Parallel Evolution of a Type IV Secretion System in Radiating Lineages of the Host-Restricted Bacterial Pathogen Bartonella

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

TA module toxins as <i>bona fide</i> substrates for conjugative type IV secretion.

<p>(A) The position of known and proposed T4SS substrates as well as proteins with a Bep-like β-hairpin are highlighted in the phylogeny of FicT toxins (adapted from our previous work [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007077#pgen.1007077.ref021" target="_blank">21</a>]; see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007077#pgen.1007077.s003" target="_blank">S3A–S3C Fig</a>). In short, the tree represents a maximum likelihood phylogeny that had been constructed from an alignment of the toxins’ FIC domains. An additional phylogeny supporting the repeated, independent recruitment of FicT toxins as T4SS substrates is presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007077#pgen.1007077.s003" target="_blank">S3B Fig</a>. FicT of <i>M</i>. <i>extorquens</i> (UniProt identifier C7CN81 (C7CN81_METED)) is shown as a candidate T4SS substrate because it carries a BID-like sequence at its C-terminus (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007077#pgen.1007077.s003" target="_blank">S3C Fig</a>). (B) The genetic arrangement of <i>vbhAT</i> at the Dtr locus of pVbh of <i>B</i>. <i>schoenbuchensis</i> R1 was compared to the locus encoding the FicTA module on p1METDI of <i>M</i>. <i>extorquens</i> DM4 (genbank accession number NC_012987). (C) The illustration shows how a PezTA module on <i>Chelativorans</i> sp. BNC1 plasmid 1 (genbank accession number CP000389.1) is encoded between the conjugative VirB T4SS and its Dtr machinery in the same genetic arrangement as the VbhTA module is encoded on pVbh.</p

A novel variant of CRAfT detects the interbacterial transfer of VbhT.

<p>(A) The scheme outlines basic principles of the new CRAfT variant that we developed as part of this study. Interbacterial protein transfer of Cre fusions is detected through the switch from spectinomycin resistance (purple) to kanamycin resistance (green). Conjugative plasmid transfer can be assayed in parallel (blue) by selection for antibiotic resistance encoded on reporter plasmid pAH188 (chloramphenicol resistance; <i>E</i>. <i>coli</i> matings) or pVbh (gentamicin resistance; <i>Bartonella</i> matings). (B) Conjugative transfer of pAH188 and CRAfT signal for the translocation of a Cre-TraI relaxase fusion through the RP4 T4SS were assayed using <i>E</i>. <i>coli</i> K-12 BW25113 (lacking EcoKI) or <i>E</i>. <i>coli</i> K-12 MG1655 (with functional EcoKI) as recipients. (C) The transfer of Cre fused to the TraA relaxase of pVbh or the catalytically inactive H136A mutant of VbhT were tested in matings of <i>B</i>. <i>schoenbuchensis</i> with <i>B</i>. <i>henselae</i> carrying the CRAfT sensor module; conjugation of pVbh was assayed in parallel. Data points and error bars in (B) and (C) represent mean and standard deviation of three independent experiments. Transconjugants are recipient cells that have received pAH188 (B) or pVbh (C), and recombinants denote recipient cells that switched resistance of the CRAfT sensor due to successful transfer of Cre fusion proteins or, much rarer, spontaneous recombination of the <i>loxP</i> sites.</p

pVbh as a conjugative replicon with composite toxin VbhT.

<p>(A) Filter matings of <i>B</i>. <i>schoenbuchensis</i> R1 donors and <i>B</i>. <i>henselae</i> recipients revealed that pVbh conjugates at a frequency of ca. 1% per donor. Conjugative transfer depended on a functional Vbh T4SS (<i>vbhB4</i> mutant) and its Dtr functions (<i>traA</i> mutant). Data points and error bars represent mean and standard deviation of at least three independent experiments. (B) The origin of conjugative DNA transfer (<i>oriT</i>, orange bar) on pVbh was inferred by comparison to closely related rhizobial plasmids where this sequence and the actual site of relaxase cleavage (<i>nic</i>) had been experimentally determined [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007077#pgen.1007077.ref016" target="_blank">16</a>]. All these plasmids invariably encode <i>oriT</i> between the <i>traA</i> relaxase and the <i>traCD</i> relaxasome components in the <i>dtr</i> region (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007077#pgen.1007077.g002" target="_blank">Fig 2</a>). (C) Domain composition and sequence alignment of the TraA relaxase of pVbh with relaxases of closely related rhizobial conjugation systems. (D) The alignment of BtrFicT, VbhT, and TraA protein sequences shows that VbhT is a composite protein with an N-terminal FIC domain closely related to FicT toxins of <i>Bartonella</i> and a C-terminal secretion signal virtually identical to homologous sequence of the TraA relaxase.</p

BtrFicTA and related FicTA modules are encoded with deteriorated conjugative T4SS machineries.

<p>Loci encoding bacterial conjugation systems consist of genes for the T4SS machinery (green), the Dtr machinery (yellow), and a type IV coupling protein (T4CP) that links these functions by recruiting the Dtr machinery to the T4SS (blue). Like VbhTA on pVbh, BtrFicTA of <i>B</i>. <i>tribocorum</i> and its orthologs are encoded directly downstream of the T4SS machineries (annotated as Vbl T4SS for “VirB-like”).</p

List and construction of all bacterial strains of this study.

<p>List and construction of all bacterial strains of this study.</p

Working model: A VbhT-like interbacterial effector as a missing link in the evolution of Beps from FicT toxins.

<p>The model illustrates the homology of genes associated with different T4SS machineries ranging from a regular conjugation system (represented by the AvhB T4SS of <i>A</i>. <i>tumefaciens</i>, bottom) to a host-targeting, effector secreting virulence factor (VirB T4SS of <i>B</i>. <i>henselae</i>, top). Core components of the T4SS machinery and the T4CP are shown in green, the Dtr machinery is shown in yellow, and TA modules or effectors are shown in red. Note that the VbhTA module of <i>B</i>. <i>schoenbuchensis</i> pVbh (second from top) displays the same domain architecture as the majority of extant Beps and likely their common ancestor. Like VbhT, the most upstream Bep (BepA) is encoded with a FicA antitoxin that is called BiaA in the context host-targeted <i>Bartonella</i> effectors [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007077#pgen.1007077.ref011" target="_blank">11</a>]. Based on the genomic islands with Vbh-like T4SS and FicTA modules like BtrFicTA (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007077#pgen.1007077.g004" target="_blank">Fig 4</a>), we infer an ancient pVbh-like plasmid in which a FicTA module was encoded between the conjugative relaxase and the T4SS machinery (second from bottom). It is clearly apparent that a DNA rearrangement fusing one BID domain of the relaxase with the FicT toxin–i.e., classical terminal reassortment–would create a VbhT-like interbacterial effector as evidenced by the clear composite architecture of this protein (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007077#pgen.1007077.g006" target="_blank">Fig 6</a>). We therefore speculate based on these homologies that an evolutionary process from the bottom to the top of this model may approximate the evolutionary history of the host-targeting VirB T4SS.</p

BtrFicTA is a regular FicTA module closely related to VbhTA.

<p>(A) Sequence alignment of YeFicT (UniProt identifier A1JNF3 (A1JNF3_YERE8)) and the FIC domain of VbhT (UniProt identifier E6Z0R3 (VBHT_BARSR)) with BtrFicT (UniParc identifier UPI00015FA8A2) and orthologs encoded by <i>Bartonella elizabethae</i> (BelFicT; UniParc identifier UPI00026E5C06) and <i>Bartonella birtlesii</i> (BbiFicT; UniParc identifier UPI00026E6E87). The FIC domain core (interpro IPR003812) is highlighted with an orange bar. BtrFicT and its orthologs have > 70% identical sequence and share 60% sequence identity with the FIC domain of VbhT. The four proteins have 28% identical sequence with YeFicT. Note that all sequences display a canonical HPFX[D/E]GNGRXXR FIC signature motif (red frame), indicating AMPylation as their molecular activity [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007077#pgen.1007077.ref034" target="_blank">34</a>]. The coloring indicates amino acid similarity according to the Blosum62 score matrix with black = 100% identity and white = <60% identity. (B) The colony forming units (c.f.u.) / ml of exponentially growing <i>E</i>. <i>coli</i> were recorded over time after the expression of BtrFicT constructs had been induced at t = 0 h with 2 mM of IPTG. Data points represent average and standard deviation of three independent experiments. (C) Autoradiograph of an AMPylation assay with lysates of <i>E</i>. <i>coli</i> that had expressed different VbhT or BtrFicT constructs. Reactions were set up by adding [α-³²P]-ATP to trace AMPylation and lysates of <i>E</i>. <i>coli</i> that expressed GST-fusions of <i>E</i>. <i>coli</i> GyrB (DNA gyrase B subunit), <i>E</i>. <i>coli</i> ParE (topo IV B subunit), or a vector control. The autoradiograph shows VbhT and BtrFicT auto-AMPylation (red arrows), AMPylation of endogenous <i>E</i>. <i>coli</i> GyrB by VbhT (green arrow), and the AMPylation of ectopically expressed GST-GyrB and GST-ParE for both constructs (orange arrows). Note that the “BtrFicT” construct included expression of a marginal amount of BtrFicA which we determined to be necessary for the generation of soluble BtrFicT (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007077#pgen.1007077.s005" target="_blank">S1 Text</a>).</p