Subtle variation within conserved effector operon gene products contributes to T6SS-mediated killing and immunity

Abstract

Type VI secretion systems (T6SS) function to deliver lethal payloads into target cells. Many studies have shown that protection against a single, lethal T6SS effector protein requires a cognate antidote immunity protein, both of which are often encoded together in a two-gene operon. The T6SS and an effector-immunity pair is sufficient for both killing and immunity. HereIn this paper we describe a T6SS effector operon that differs from conventional effector-immunity pairs in that eight genes are necessary for lethal effector function, yet can be countered by a single immunity protein. In this study, we investigated the role that the PefE T6SS immunity protein plays in recognition between two strains harboring nearly identical effector operons. Interestingly, despite containing seven of eight identical effector proteins, the less conserved immunity proteins only provided protection against their native effectors, suggesting that specificity and recognition could be dependent on variation within an immunity protein and one effector gene product. The variable effector gene product, PefD, is encoded upstream from pefE, and displays toxic activity that can be countered by PefE independent of T6SS-activity. Interestingly, while the entire pef operon was necessary to exert toxic activity via the T6SS in P. mirabilis, production of PefD and PefE alone was unable to exert this effector activity. Chimeric PefE proteins constructed from two P. mirabilis strains were used to localize immunity function to three amino acids. A promiscuous immunity protein was created using site-directed mutagenesis to change these residues from one variant to another. These findings support the notion that subtle differences between conserved effectors are sufficient for T6SS-mediated kin discrimination and that PefD requires additional factors to function as a T6SS-dependent effector.

Fig 1. Organization of and requirement for multiple genes within T6SS effector operons in P. mirabilis.

(A) T6SS effector operons in P. mirabilis HI4320 all include hcp, vgrG, and between five and nine additional genes. (B) Organization of the hcp-vgrG1 primary effector (pef) operon in P. mirabilis HI4320. Position of insertion mutations are indicated by triangles. (C) Schematic of the predicted nuclease toxin PefD. The position of the conserved domains are indicated; PAAR, Phasin2, and Tox GHH2. (D) Parental wild-type P. mirabilis HI4320 forms a Dienes line with pefE mutant bacteria (black arrows) and mutants lacking single genes pefD, pefE (9C1), pefF (12B5), or pefG each fail to form the boundary with the immunity mutant (center). (E) Complementation restores T6SS-dependent boundary formation, indicating the requirement for multiple T6SS genes for effector function. In (A) and (B) predicted conserved domains are color-coded and include DUF2169, DUF4150, DUF4123, PAAR-containing domains, lipoprotein, lipase, and RHS domains. The known immunity-encoding gene pefE is in yellow.

Fig 2. PefD and PefE can function as a toxin/anti-toxin independent of T6SS activity.

(A) Introduction of the entire pef operon from strain HI4320 into P. mirabilis BB2000 is sufficient to form a Dienes line with wild-type parental strain BB2000 (black arrow), while PefD and PefE alone are not sufficient for T6SS-dependent effector activity in P. mirabilis BB2000 and produce a result identical to the lack of a boundary between isogenic swarms of wild-type BB2000 (white arrows). (B) Serial dilutions of E. coli containing PefD and PefE, or the predicted nuclease domain of PefD (PefD-Tox) and PefE, on LB agar (uninduced), agar containing IPTG to induce PefD or PefD-Tox, and agar containing IPTG and L-arabinose to additionally induce PefE. Toxicity is observed when PefD or PefD-Tox are induced in the absence of PefE induction.

Fig 3. P. mirabilis HI4320 encodes two functionally distinct PefE proteins.

(A) The primary effector operon; hcp, vgrG, pefABCDEFG, in HI4320 (grey arrows). Downstream genes from the pef operon include a second copy of pefE (pefE2, black arrow) located next to two transposase pseudogenes (PMI0759 and PMI0760) and a recombination hot-spot (rhs) gene fragment (PMI0761) depicted as blue arrows. (B) Alignment of HI4320 PefE and PefE2 predicted amino acid sequences. Amino acids shaded in red indicate residue similarities (284/320 amino acids, 89% identity) between HI4320 PefE and PefE2. (C) Dienes line formation is observed under uninduced conditions (-) with 9C1 containing empty vector, 9C1 containing pefE, and 9C1 containing pefE2 against parent strain, HI4320. Following induction on 10 mM L-arabinose (+), expression of pefE2 in 9C1 does not complement the immunity defect caused by disruption of pefE (black arrow) against parent strain HI4320. Only pefE expressed in 9C1 restores immunity (white arrow) against HI4320. (D) Western blot of 6xHIS tagged PefE and 6xHIS tagged PefE2 expressed during growth in LB broth with (+) and without (-) 10 mM L-arabinose.

Fig 4. Comparison of protein sequences of PefE2 of P. mirabilis HI4320 and PefE of BA6163 shows high level of conservation.

(A) Amino acid sequence alignments indicate that PefE of BA6163 is more similar to PefE2 than PefE of HI4320 with 315/320 shared amino acids (98% identity). Amino acids shaded in red indicate residue similarities between BA6163 PefE and HI4320 PefE2 that differ from PefE of HI4320. (B) Arrows represent the gene products of the primary effector operon in HI4320 and BA6163. Percent identity of the amino acid sequences comparison and number of conserved amino acids are listed for each protein. PefE (shaded black) has the lowest percent identity, 89.6%, of the two strains. (C) Following induction on 10 mM L-arabinose, 9C1 containing the BA6163 pef operon (Pef_BA) was unable to complement 9C1 immunity resulting in a visible Dienes line (black arrow) with parent strain, HI4320, while 9C1 containing the HI4320 pef operon (Pef_HI) complements immunity against HI4320 (white arrow). The boxed area in (C) indicates that 9C1 containing Pef_HI and Pef_BA are restored for killing and are not immune to one another.

Fig 5. The pef operon is sufficient to create new Dienes-types in P. mirabilis BB2000 and immunity can be conferred by the cognate PefE immunity protein.

(A) Introduction of the entire HI4320 pef operon (Pef_HI) or the BA6163 pef operon (Pef_BA) into isolate BB2000, which lacks a pef operon, creates strains that form Dienes lines with parental BB2000 (black arrows). A Dienes line also forms between BB2000 containing the HI4320 pef operon and BB2000 containing the BA6163 pef operon (white arrow) because they encode different pefE alleles. (B) Dienes line formation is not observed between BB2000 containing the same pef operon (black arrows). (C) Expression of pefE2 provided immunity for BB2000 against BB2000 containing the BA6163 pef operon (Pef_BA) (black arrow). BB2000 containing pefE2 is not immune to BB2000 containing the HI4320 pef operon (Pef_HI). (D) BB2000 expressing pefE is immune to BB2000 containing the HI4320 pef operon (black arrow), while pefE does not provide immunity against BB2000 containing the BA6163 pef operon.

Fig 6. Chimeric immunity proteins constructed from PefE and PefE2 reveal amino acid residues responsible for specificity.

(A) P. mirabilis HI4320 pefE (black) and pefE2 (grey) were PCR-amplified to construct chimeric immunity proteins to determine regions within PefE and PefE2 responsible for immunity function. (B) Nine chimeras (C1-C9) composed of partial gene fragments from pefE and pefE2 were created using conserved restriction sites (XbaI, BamHI, NspI, EcoRI, and EcoRV) located within the genes and ligated into pBAD-MycHisA. The amino acid sequence number of the 3’ end pefE or pefE2 gene fragment is indicated below each restriction enzyme site and also depicts the point of fragment ligation. (C) Following induction by 10 mM L-arabinose, 9C1 expressing pefE restored immunity function against parent strain HI4320 (no Dienes line) in contrast to 9C1 containing pBAD empty vector or 9C1 expressing pefE2, which were unable to complement the immunity defect and thus result in formation of a Dienes line. Chimeric immunity proteins C2, C3, C4, C7, and C9 restored immunity against HI4320 (no Dienes line; white arrows). (D) Amino acid sequences of residues 130–150 from PefE and PefE2 are highly conserved except for residues 137, 138, and 140 suggesting that these residues are responsible for specificity of immunity.

Fig 7. Alignment of PefE immunity proteins identifies five variable regions.

(A) Alignment of the 320-amino acid PefE from 16 P. mirabilis isolates identified conserved regions (black) interspersed with 5 variable regions (VR1, VR2, VR3, VR4, VR5) that were less conserved. Within each variable region, 2–3 variants were identified. Assembly of these variant modules led to 7 versions of the PefE immunity protein among 16 clinical isolates. Three of these isolates encode 2 versions of PefE (HI4320, BA6163, PL105). (B) Phyre-predicted 3D structure of PefE is depicted as a ribbon diagram with the 5 variable regions superimposed and visualized by color. Variable regions predominate on one ‘side’ of the PefE model which adopts a beta-sheet rich bladed propeller fold. The colors in (B) correspond to the labeled variable regions from (A). (C) Space-filling model of PefE generated by PyMOL. The colors in (C) correspond to the labeled variable regions from (A and B). (D) Kin recognition is dependent on three amino acid residues from VR3. PefE from P. mirabilis isolate HU1069 was cloned and expressed in 9C1 (PefE_HU1069) under non-inducing conditions (-) and induced with 10 mM L-arabinose (+).