall summed abundance of all gut associated CAZyme families does not show any correlation with the BMI of the individuals. However, obtaining the correlation of the individual CAZyme families identified 10 such families (listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142038#pone.0142038.t001" target="_blank">Table 1</a>) having a significantly positive correlation with BMI. The correlation of the summed abundances of these 10 families (computed using a sliding window based approach explained in the Methods section) with the BMI was observed to be even more significant (R^2 = 0.44, P < 0.01).</p

Taxonomic origins of the 10 CAZyme families showing significant positive correlation with BMI.

<p>Distribution of contributing clades at (<b>a)</b> Phylum level and <b>(b)</b> Genus level. Several genera belonging to Firmicutes phylum, like Roseburia, Faecalibacterium, Ruminococcus and Eubacterium, were observed to harbor these CAZyme families, two genera, namely Bifidobacterium (belonging to Actinobacteria) and Bacteroides (belonging to Bacteroidetes) accounted for almost half the proportion of such CAZymes.</p

Phylum specific signatures of CAZyme family abundances.

<p>Heatmap of relative percentage abundance of the different digestive CAZyme families across top contributing phyla. The marker CAZyme families identified significantly abundant in the CAZotype-1, CAZotype-2 and CAZotype-3 are highlighted in Blue, Orange and Green text boxes, respectively. Distinct phylum specific signatures in the abundances of different CAZyme families are observed. Further, mapping of marker CAZyme families of the three CAZotypes on to the heatmap indicates that enzymes specific to each CAZotype show distinct phylum specific abundances. For example, a driver enzyme from CAZotype-1 maps perfectly well in the ‘Bacteroidetes specific’ region of the heatmap.</p

Identification of CAZotypes.

<p><b>(a)</b> Distinct clustering of gut microbiomes based on similarities in the CAZyme profiles obtained using the BCA analysis. Each cluster represents a ‘CAZotype’. <b>(b)</b> CAZotype distributions across various nationalities. The CAZyme profiles of the 448 gut microbiomes could be grouped into three distinct clusters, referred to as CAZotypes. Further, the gut microbiomes from individuals of various geographies, were observed to have differential preferences to belong to one of the three CAZotypes.</p

Taxonomic lineage of key drivers of the CAZotypes.

<p>Abundances of the different taxonomic lineages specific to the different CAZotypes. While CAZotype-1 is characterized by the over-representation of the genus Bacteroides lineage (phylum Bacteroidetes), CAZotype-2 has an over-representation of the genera Eubacterium, Ruminococcus and Roseburia (belonging to the Firmicutes phyla) and the genera Escherichia (belonging to Proteobacteria). The CAZotype-3, dominated by infant gut microbiomes, was observed to be dominated by two genera namely, Lactobacillus (phyla Firmicutes; class Bacilli) and Bifidobacterium (phyla Actinobacteria).</p