The genome of the amoeba symbiont "Candidatus Amoebophilus asiaticus" reveals common mechanisms for host cell interaction among amoeba-associated bacteria

Abstract

Protozoa play host for many intracellular bacteria and are important for the adaptation of pathogenic bacteria to eukaryotic cells. We analyzed the genome sequence of "Candidatus Amoebophilus asiaticus," an obligate intracellular amoeba symbiont belonging to the Bacteroidetes. The genome has a size of 1.89 Mbp, encodes 1,557 proteins, and shows massive proliferation of IS elements (24% of all genes), although the genome seems to be evolutionarily relatively stable. The genome does not encode pathways for de novo biosynthesis of cofactors, nucleotides, and almost all amino acids. "Ca. Amoebophilus asiaticus" encodes a variety of proteins with predicted importance for host cell interaction; in particular, an arsenal of proteins with eukaryotic domains, including ankyrin-, TPR/SEL1-, and leucine-rich repeats, which is hitherto unmatched among prokaryotes, is remarkable. Unexpectedly, 26 proteins that can interfere with the host ubiquitin system were identified in the genome. These proteins include F- and U-box domain proteins and two ubiquitin-specific proteases of the CA clan C19 family, representing the first prokaryotic members of this protein family. Consequently, interference with the host ubiquitin system is an important host cell interaction mechanism of "Ca. Amoebophilus asiaticus". More generally, we show that the eukaryotic domains identified in "Ca. Amoebophilus asiaticus" are also significantly enriched in the genomes of other amoeba-associated bacteria (including chlamydiae, Legionella pneumophila, Rickettsia bellii, Francisella tularensis, and Mycobacterium avium). This indicates that phylogenetically and ecologically diverse bacteria which thrive inside amoebae exploit common mechanisms for interaction with their hosts, and it provides further evidence for the role of amoebae as training grounds for bacterial pathogens of humans.

FIG. 1.

Ultrastructure of “Ca. Amoebophilus asiaticus” 5a2 within its Acanthamoeba host cell. (A) Transmission electron micrograph showing the distribution of “Ca. Amoebophilus asiaticus” in its Acanthamoeba host cell. (B and C) An association of “Ca. Amoebophilus asiaticus” with host cell ribosomes (a selected ribosome is marked by an arrow) is clearly visible. No indications for the presence of a membrane surrounding “Ca. Amoebophilus asiaticus” are evident. Host cell nucleus (“n”) and selected mitochondria (“m”) are labeled. Bar represents 5 μm (A), 500 nm (B), or 200 nm (C).

FIG. 2.

Abundance of proteins with eukaryotic domains in proteomes of “Ca. Amoebophilus asiaticus” and other bacteria. Proteins with eukaryotic domains are most abundant in intracellular bacteria. The percentage of proteins with eukaryotic domains in complete proteomes is shown for selected intracellular and extracellular bacteria, which demonstrates an unprecedented abundance of these proteins in “Ca. Amoebophilus asiaticus”. Note that several proteins contain more than one type of eukaryotic domain. For further details, see Table S7 in the supplemental material.

FIG. 3.

Enrichment of protein domains among proteomes of amoeba-associated bacteria. The percentage of InterPro domains in proteomes of bacteria which have been demonstrated to be able to replicate in amoebae was compared against those of 480 other bacterial proteomes. For the 59 listed domains, P values (Fisher's test with FDR correction) indicate a highly significant enrichment in amoeba-associated bacteria (see Table S9 in the supplemental material). Most of these domains are proposed to be involved in host cell interaction. InterPro domains affiliating with the same eukaryotic domain are shown in the same color: Leucine-rich repeats, dark blue; TPR/SEL1 repeats, orange; Ankyrin repeats, purple; F box, red; U box, light blue. For further details, see Table S10.

FIG. 4.

Phylogenetic relationships of Aasi_1236 and other CDP-diacylglycerol-inositol 3-phosphatidyltransferases. The phylogenetic tree is based on CDP-diacylglycerol-inositol 3-phosphatidyltransferase (EC number 2.7.8.11) amino acid sequences which were aligned using MAFFT; the tree was calculated with MEGA4 using the neighbor-joining algorithm and 1,000× bootstrapping. GenBank accession numbers are indicated. Neighbor-joining bootstrap values higher than 80% are indicated above the respective nodes. In addition, a maximum-likelihood tree was calculated using RAxML with the JTT amino acid substitution model and 1,000× bootstrapping. RAxML likelihood values higher than 80% are shown below the respective nodes. The bar represents 10% estimated evolutionary distance. Bacterial CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase (EC number 2.7.8.5) sequences were used as an outgroup.