Parallel evolution of a type IV secretion system in radiating lineages of the host-restricted bacterial pathogen Bartonella

Abstract

Adaptive radiation is the rapid origination of multiple species from a single ancestor as the result of concurrent adaptation to disparate environments. This fundamental evolutionary process is considered to be responsible for the genesis of a great portion of the diversity of life. Bacteria have evolved enormous biological diversity by exploiting an exceptional range of environments, yet diversification of bacteria via adaptive radiation has been documented in a few cases only and the underlying molecular mechanisms are largely unknown. Here we show a compelling example of adaptive radiation in pathogenic bacteria and reveal their genetic basis. Our evolutionary genomic analyses of the α-proteobacterial genus Bartonella uncover two parallel adaptive radiations within these host-restricted mammalian pathogens. We identify a horizontally-acquired protein secretion system, which has evolved to target specific bacterial effector proteins into host cells as the evolutionary key innovation triggering these parallel adaptive radiations. We show that the functional versatility and adaptive potential of the VirB type IV secretion system (T4SS), and thereby translocated Bartonella effector proteins (Beps), evolved in parallel in the two lineages prior to their radiations. Independent chromosomal fixation of the virB operon and consecutive rounds of lineage-specific bep gene duplications followed by their functional diversification characterize these parallel evolutionary trajectories. Whereas most Beps maintained their ancestral domain constitution, strikingly, a novel type of effector protein emerged convergently in both lineages. This resulted in similar arrays of host cell-targeted effector proteins in the two lineages of Bartonella as the basis of their independent radiation. The parallel molecular evolution of the VirB/Bep system displays a striking example of a key innovation involved in independent adaptive processes and the emergence of bacterial pathogens. Furthermore, our study highlights the remarkable evolvability of T4SSs and their effector proteins, explaining their broad application in bacterial interactions with the environment.

Figure 1. Phylogeny of Bartonella based on a genome-wide dataset.

Maximum likelihood analysis using an alignment of 478 genes (515,751 nt) of ten sequenced Bartonella species (indicated by bold and color type) and Brucella abortus. Based on sequence data from rpoB, gltA, ribC, and groEL genes, additional Bartonella species were included in the analysis. Numbers above the branches represent maximum likelihood bootstraps (100 replicates); numbers below represent Bayesian posterior probabilities. Values ≥80% are shown. Lineages harboring the VirB T4SS are shaded in gray; the primary mammalian hosts are indicated for each species. Phylogenetic trees inferred from either the genomic data set excluding non-sequenced bartonellae or the sequences from only rpoB, gltA, ribC, and groEL genes revealed the same four lineages for the Bartonella ingroup (Figure S1). Estimates of average evolutionary divergence over sequence pairs within lineages and between lineages are presented in Table S1. l1, lineage 1.

Figure 2. Gene-wide dn/ds analysis of the core genomes of the two radiating lineages.

dn/ds analysis for lineage 3 (A) and for lineage 4 (B). The mean values plotted on the y-axis were obtained from the dn/ds values of pairs of orthologs. Genes are ordered according to their position in the genome of Bc (A) and Bq (B). Genes with elevated dn/ds values homologous to known colonization factors of Bartonella (based on previously published results , –[45]), are depicted by colors. at, putative autotransporter, bep, Bartonella effector protein, hbp, hemin-binding protein, iba, inducible Bartonella autotransporter, virB, VirB T4SS protein, trw, Trw T4SS protein. Genes encoding hypothetical proteins known to be important for colonization are depicted by open black squares (one in lineage 3, four in lineage 4). In lineage 3, three genes exhibit average dn/ds values ≫1 (marked by crosses). This is explained by the fact that between Br and B1-1C, only non-synonymous mutations but no synonymous mutations have been detected in these three genes. As all three genes revealed ω<1 in the other pair-wise comparisons, they were treated as outliers and excluded from further analysis.

Figure 3. Genomic organization of the virB T4SS and bep gene loci in the two radiating lineages.

Synteny plot of Bc (lineage 3) and Bq (lineage 4) generated in MaGe and genetic organization of the virB T4SS loci. Syntenic relationships comprising at least five genes are indicated by violet and blue lines for genes found on the same or the opposite strand, respectively. The genomic integration sites of virB loci are indicated. Additional bep loci in lineage 3 are marked by arrows. For the depicted genomic loci, orthologous genes are connected via gray boxes. For bep genes, connections are drawn if they belong to the same Bep clade (Figure 4) or if they are top blast hits of one another. The glutamine syntethase I gene (glnA) and its fragments which are flanking the two inverted virB T4SS copies are colored in green. The fragments are indicated by an asterisk.

Figure 4. Gene tree of Bartonella effector proteins.

The maximum likelihood tree is based on the BID domain and the C-terminal extension of all bep genes. Numbers above the branches represent maximum likelihood bootstraps (100 replicates); numbers below Bayesian posterior probabilities. Values ≥80% are shown. The independent clustering of the bep genes from lineage 3 and lineage 4 is indicated by blue and green shading, respectively. Different bep genes are named with locus_tag and gene name (if existing) and they are color-coded species-specifically according to the legend. Bold type highlights phylogenetic branches with dn/ds values >1 (the estimated dn/ds value is indicated in red color type). Sub-clades comprising orthologous bep genes are indicated (Bep clades). In lineage 3, BARCL_0629 and BARCL_0635 are not included in any clade since their phylogenetic position is neither supported by bootstraps nor posterior probabilities. In both lineages, clades of bep genes encoding tandem-repeated tyrosine-phosphorylation motifs are indicated by gray shading and an encircled ‘P’ symbol.

Figure 5. Parallel evolution of Bartonella effector proteins harboring tandem-repeated tyrosine motifs phosphorylated in host cells.

(A) The lineage-specific amplification of effector proteins harboring the ancestral domain structure was followed by secondary recombination events in both lineages. This resulted in the independent emergence of Beps with a derived domain structure (see also Figure S5). In silico predictions for tyrosine-phosphorylation motifs consistently detected high numbers of tandem-repeated phosphorylation sites in independently derived Beps of both lineages (here shown for two Beps of Bep clade 9 [BARRO_80017 of Br and BARCL_1034 of Bc] from lineage 3 and BepD and BepE of Bh from lineage 4). The depicted motifs were identified with NetPhos2.0 with a score ≥0.8 (for predictions of all motifs in lineage 3 see Table S4). The consensus sequence of tandem-repeated motifs (color-coded) is shown for each of the four Beps by WebLogos (http://weblogo.berkeley.edu/logo.cgi). (B) and (C), Immunoprecipitation/Western blot analysis of Beps with predicted tyrosine-phosphorylation motifs ectopically expressed in HEK293T cells. Immunoprecipitations were performed with HA antibody-coated agarose beads. (B) Western blot analysis was performed with anti-phosphotyrosine antibody. (C) Western blot analysis was performed with anti-GFP antibody. The tested constructs correspond to the Beps of Bep clade 9 (BARCL_1034, 70.8 kDa, and BARRO_80017, 113.1 kDa) and BepE of B. henselae (BH13420, 81.4 kDa) shown in (A). HA-GFP was used as a control (28.7 kDa).