The Skin Microbiome of the Neotropical Frog Craugastor fitzingeri: Inferring Potential Bacterial-Host-Pathogen Interactions From Metagenomic Data

Abstract

Skin symbiotic bacteria on amphibians can play a role in protecting their host against pathogens. Chytridiomycosis, the disease caused by Batrachochytrium dendrobatidis, Bd, has caused dramatic population declines and extinctions of amphibians worldwide. Anti-Bd bacteria from amphibian skin have been cultured, and skin bacterial communities have been described through 16S rRNA gene amplicon sequencing. Here, we present a shotgun metagenomic analysis of skin bacterial communities from a Neotropical frog, Craugastor fitzingeri. We sequenced the metagenome of six frogs from two different sites in Panamá: three frogs from Soberanía (Sob), a Bd-endemic site, and three frogs from Serranía del Sapo (Sapo), a Bd-naïve site. We described the taxonomic composition of skin microbiomes and found that Pseudomonas was a major component of these communities. We also identified that Sob communities were enriched in Actinobacteria while Sapo communities were enriched in Gammaproteobacteria. We described gene abundances within the main functional classes and found genes enriched either in Sapo or Sob. We then focused our study on five functional classes of genes: biosynthesis of secondary metabolites, metabolism of terpenoids and polyketides, membrane transport, cellular communication and antimicrobial drug resistance. These gene classes are potentially involved in bacterial communication, bacterial-host and bacterial-pathogen interactions among other functions. We found that C. fitzingeri metagenomes have a wide array of genes that code for secondary metabolites, including antibiotics and bacterial toxins, which may be involved in bacterial communication, but could also have a defensive role against pathogens. Several genes involved in bacterial communication and bacterial-host interactions, such as biofilm formation and bacterial secretion systems were found. We identified specific genes and pathways enriched at the different sites and determined that gene co-occurrence networks differed between sites. Our results suggest that skin microbiomes are composed of distinct bacterial taxa with a wide range of metabolic capabilities involved in bacterial defense and communication. Differences in taxonomic composition and pathway enrichments suggest that skin microbiomes from different sites have unique functional properties. This study strongly supports the need for shotgun metagenomic analyses to describe the functional capacities of skin microbiomes and to tease apart their role in host defense against pathogens.

Keywords: Batrachochytrium dendrobatidis; amphibians; host-bacteria interactions; shotgun metagenomics; skin microbiome.

Figure 1

(A) Pie charts of the most abundant genera obtained with metaphlan for each frog microbiome sample. An UPGMA on the left shows grouping of similar samples based on the relative abundance of bacterial taxa. (B) Procrustes analysis comparing the relative abundance of bacterial taxa (Bray-Curtis dissimilarity matrices) of the 16S rRNA gene amplicon data (arrowheads) and the shotgun metagenome data (black circles). A p-value of 0.006 (PROTEST test) indicates that the matrices are more similar than expected by random association.

Figure 2

Read level analysis (A) Class abundances normalized with MUSiCC. (B) Heatmap showing the relative abundance of KOs across samples. Rows are individual KOs and columns are frog samples. Dendogram at the bottom indicates clustering of samples based on Bray-Curtis distances. Colors indicate the relative abundance (proportions) of KOs across samples (see color legend on the right hand side of the figure).

Figure 3

Contig level analysis. Gene abundance stacked graphs of five functional categories from KEGG that are involved in bacterial communication and bacterial-host-pathogen interactions. Each category represents the gene abundance for Sapo (N = 3) and Sob (N = 3) sites.

Figure 4

Contig level analysis. Gene abundance plots of significant pathways enriched in Sob or Sapo sites within five functional classes from KEGG involved in bacterial communication and bacterial-host-pathogen interactions. Each bar represents the gene abundances for Sapo (N = 3) and Sob (N = 3) sites. Colors indicate the taxonomic assignment.