Bacterial Competition Systems Share a Domain Required for Inner Membrane Transport of the Bacteriocin Pyocin G from Pseudomonas aeruginosa

Abstract

Bacteria exploit a variety of attack strategies to gain dominance within ecological niches. Prominent among these are contact-dependent inhibition (CDI), type VI secretion (T6SS), and bacteriocins. The cytotoxic endpoint of these systems is often the delivery of a nuclease to the cytosol. How such nucleases translocate across the cytoplasmic membrane of Gram-negative bacteria is unknown. Here, we identify a small, conserved, 15-kDa domain, which we refer to as the inner membrane translocation (IMT) domain, that is common to T6SS and bacteriocins and linked to nuclease effector domains. Through fluorescence microscopy assays using intact and spheroplasted cells, we demonstrate that the IMT domain of the Pseudomonas aeruginosa-specific bacteriocin pyocin G (PyoG) is required for import of the toxin nuclease domain to the cytoplasm. We also show that translocation of PyoG into the cytosol is dependent on inner membrane proteins FtsH, a AAA+ATPase/protease, and TonB1, the latter more typically associated with transport of bacteriocins across the outer membrane. Our study reveals that the IMT domain directs the cytotoxic nuclease of PyoG to cross the cytoplasmic membrane and, more broadly, has been adapted for the transport of other toxic nucleases delivered into Gram-negative bacteria by both contact-dependent and contact-independent means. IMPORTANCE Nuclease bacteriocins are potential antimicrobials for the treatment of antibiotic-resistant bacterial infections. While the mechanism of outer membrane translocation is beginning to be understood, the mechanism of inner membrane transport is not known. This study uses PyoG as a model nuclease bacteriocin and defines a conserved domain that is essential for inner membrane translocation and is widespread in other bacterial competition systems. Additionally, the presented data link two membrane proteins, FtsH and TonB1, with inner membrane translocation of PyoG. These findings point to the general importance of this domain to the cellular uptake mechanisms of nucleases delivered by otherwise diverse and distinct bacterial competition systems. The work is also of importance for the design of new protein antibiotics.

Keywords: P. aeruginosa; antibiotic; bacterial competition; cell envelope; protein import; pyocin.

FIG 1

IMT domain is a conserved structural domain found in multiple orders within Gammaproteobacteria and associated with multiple toxin systems. (A) Structure of the conserved beta-sheets of the IMT domain from colicin D (blue, PDB entry 5ZNM) and a T6SS effector (gold, PDB entry 3MFB). (B) Taxonomy of Gammaproteobacteria within uniref100. Ticks indicate the presence of at least 1 IMT domain-containing protein (blue) and predicted T6SS effector (gold). IMT domain proteins are prevalent in Gammaproteobacteria though restricted to certain orders (unlabeled taxa are Cellvibrionales, gray; Thiotrichales, peach; Pasteurellaceae, purple; Legionellales, green; Aeromonadales, blue; Methylococcales, yellow). (C) Alignment of the IMT domain from bacterial toxins including colicins and other nuclease bacteriocins (colicin E9, colicin D, klebicin B, pyocin S2) and T6SS effectors (ECA1669 protein from Erwinia carotovora, PDB entry 3MFB; Usp, uropathic specific protein). (D) Pfam domains that cooccur in proteins with IMT domain. IMT domain is found in proteins that contain numerous toxin effectors or structural domains. Colicin-DNase, common HNH nuclease found in DNase bacteriocins and T6SS effectors; cytotoxic, rRNase toxin found in bacteriocins and T6SS effectors; Colicin_D, tRNase found in bacteriocins and T6SS effectors; PAAR motif, cloacin, DUF796 (T6SS_HCP), structural motif found in T6SS effectors; E2R135, receptor domain for certain colicins; Colicin_Pyocin, a domain found in the immunity proteins for many DNase bacteriocins. Phage_base_V/Phage_GPD, phage/T6SS structural proteins; Colicin_C, tRNase found in bacteriocins and T6SS effectors; T6SS_Vgr, structural domain of the T6SS; DUF2345, domain of unknown function associated with the T6SS; colicin, pore-forming domain found in many colicins, all proteins identified with this domain had similarity to colicin B, GH-E, HNH family of nucleases. (E) The domain organization of PyoG (5). For comparison, the position of the IMT domain is shown for a T6SS nuclease effector (Usp) and a nuclease colicin (ColE9). The unstructured N terminus of bacteriocins is represented with a dotted line.

FIG 2

Import and localization of fluorescent PyoG constructs. (A) A fluorescence microscopy experiment setup, used for localization of PyoG constructs in P. aeruginosa cells. All constructs are conjugated to AF488 (represented with a green asterisk) via a C-terminal cysteine. Intact cells, or spheroplasts generated by lysozyme/EDTA treatment, are exposed to 2 μM fluorescent PyoG. Unbound and untranslocated pyocin constructs are removed by trypsin treatment. (B) Representative micrographs before and after trypsin treatment are shown. All snapshots were adjusted to the same contrast value. Tested constructs are full-length PyoG, PyoG^1–485 lacking the cytotoxic domain, and PyoG^1–255 lacking the cytotoxic domain and the IMT domain. (C) Average fluorescence intensities were measured for 150 cells per condition. Means from three biological replicates with standard deviations are shown. Fluorescence intensities for labeled and trypsin-treated groups under each condition are compared to the unlabeled control. ****, P value below 0.0001 in the Kruskal-Wallis Test; ns represents no significant difference or lack of fluorescent labeling.

FIG 3

Inner membrane translocation of PyoG requires FtsH and binding to TonB1. PyoG^1–485 is conjugated to AF488 via a C-terminal cysteine and added to P. aeruginosa spheroplasts at 2 μM. Nontranslocated pyocin is removed by trypsin treatment. (A and B) FtsH is required for inner membrane translocation of PyoG. PyoG is trypsin protected in intact ΔftsH cells but not in ΔftsH spheroplasts. This phenotype is complemented with FtsH expressed from a plasmid (pFtsH). No PyoG translocation is measured if the protease activity of FtsH has been disrupted with a point mutation (FtsH H416Y). (C and D) Labeling of ΔtonB1 spheroplasts with fluorescent PyoG. No residual labeling is measured after trypsin treatment, which indicates that TonB1 is required for inner membrane translocation of PyoG. PyoG import into spheroplasts is restored if TonB1 is expressed from a plasmid (pTonB1). (E and F) The TonB binding box of PyoG, located in the unstructured N terminus, is required for PyoG import. PyoG^31–485 lacks the first 30 residues of PyoG and is conjugated to AF488 via a cysteine in the C terminus. This deletion disrupts both the outer and the inner membrane import of PyoG, since the PyoG^31–485 label is not trypsin protected in both intact cells and P. aeruginosa spheroplasts. (A, C, E, and G) Representative micrographs before and after trypsin treatment are shown. All snapshots were adjusted to the same intensity scale. (B, D, F, and H) Average fluorescence intensities were measured for 150 cells per condition. Means from three biological replicates with standard deviations are shown. Fluorescence intensities for labeled and trypsin treated groups under each condition are compared to the unlabeled control. ****, P value below 0.0001 in the Kruskal-Wallis Test; ns represents no significant difference or lack of fluorescent labeling.

FIG 4

Probable import mechanism of nuclease pyocin G and a T6SS effector. (A) PyoG uses Hur, a TBDT, as its outer membrane receptor and translocator (5). The pyocin is composed of an unstructured N terminus (dashed line), a receptor binding domain (R), a conserved inner membrane translocation domain (IMT domain), and a cytotoxic nuclease domain (C). Like in the case of nuclease colicins (29), the immunity protein (Im) probably disassociates from the C domain during translocation. The plug domain of Hur interacts with the periplasmic domain of TonB1 (5). The binding of TonB1 and the pyocin R domain to the TBDT induces a conformational change that dislocates the plug of the TBDT, allowing the passage of the pyocin through the TBDT (10). The TonB box, located in the N terminus of PyoG, is essential for this translocation step, probably because TonB1 pulls PyoG through Hur and into the periplasm (9, 10). (B) Translocation of PyoG from the periplasm into the cytoplasm also requires TonB1 binding. The exact role of TonB1 in inner membrane transport remains unknown. Potentially, TonB1 positions the pyocin on the surface of the inner membrane so it can interact with other proteins involved in inner membrane transport. This translocation step requires the IMT domain, a conserved domain present in all nuclease bacteriocins. Inner membrane transport of PyoG requires FtsH, an inner membrane ATPase/protease previously associated with the killing activity of PyoG (5) and nuclease colicins (10). FtsH must be proteolytically active for PyoG to translocate into the cytoplasm. Inner membrane transport of PyoG could be coupled with FtsH-dependent proteolytic processing that releases the C domain into the bacterial cytoplasm, as previously demonstrated for nuclease colicins (15, 19). (C) Like nuclease bacteriocins, nuclease T6SS effectors contain the IMT domain (C, cytotoxic domain; IMT, inner membrane translocation domain; N, other domains and motifs in the N terminus). The needle and needle tail proteins can deliver these effectors to the cis side of the peptidoglycan layer or directly to the bacterial cytoplasm (26). Inner membrane proteins required for the inner membrane transport of T6SS effectors are currently unknown. It is possible that, as in the case of nuclease PyoG, this translocation step depends on the IMT domain.