A widespread bacterial type VI secretion effector superfamily identified using a heuristic approach

Abstract

Sophisticated mechanisms are employed to facilitate information exchange between interfacing bacteria. A type VI secretion system (T6SS) of Pseudomonas aeruginosa was shown to deliver cell wall-targeting effectors to neighboring cells. However, the generality of bacteriolytic effectors and, moreover, of antibacterial T6S remained unknown. Using parameters derived from experimentally validated bacterial T6SS effectors we identified a phylogenetically disperse superfamily of T6SS-associated peptidoglycan-degrading effectors. The effectors separate into four families composed of peptidoglycan amidase enzymes of differing specificities. Effectors strictly co-occur with cognate immunity proteins, indicating that self-intoxication is a general property of antibacterial T6SSs and effector delivery by the system exerts a strong selective pressure in nature. The presence of antibacterial effectors in a plethora of organisms, including many that inhabit or infect polymicrobial niches in the human body, suggests that the system could mediate interbacterial interactions of both environmental and clinical significance.

Figure 1. Identification of B. thailandensis T6SS-1 substrates

(A–C) Comparison of individual protein abundance in wild-type versus ΔT6SS-1 (A), ΔT6SS-5 (B), and ΔT6S (C) B. thailandensis secretomes. Proteins absent from ΔT6SS -1 are indicated in each panel by filled red spheres. (D) Organization of genes encoding B. thailandensis proteins (boxed red) that specifically require T6SS-1 for export. Color indicates homology.

Figure 2. B. thailandensis BTH_I0068 and BTH_I0069 are a T6S amidase effector–immunity pair

(A) Sequence alignment of conserved catalytic motifs share between BTH_I0068 and characterized cell wall amidase enzymes. SWISS-PROT entry names for the proteins shown are: BTH_I0068 (Q2T2K7_BURTA), Spy_1438 (Q99Z24_STRP1), LytA (LYTA_BACSU), and Tse1 (Q9I2Q1_PSEAE). (B) BTH_I0068 acts as a peptidoglycan amidase with specificity toward the m-DAP-D-alanine DD-bond. Partial HPLC chromatograms of sodium borohydride-reduced soluble E. coli peptidoglycan products resulting from digestion with BTH_I0068 and subsequent cleavage with cellosyl. (C) Growth of E. coli harboring one (top panels) or two (bottom panel) vectors expressing the indicated genes. A dash indicates the empty vector. From left to right are increasing serial ten-fold dilutions. Expression data for this experiment are shown in Figure S2. (D) BTH_I0068 and BTH_I0069 act between cells as a T6S-dependent toxin–immunity pair between B. thailandensis cells. Growth competition assays between the indicated B. thailandensis donor and recipient strains under T6S-conducive conditions. The ΔclpV1 strain is a T6S-deficient control. Asterisks mark significantly different competition outcomes (p < 0.01). Error bars represent ± s.d. n=6.

Figure 3. Identification of a T6S effector superfamily

(A) Overview of shared T6S EI pair properties. Depicted at left are the four bicistrons encoding all characterized EI pairs (this study and (Hood et al., 2010; Russell et al., 2011)). Properties are divided among those shared by all EI pairs (orange), periplasmically-targeted pairs (blue), and amidase pairs (brown). Sequences encoding signal peptides within immunity proteins are represented in blue. (B) Schematic of informatic effector identification workflow. Key steps in the workflow are indicated: 1) filter by constraints depicted in (A), 2) application of structure prediction criteria, 3) expansion by homology searching of the non-redundant nucleotide database. (C) Phylogenetic tree of T6S effectors identified by the methods depicted in A and B. The tree was based on alignment of catalytic motifs (Figure S3). Effectors distribute into four branches, referred to as Families 1–4. The background colors assigned to the families are used henceforth. Critical boostrap values are indicated (n = 100). The tree was rooted using equivalent catalytic motifs of papain-like fold enzymes (Pfam clan, CL0125). Scale bar indicates evolutionary distance in amino acid substitutions per site.

Figure 4. Representatives of Families 3 and 4 are T6S amidase EI pairs

(A) Tae3^TY and Tae4^TM are peptidoglycan amidases with specificity for the m-DAP-D-alanine DD-bond and the γ-D-glutamyl-L-m-DAP bond, respectively. Partial HPLC chromatograms of sodium borohydride-reduced soluble E. coli peptidoglycan products resulting from digestion with Tae3^TY or Tae4^TM and subsequent cleavage with cellosyl. The control sample was digested with cellosyl alone. (B) Simplified representation of Gram-negative peptidoglycan showing cleavage sites of effector families 1–4 (F1-4) based on HPLC data. Cleavage specificity on peptidoglycan with tetrapeptide (left) and pentapeptide (right) stems are depicted. Tse1 activity against pentapeptide-rich peptidoglycan has not been tested, as indicated by yellow stars. Abbreviations: GlcNAc, N-acetyl-glucosamine, MurNAc, N-acetyl-muramic acid. (C) Tae3^TY and Tae4^TM are toxic in the periplasm and this toxicity is rescued specifically by cognate immunity proteins, Tai3^TY and Tai4^TM, respectively. Growth of E. coli harboring one (top panels) or two (bottom panel) vectors expressing the indicated genes. A dash indicates the empty vector. From left to right are increasing serial ten-fold dilutions. Expression data for this experiment are shown in Figure S2. (D) Tae3^PF is secreted in a T6SS-dependent manner. Western blot analysis of supernatant (Sup) and cell-associated (Cell) fractions of the indicated P. fluorescens strains expressing vesicular stomatitis virus glycoprotein (VSV-G) tagged Tae3^PF (Tae3^PF –V).

Figure 5. Immunity proteins display varying non-cognate effector neutralization

(A) Tai3^TY is an exception to a simple cognate effector–immunity protection model. All panels in this figure show the growth of E. coli harboring vectors co-expressing the indicated effector and immunity proteins. Immunity proteins were induced identically in all panels. Error bars represent ± s.d. (n=3). Expression data for all experiments are provided in Figure S2. Asterisks in (A), (B), and (C) indicate immunity proteins that provided significant protection above the empty vector control (p < 0.05). (B) The immunity provided by Tai3^TY against Tae2^BT does not extend to all family 2 effectors. Data demonstrating the catalytic activity of Tae2^TY on peptidoglycan are shown in Figure S5. (C, D) Effector proteins of the same family are not always recognized by all immunity proteins of that family. (C) Co-expression of either Tae2^BT or Tae2^TY with Tai2^BT or Tai2^TY. (D) Co-expression of Tse1^PA with Tsi1^PA or Tsi1^BP with either lower (10μM IPTG) or higher (25μM IPTG) induction of Tse1^PA. Asterisks denote instances in which immunity proteins provided significantly lower protection against Tse1^PA at higher induction levels of the effector (p < 0.05).

Figure 6

T6S cell wall amidase effector and immunity proteins are broadly distributed. Phylogenetic tree depicting the distribution of select effector and immunity proteins in Families 1–4. Trees are based on the 16s rRNA tree of life from Silva’s Living Tree project (http://www.arb-silva.de/projects/living-tree/). For each species all effector and immunity proteins present in any genome are noted, however due to variability at the species level, not all members organisms in the group may have all effector and immunity proteins shown. Additional data concerning the genomic context of effectors and immunity proteins are found in Figure S6.