Differential processing of VesB by two rhomboid proteases in Vibrio cholerae

Abstract

Rhomboid proteases are universally conserved and facilitate the proteolysis of peptide bonds within or adjacent to cell membranes. While eukaryotic rhomboid proteases have been demonstrated to harbor unique cellular roles, prokaryotic members have been far less characterized. For the first time, we demonstrate that Vibrio cholerae expresses two active rhomboid proteases that cleave a shared substrate at distinct sites, resulting in differential localization of the processed protein. The rhomboid protease rhombosortase (RssP) was previously found to process a novel C-terminal domain called GlyGly-CTERM, as demonstrated by its effect on the extracellular serine protease VesB during its transport through the V. cholerae cell envelope. Here, we characterize the substrate specificity of RssP and GlpG, the universally conserved bacterial rhomboid proteases. We show that RssP has distinct cleavage specificity from GlpG, and specific residues within the GlyGly-CTERM of VesB target it to RssP over GlpG, allowing for efficient proteolysis. RssP cleaves VesB within its transmembrane domain, whereas GlpG cleaves outside the membrane in a disordered loop that precedes the GlyGly-CTERM. Cleavage of VesB by RssP initially targets VesB to the bacterial cell surface and, subsequently, to outer membrane vesicles, while GlpG cleavage results in secreted, fully soluble VesB. Collectively, this work builds on the molecular understanding of rhomboid proteolysis and provides the basis for additional rhomboid substrate recognition while also demonstrating a unique role of RssP in the maturation of proteins containing a GlyGly-CTERM.

Importance: Despite a great deal of insight into the eukaryotic homologs, bacterial rhomboid proteases have been relatively understudied. Our research aims to understand the function of two rhomboid proteases in Vibrio cholerae. This work is significant because it will help us better understand the catalytic mechanism of rhomboid proteases as a whole and assign a specific role to a unique subfamily whose function is to process a subset of effector molecules secreted by V. cholerae and other pathogenic bacteria.

Keywords: T2SS; Vibrio cholerae; posttranslational modification; proteases; protein secretion; rhomboid; type II secretion system.

Fig 1

RssP uniquely targets active VesB to the cell surface and outer membrane vesicles. (A) Cell and supernatant fractions from overnight cultures of WT and indicated rhomboid mutant strains with empty vector (p) or plasmid encoding active or inactive (S > A) rhomboid proteases RssP or GlpG were assessed for natively expressed serine protease activity assayed against the fluorogenic peptide Boc-Gln-Ala-Arg-AMC. Data represent mean ± SD of n = 3 experiments. The same samples were also run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane, and blotted with anti-VesB antibodies. Arrows indicate active VesB with its propeptide removed (single arrow or bottom) and inactive VesB (top). (B) Cell and supernatant fractions from overnight cultures of WT and the double rhomboid mutant strain with empty vector (p) or plasmid encoding active rhomboid proteases RssP or GlpG were assessed for natively expressed serine protease activity assayed against the fluorogenic peptide Boc-Gln-Ala-Arg-AMC. Data represent mean ± SD of n = 3 experiments. The same samples were also run on SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with anti-VesB antibodies. Arrows indicate active VesB with its propeptide removed (single arrow or bottom) and inactive VesB (top). (C) VesB was ectopically overexpressed (50 µM) in the ΔvesABC mutant strain and in the same mutant strain with either glpG deleted (ΔglpG) or rssP disrupted (rssP::kan). Cell fractions were isolated after growth in M9 media supplemented with casamino acids and glucose, and the surface exposed VesB was probed by incubating with anti-VesB followed by incubation with goat anti-rabbit IgG coupled with Alexa Fluor 488 and scanning for fluorescence emission intensity in 96-well format. Fold change was determined by dividing the fluorescence intensity produced by VesB-expressing strains over control strains containing empty vector (mean ± SD of n = 3). (D) Filtered supernatants from overnight cultures of WT, ΔvesABC, ΔglpG, or rssP::kan mutant strains were subjected to high-speed centrifugation to separate crude outer membrane vesicles from the cleared supernatant. The pelleted fraction was resuspended in Luria-Bertani and subjected to serine protease activity determination and Western blotting as described in panel A (mean ± SD of n = 3). For the blot, 10 times the concentration compared to fractions assessed for activity was analyzed. As a control, samples were also blotted for OmpU (lower). (E) VesB was ectopically overexpressed (50 µM isopropyl β-d-1-thiogalactopyranoside [IPTG]) in the ΔvesABC mutant strain with and without glpG deleted (ΔglpG) or rssP disrupted (rssP::kan). Culture supernatants were assayed for activity using the same fluorogenic peptide as in panel A (mean ± SD of n = 3). Supernatants were also probed for VesB using anti-VesB antibodies. Arrow indicates active VesB. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by one-way ANOVA analysis with Dunnett’s multiple comparisons test (A and B) and one-way ANOVA analysis with Tukey’s multiple comparisons test (C–E). Representative Western blots are shown from at least two blots performed on biological replicas.

Fig 2

RssP cleaves VesB after glycine 383 and requires both a helix kinking residue and conserved leucine residue with the TMD of VesB. (A) Schematic of VesB is shown with a focus on the GlyGly-CTERM domain. GlpG and RssP cleavage sites are indicated as determined in this study. The disordered loop, transmembrane domain, and positively charged tail are labeled along with important amino acids for RssP-mediated cleavage. (B) A predicted interaction of RssP (gray) and VesB (blue) was generated using AlphaFold2. RssP and the GlyGly-CTERM of VesB are shown, including the active site residues (yellow) and TMDs of RssP and the GlyGly signal (red) of VesB. (C) A close-up of the predicted structure in panel B shows the active site of RssP with VesB threaded through. Predicted alignment error is shown in Fig. S3A. (D) Overnight cultures of the mutant strain ΔvesABC containing empty vector (p) or plasmids encoding WT VesB or VesB with di-alanine (AA) and di-tyrosine (YY) substitutions of the di-glycine motif were separated into cell and supernatant fractions and assessed for serine protease activity using the fluorogenic peptide substrate Boc-Gln-Ala-Arg-AMC. Data represent mean ± SD of n = 3 experiments. Fractions were also run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane, and blotted with anti-VesB antibodies. (E) Cultures of the mutant strain ΔvesABCΔglpG containing empty vector (p) or plasmids encoding VesB constructs were separated into cell and supernatant fractions and run on SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with anti-VesB antibodies. (F) VesB with G389V and P395 substitutions were compared to WT VesB and similarly processed as in panel D (mean ± SD of n = 3). (G) VesB with valine substitutions in the leucine-rich region was compared to WT VesB and processed as in panel D (mean ± SD of n = 3). Arrows indicate active (bottom) and inactive (top) VesB. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test. Representative Western blots are shown from at least two blots performed on biological replicas.

Fig 3

The highly conserved “SGGS” motif within the GlyGly-CTERM is sensitive to substitutions. Cultures from individual colonies of ΔvesABC ectopically expressing either WT VesB or VesB variants were grown overnight in Luria-Bertani without induction in a 96-well format. Wells were directly assessed for serine protease activity using the fluorogenic peptide substrate Boc-Gln-Ala-Arg-AMC without separating cells and culture supernatants. First, 24 colonies each of ΔvesABC ectopically expressing either VesB or the active site mutant (VesB-S221A) were tested (WT). Each data point represents the activity from an overnight culture of an individual colony. Mean (12.0 pmol AMC/min/OD) ± SD (1.25 pmol/min/OD) for the 24 cultures expressing WT VesB is shown and indicated with dotted lines. Next, a library of VesB plasmids was generated with degenerate primers “NNK” at the indicated amino acid position before conjugation into the ΔvesABC mutant strains where cultures of individual colonies were grown and processed for activity (indicated mutant). Each point represents a culture from an individual colony, and the activity is represented as a percentage of the average activity of WT VesB expressed in the ΔvesABC strain. The dotted line indicates four standard deviations below the normalized 100% WT VesB activity. For each amino acid position, cultures of 100 colonies were analyzed.

Fig 4

GlpG cleaves VesB within the disordered loop of the GlyGly-CTERM when RssP is absent. (A) Predicted AlphaFold2 interaction of GlpG (gray) with VesB (blue) (upper). The TMDs and catalytic residues (yellow) of GlpG are indicated as well as the GlyGly signal of VesB (red). (B) A close-up of panel A showing the GlpG active site and VesB disordered loop is shown (lower). Predicted alignment error is shown in Fig. S3C. (C) Supernatants from cultures of either ΔvesABC, ΔvesABCΔglpG, and ΔvesABC rssP::kan strains containing pVesB or pVesBΔ373–379, which codes for a VesB construct with residues F375-A379 deleted, were run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane, and blotted with anti-VesB antibodies. The arrow indicates active VesB. (D) Supernatants from cultures induced with 50 µM IPTG of the ΔvesABC rssP::kan mutant strain containing either empty vector, pVesB, or indicated plasmids with VesB substitutions were assessed for serine protease activity using the fluorogenic peptide Boc-Gln-Ala-Arg-AMC control (mean ± SD of n = 3). *P < 0.05, **P < 0.005, and ***P < 0.0005 by one-way ANOVA with Dunnett’s multiple comparisons test. Fractions were also run on SDS-PAGE, transferred to a nitrocellulose membrane, and blotted with anti-VesB antibodies. Arrows indicate active (bottom) and inactive (top) VesB. (E) Supernatants from cultures induced with 50 µM IPTG of the ΔvesABC rssP::kan mutant containing empty vector, pVesB, and plasmids with VesB A379E or A379D substitutions were probed by Western blotting. (F) Samples are similarly prepared as in panel D but VesB F375A and F375I substitutions are shown. The arrow indicates active VesB. Representative Western blots are shown from at least two blots performed on biological samples. (G) The GlyGly-CTERM from VesA, B, and C were aligned to identify possible GlpG-cleavage sites in VesA and VesC. Each GlyGly-CTERM was blasted using the NCBI clustered database where all sequences were extracted, and a sequence logo of the desired positions was generated. VesB P4-P2′ cleavage positions of GlpG are listed. The equivalent residues are shown for VesA and VesC.

Fig 5

The TMD of VesB drives substrate recognition and protein targeting by the rhomboid proteases in V. cholerae. Culture supernatants of either ΔvesABC (A) or ΔvesABCΔglpG (B) strains containing pVesB or pVesB-HybA, which codes for a VesB construct with the TMD residues of VesB swapped for those of the TMD of HybA from S. sonnei, were assessed for serine protease activity using the fluorogenic peptide Boc-Gln-Ala-Arg-AMC. Data represent mean ± SD of n = 3 experiments. Fractions were also run on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and blotted with anti-VesB antibodies. (C) Supernatants from cultures induced with 10 µM IPTG of ΔvesABC mutant strain containing empty vector (p), pVesB, pVesB-YY, pVesBΔ20, or pVesB-HybA were sterile filtered and subjected to high-speed centrifugation to separate crude outer membrane vesicles from the cleared supernatant. The pelleted fractions were resuspended in Luria-Bertani and subjected to serine protease activity and Western blotting as described in panel A (mean ± SD of n = 3). Pelleted fractions were loaded at 10 times the concentration of total supernatant. As a control, samples were also blotted for OmpU (bottom). *P < 0.05, ***P < 0.0001, and ****P < 0.0001 by one-way ANOVA with Dunnett correction comparison to WT. Representative Western blots are shown from at least two blots performed on biological replicas.

Fig 6

Biogenesis of VesB in V. cholerae. With the aid of its signal peptide, VesB is first transported from the cytoplasm to the periplasm via the Sec pathway (not shown). The GlyGly-CTERM anchors VesB to the inner membrane (IM) where rhombosortase (RssP) cleaves after G383 resulting in a posttranslational modification of the newly generated C-terminus denoted by the black cylinder addition. Modified VesB is trafficked to the extracellular space by the T2SS followed by surface retention and autoactivation denoted by the color change. A fraction of VesB is then released to the extracellular space either as a soluble protein or in association with outer membrane vesicles. Cleavage by RssP requires both a conserved leucine residue and a helix-breaking residue, proline in the TMD. Alternatively, in the absence of RssP, GlpG (or a VesB chimera that contains the TMD from HybA) cleaves VesB at an alternate site resulting in an inactive VesB variant that is freely released to the extracellular space following translocation by the T2SS. Autoactivation of GlpG-cleaved VesB can occur with increased VesB expression or accumulation (denoted by the color change). In the absence of either rhomboid protease, VesB remains cell associated, likely trapped in the inner membrane where it is subject to degradation indicated by an X.