c-di-GMP inhibits LonA-dependent proteolysis of TfoY in Vibrio cholerae

Abstract

The LonA (or Lon) protease is a central post-translational regulator in diverse bacterial species. In Vibrio cholerae, LonA regulates a broad range of behaviors including cell division, biofilm formation, flagellar motility, c-di-GMP levels, the type VI secretion system (T6SS), virulence gene expression, and host colonization. Despite LonA's role in cellular processes critical for V. cholerae's aquatic and infectious life cycles, relatively few LonA substrates have been identified. LonA protease substrates were therefore identified through comparison of the proteomes of wild-type and ΔlonA strains following translational inhibition. The most significantly enriched LonA-dependent protein was TfoY, a known regulator of motility and the T6SS in V. cholerae. Experiments showed that TfoY was required for LonA-mediated repression of motility and T6SS-dependent killing. In addition, TfoY was stabilized under high c-di-GMP conditions and biochemical analysis determined direct binding of c-di-GMP to LonA results in inhibition of its protease activity. The work presented here adds to the list of LonA substrates, identifies LonA as a c-di-GMP receptor, demonstrates that c-di-GMP regulates LonA activity and TfoY protein stability, and helps elucidate the mechanisms by which LonA controls important V. cholerae behaviors.

Fig 1. TfoY is significantly enriched in the ΔlonA mutant relative to wild-type.

A volcano plot of proteins enriched in WT and ΔlonA mutant after translational inhibition. The proteomes of WT and ΔlonA strains (n = 5) that had been grown to an OD₆₀₀ = 1.0 and exposed to the translational inhibitor chloramphenicol for 1-hour were analyzed by TMT-labeling and LC-MS/MS. A student’s t-test using a Benjamini-Hochberg FDR cutoff of 5% was used to identify proteins that were statistically significantly enriched. Proteins enriched in ΔlonA relative to WT are shown in pink. Proteins enriched in WT relative to ΔlonA are shown in blue.

Fig 2. TfoY stability depends on the LonA protease.

In vivo stability of TfoY after translational inhibition. TfoY was overproduced from the Tn7 locus in WT and a ΔlonA strain. Stability of TfoY was analyzed by western blot using an antibody against TfoY 1-hour post translational inhibition via chloramphenicol. RNAP was used as a biomass loading control.

Fig 3. LonA represses motility and the T6SS through TfoY.

Quantification of flagellar motility and T6SS killing experiments. For motility assays, single colonies were stabbed into LB soft agar plates (0.3% agar) and incubated at 30°C for approximately 18 hours. (A) Swimming motility phenotypes of WT, ΔfliA (negative control) and various ΔlonA and/or ΔtfoY deletions as well as their complementation strains from the Tn7 site. (B) Overexpression of tfoY from the Ptac promoter in plates with and without IPTG. (C) The T6SS killing phenotypes of various tfoY and lonA deletion mutants as well as their complementation strains were analyzed. T6SS killing was determined by enumerating the survival of E. coli strain MC4100, which is susceptible to T6SS attack. In addition, hcp was included as a negative control for T6SS dependent killing and lonA(S678A) as a control for LonA-dependent proteolysis. (D) Overexpression of tfoY from the Ptac promoter on plates with or without IPTG. Motility and T6SS-dependent killing experiments represent the average and SD of at least three independent experiments. Statistical analysis was performed using an unpaired Student’s t-test. Statistical values indicated are (**p<0.01, ***p < .001, and ****p < .0001). (E) Abundance of natively produced TfoY as well as overexpressed TfoY from the Ptac promoter.

Fig 4. LonA regulates biofilm formation, cellular c-di-GMP levels, and intestinal colonization through TfoY independent mechanisms.

Analysis of biofilm formation by CLSM, cellular c-di-GMP levels by LC-MS/MS, and intestinal colonization by in vivo competition assays. (A) Top-down and orthogonal views of mature biofilms formed by WT, ΔtfoY, ΔlonA, ΔlonAΔtfoY mutants that contained gfp at the Tn7 locus. Scale bars are 40μm. (B) Cellular levels of c-di-GMP in WT, ΔtfoY, ΔlonA, ΔlonAΔtfoY that had been grown to exponential phase (OD₆₀₀ = 0.4) and analyzed for global c-di-GMP by LC-MS/MS. (C) Competitive index of V. cholerae strains. Otherwise WT strain (ΔlacZ) was co-inoculated with the strains indicated at a 1:1 ratio into 5-day old infant mice. The number of bacteria per intestine was determined 20 to 22 h post inoculation. The competitive index (CI) was determined as the output ratio of mutant to WT cells divided by the input ratio of mutant to WT cells per gram of intestine. Statistical analysis for panel B used a One-way ANOVA with Tukey’s post-hoc analysis. Statistical analysis for panel C used Wilcoxon’s signed rank test (*p<0.05, **p<0.01).

Fig 5. TfoY stability is influenced by c-di-GMP.

In vivo abundance and stability of TfoY in high and low c-di-GMP genetic backgrounds relative to WT. (A) Abundance of natively produced TfoY in WT, ΔlonA, a mutant lacking two phosphodiesterases (Δ2PDE; ΔrocSΔcdgJ) as well as a strain lacking four diguanylate cyclases (Δ4DGC; ΔcdgDΔcdgHΔcdgKΔcdgL). (B) Abundance of TfoY in WT, ΔlonA, ΔlonAΔ2PDE, ΔlonAΔ4DGC strains. (C) Abundance of LonA in WT, Δ2PDE, and Δ4DGC strains. (D) TfoY was overproduced from the Tn7 locus in WT, Δ2PDE and Δ4DGC strains. Overproduction of TfoY was achieved via the addition of 0.1mM IPTG for 2 hours. Levels of TfoY were assessed immediately before and after translational inhibition via chloramphenicol. (E) Prior to translational inhibition, 40mLs of culture was spun down and analyzed for global c-di-GMP by LC-MS/MS. A One-way ANOVA using Dunnet’s multiple comparisons test was used for statistical analysis (**p<0.01, ***p < .001). Abundance and stability of TfoY was analyzed by western blot using an αTfoY antibody. RNAP was used as a control for sample loading in all western blots. Levels of LonA were analyzed by western blot using a αLonA antibody. (F) Swimming motility phenotypes of ΔtfoY and ΔlonA deletions in WT, Δ2PDE, and Δ4DGC strains. (G) Overexpression of tfoY from the Ptac promoter in plates with (+) and without (-) IPTG. For motility assays, single colonies were stabbed into LB soft agar plates (0.3% agar) and incubated at 30°C for approximately 18 hours. (H) The T6SS killing phenotypes of ΔtfoY and ΔlonA deletions in WT, Δ2PDE, and Δ4DGC strains. (I) Overexpression of tfoY from the Ptac promoter in liquid culture and on plates (++) relative to uninduced (-). (J) Overexpression of tfoY from the Ptac promoter in liquid culture (+). Cells were then washed to remove the inducer and spotted onto plates lacking IPTG. T6SS-dependent killing was determined by enumerating the survival of E. coli strain MC4100, which is susceptible to T6SS attack. Statistical analysis was performed using an unpaired Student’s t-test. Statistical values indicated are (*p<0.05, **p<0.01, ***p < .001, and ****p < .0001).

Fig 6. LonA proteolysis is directly regulated by c-di-GMP.

In vitro proteolysis and protein ligand binding assays are shown. (A) Proteolysis of FITC-casein by purified LonA results in increased fluorescent signal. (B) Initial rate of substrate degradation as a function of c-di-GMP. (C) ATP hydrolysis for LonA alone and in the presence of c-di-GMP was monitored by loss of NADH, which is consumed stoichiometrically with ATP. (D) Rate of ATP hydrolysis as a function of c-di-GMP. Representative curves for (A) and (C) are shown with different concentrations of c-di-GMP. (E) Quantification of fraction bound of ³²P-c-di-GMP to LonA by DRaCALA. Binding data shown represent the average and SD of triplicate independent experiments. Fraction bound by LonA and Alg44 were compared to buffer control by Student’s t-test. *** indicate p value of 0.0005, respectively. (F) Purified LonA or heat denatured Lon (boiled) were incubated with MANT-c-di-GMP. Representative emission fluorescence spectrum (excitation at 355 nm) is shown. Emission spectrum of MANT-c-di-GMP alone is also shown as a control.

Fig 7. Model of LonA and c-di-GMP regulation of TfoY.

Regulation of tfoY by c-di-GMP takes place at multiple regulatory points. TfoY production is regulated at the transcriptional level via a c-di-GMP binding transcriptional factor; at the post-transcriptional level via a c-di-GMP dependent riboswitch; and the post-translational level via c-di-GMP modulated proteolysis. The upstream regulatory region of tfoY contains 4 promoters. Promoters P1 and P2 (black bent arrows) produce transcript that contains the Vc2 riboswitch (close stem loop), which functions as an off switch when bound by c-di-GMP (yellow circle) and prevents translation [24,26,28]. In contrast, transcripts produced from promoters P3 and P4 (red bent arrows), driven by the c-di-GMP binding transcriptional activator VpsR, do not contain the Vc2 riboswitch [26]. In this model, we present our current understanding of post-transcriptional and post-translational regulation of TfoY (orange circles) by LonA (purple barrel) and c-di-GMP. At high c-di-GMP levels, transcripts without the Vc2 riboswitch accumulate at high levels. Transcript containing the Vc2 riboswitch are not translated. This results in moderate levels of TfoY. At the same time, high levels of c-di-GMP greatly reduce LonA-dependent proteolysis of TfoY, which results in TfoY activation of motility and T6SS-dependent killing. At intermediate c-di-GMP concentrations, transcripts with and without the Vc2 aptamer are present and LonA activity is reduced. This leads to low levels of TfoY production and the activation of motility and T6SS-dependent killing. At low c-di-GMP conditions, transcripts with the Vc2 aptamer are not inhibited by c-di-GMP. In addition, LonA activity is also not repressed by c-di-GMP, thereby leading to increased TfoY degradation. Under these conditions, TfoY activates T6SS-dependent killing but not motility.