Analysis of non-typeable Haemophilous influenzae VapC1 mutations reveals structural features required for toxicity and flexibility in the active site

Abstract

Bacteria have evolved mechanisms that allow them to survive in the face of a variety of stresses including nutrient deprivation, antibiotic challenge and engulfment by predator cells. A switch to dormancy represents one strategy that reduces energy utilization and can render cells resistant to compounds that kill growing bacteria. These persister cells pose a problem during treatment of infections with antibiotics, and dormancy mechanisms may contribute to latent infections. Many bacteria encode toxin-antitoxin (TA) gene pairs that play an important role in dormancy and the formation of persisters. VapBC gene pairs comprise the largest of the Type II TA systems in bacteria and they produce a VapC ribonuclease toxin whose activity is inhibited by the VapB antitoxin. Despite the importance of VapBC TA pairs in dormancy and persister formation, little information exists on the structural features of VapC proteins required for their toxic function in vivo. Studies reported here identified 17 single mutations that disrupt the function of VapC1 from non-typeable H. influenzae in vivo. 3-D modeling suggests that side chains affected by many of these mutations sit near the active site of the toxin protein. Phylogenetic comparisons and secondary mutagenesis indicate that VapC1 toxicity requires an alternative active site motif found in many proteobacteria. Expression of the antitoxin VapB1 counteracts the activity of VapC1 mutants partially defective for toxicity, indicating that the antitoxin binds these mutant proteins in vivo. These findings identify critical chemical features required for the biological function of VapC toxins and PIN-domain proteins.

Figure 1. Characteristics of NTHi VapBC1 toxin-antitoxin system in vivo.

A. HMM Logo analysis of 8807 VapC sequences in the PF01850 Pfam family database. The height of letters at each position represents the deviation of that letter's frequency from the background frequency of that letter . B. Diagram of vapC1 or vapBC1 sequences cloned into pBAD-MycHisB plasmids under control of the L-arabinose inducible pBAD promoter. C. Analysis of the growth of LMG194 cells in M9 glycerol with 50 µg/ml ampicillin after induction by addition of L-arabinose to a final concentration of 0.02%. The curves are representative examples of multiple experiments that yielded the same results. D. Growth of LMG194 carrying the indicated plasmids after spotting ten-fold dilutions of cells on LB or LB +0.02% arabinose plates that were incubated at 37°C for 16 hours.

Figure 2. Growth characteristics of VapC1-eGFP fusions.

A. Diagram of vapC1 or vapBC1 sequences cloned as vapC1-eGFP fusions into pBAD-MycHisB plasmids under control of the L-arabinose inducible pBAD promoter. B. Analysis of the growth of LMG194 cells in M9 glycerol with 50 µg/ml ampicillin after induction by addition of L-arabinose to a final concentration of 0.02%. The curves are representative examples of multiple experiments that yielded the same results. C. Western blot analysis of VapC1-eGFP from cells grown as in B after a 30-minute induction with L-arabinose to a final concentration of 0.02%. Each lane contains lysate from an equal number of cells.

Figure 3. Workflow for isolation of VapC1 loss of function mutations.

VapC1 DNA was amplified by PCR with Taq polymerase and inserted in frame with eGFP in pBAD-eGFP-MycHisB using standard DNA cloning techniques. DNA was transformed into Top10 and plated on Luria Broth + ampicillin (50 µg/ml; LBA) plates containing 0.02% L-arabinose. After incubation of the plates at 37°C for 16 hours, colonies were visualized on a Typhoon 9410 imager (excitation at 488 nm; 520BP40 emission filter). Colonies were then screened on LBA plates with and without arabinose, and colonies that grew on both plates and exhibited fluorescence above background were chosen for further analysis.

Figure 4. VapC1 mutations cause a spectrum of toxicity defects.

A. Fluorescence yield from LMG194 cells carrying VapC1 mutants grown in M9 glycerol with 50 µg/ml ampicillin as a function of time after induction with arabinose at a final concentration of 0.02%. Fluorescence was measure in triplicate in a Typhoon 9410 Imager and normalized to the OD₆₀₀ of the culture. B. Western blot analysis of VapC1 mutant proteins isolated 60 minutes after induction with arabinose at a final concentration of 0.02%. Blots were probed with anti-myc antibody for VapC1 proteins and anti-GroEL as a loading control. The first four lanes in each panel show a two-fold dilution series of VapC1-sfGFP as a control for signal linearity. C. Analysis of the growth of LMG194 cells expressing the indicated VapC1 mutants in M9 glycerol with 50 µg/ml ampicillin after induction by addition of L-arabinose to a final concentration of 0.02%. The curves are one set of examples of two biological replicates whose average doubling time and standard error is shown in (D). D. Doubling times for cells expressing VapC1 mutants. Values represent the doubling times calculated for the first 510 minutes of growth and are the average of two independent biological replicates, shown with error bars representing the standard error. E. Analysis of the growth of LMG194 cells expressing the indicated VapC1 mutants in M9 glycerol with 50 µg/ml ampicillin after induction by addition of L-arabinose to a final concentration of 0.02%. Time points on the curves are the average of three biological replicates with error bars representing the standard deviation.

Figure 5. Structural analysis of VapC1 mutations.

A. Position of mutations affecting activity of NTHi VapC1. NTHi VapC1 was modeled to Shigella flexneri VapBC (MMDB ID: 94821; PDB ID: 3TND) using Phyre . Modeling confidence was 100% for 98% of the VapC1 sequence. Arrows indicate mutated NTHi VapC1 side chains. Colors correspond to; white (peptide backbone), grey (amino acid side chains), blue (nitrogen), yellow (sulfur) and red (oxygen). B. Comparison of NTHi VapC proteins with several VapC proteins from the listed species. Asterisks indicate the position of NTHi VapC1 mutations described herein. Black boxes indicate identity in at least four of seven species and gray boxes indicate similarity in at least four of seven species.

Figure 6. N117D suppresses the E120G defect.

A. Comparison of the amino acid sequence of VapC1 and mutants from positions 113 to 120. Mutations are indicated in bold. B. Analysis of the growth of LMG194 cells expressing the indicated alleles of VapC1 in M9 glycerol with 50 µg/ml ampicillin after induction by addition of L-arabinose to a final concentration of 0.02%. Time points on the curves are the average of three biological replicates with error bars representing the standard deviation. C. Western blot analysis of VapC1 mutant proteins isolated 60 minutes after induction with arabinose at a final concentration of 0.02%. Blots were probed with anti-myc antibody for VapC1 proteins and anti-GroEL as a loading control. The first four lanes in the panel show a two-fold dilution series of VapC1-sfGFP as a control for signal linearity.

Figure 7. Ability of antitoxin VapB1 to relieve toxicity of VapC1 mutants.

A. Analysis of the growth of LMG194 cells expressing the indicated alleles of VapC1 in M9 glycerol with 50 µg/ml ampicillin after induction by addition of L-arabinose and/or IPTG to a final concentration of 0.02% and 0.5 mM, respectively. Time points on the curves are the average of two biological replicates with error bars representing the standard error.