A bacterial toxin-antitoxin module is the origin of inter-bacterial and inter-kingdom effectors of Bartonella

Abstract

Host-targeting type IV secretion systems (T4SS) evolved from conjugative T4SS machineries that mediate interbacterial plasmid transfer. However, the origins of effectors secreted by these virulence devices have remained largely elusive. Previous work showed that some effectors exhibit homology to toxins of bacterial toxin-antitoxin modules, but the evolutionary trajectories underlying these ties had not been resolved. We previously reported that FicT toxins of FicTA toxin-antitoxin modules disrupt cellular DNA topology via their enzymatic FIC (filamentation induced by cAMP) domain. Intriguingly, the FIC domain of the FicT toxin VbhT of Bartonella schoenbuchensis is fused to a type IV secretion signal-the BID (Bep intracellular delivery) domain-similar to the Bartonella effector proteins (Beps) that are secreted into eukaryotic host cells via the host-targeting VirB T4SS. In this study, we show that the VbhT toxin is an interbacterial effector protein secreted via the conjugative Vbh T4SS that is closely related to the VirB T4SS and encoded by plasmid pVbh of B. schoenbuchensis. We therefore propose that the Vbh T4SS together with its effector VbhT represent an evolutionary missing link on a path that leads from a regular conjugation system and FicTA toxin-antitoxin modules to the VirB T4SS and the Beps. Intriguingly, phylogenetic analyses revealed that the fusion of FIC and BID domains has probably occurred independently in VbhT and the common ancestor of the Beps, suggesting parallel evolutionary paths. Moreover, several other examples of TA module toxins that are bona fide substrates of conjugative T4SS indicate that their recruitment as interbacterial effectors is prevalent and serves yet unknown biological functions in the context of bacterial conjugation. We propose that the adaptation for interbacterial transfer favors the exaptation of FicT and other TA module toxins as inter-kingdom effectors and may thus constitute an important stepping stone in the evolution of host-targeted effector proteins.

Fig 1. BtrFicTA is a regular FicTA module closely related to VbhTA.

(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 Bartonella elizabethae (BelFicT; UniParc identifier UPI00026E5C06) and Bartonella birtlesii (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 [34]. 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 E. coli 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 E. coli that had expressed different VbhT or BtrFicT constructs. Reactions were set up by adding [α-³²P]-ATP to trace AMPylation and lysates of E. coli that expressed GST-fusions of E. coli GyrB (DNA gyrase B subunit), E. coli ParE (topo IV B subunit), or a vector control. The autoradiograph shows VbhT and BtrFicT auto-AMPylation (red arrows), AMPylation of endogenous E. coli 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 S1 Text).

Fig 2. BtrFicTA and related FicTA modules are encoded with deteriorated conjugative T4SS machineries.

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 B. tribocorum and its orthologs are encoded directly downstream of the T4SS machineries (annotated as Vbl T4SS for “VirB-like”).

Fig 3. pVbh encodes a multitude of TA modules but lacks any recognizable cargo genes.

All genes on pVbh of B. schoenbuchensis R1 (genbank accession number CP019790.1) were manually annotated and categorized as belonging to the Vbh conjugation system (green; see also Fig 2), plasmid replication and partitioning functions (light blue), TA modules (red), or as encoding proteins of unknown function with (light pink) or without (dark pink) known protein domains. The fourteen predicted TA modules and four orphan antitoxins as well as the loci encoding the Vbh T4SS (VbhB2-11), Dtr functions (TraA-D), and T4CP (TraG) were highlighted in detail. Annotations of every gene on pVbh are available in S1 Fig.

Fig 4. pVbh as a conjugative replicon with composite toxin VbhT.

(A) Filter matings of B. schoenbuchensis R1 donors and B. henselae recipients revealed that pVbh conjugates at a frequency of ca. 1% per donor. Conjugative transfer depended on a functional Vbh T4SS (vbhB4 mutant) and its Dtr functions (traA mutant). Data points and error bars represent mean and standard deviation of at least three independent experiments. (B) The origin of conjugative DNA transfer (oriT, orange bar) on pVbh was inferred by comparison to closely related rhizobial plasmids where this sequence and the actual site of relaxase cleavage (nic) had been experimentally determined [16]. All these plasmids invariably encode oriT between the traA relaxase and the traCD relaxasome components in the dtr region (see Fig 2). (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 Bartonella and a C-terminal secretion signal virtually identical to homologous sequence of the TraA relaxase.

Fig 5. A novel variant of CRAfT detects the interbacterial transfer of VbhT.

(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; E. coli matings) or pVbh (gentamicin resistance; Bartonella 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 E. coli K-12 BW25113 (lacking EcoKI) or E. coli 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 B. schoenbuchensis with B. henselae 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 loxP sites.

Fig 6. TA module toxins as bona fide substrates for conjugative type IV secretion.

(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 [21]; see also S3A–S3C Fig). 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 S3B Fig. FicT of M. extorquens (UniProt identifier C7CN81 (C7CN81_METED)) is shown as a candidate T4SS substrate because it carries a BID-like sequence at its C-terminus (S3C Fig). (B) The genetic arrangement of vbhAT at the Dtr locus of pVbh of B. schoenbuchensis R1 was compared to the locus encoding the FicTA module on p1METDI of M. extorquens DM4 (genbank accession number NC_012987). (C) The illustration shows how a PezTA module on Chelativorans 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.

Fig 7. Working model: A VbhT-like interbacterial effector as a missing link in the evolution of Beps from FicT toxins.

The model illustrates the homology of genes associated with different T4SS machineries ranging from a regular conjugation system (represented by the AvhB T4SS of A. tumefaciens, bottom) to a host-targeting, effector secreting virulence factor (VirB T4SS of B. henselae, 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 B. schoenbuchensis 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 Bartonella effectors [11]. Based on the genomic islands with Vbh-like T4SS and FicTA modules like BtrFicTA (Fig 4), 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 (Fig 6). 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.