Dps-dependent in vivo mutation enhances long-term host adaptation in Vibrio cholerae

Abstract

As one of the most successful pathogenic organisms, Vibrio cholerae (V. cholerae) has evolved sophisticated regulatory mechanisms to overcome host stress. During long-term colonization by V. cholerae in adult mice, many spontaneous nonmotile mutants (approximately 10% at the fifth day post-infection) were identified. These mutations occurred primarily in conserved regions of the flagellar regulator genes flrA, flrC, and rpoN, as shown by Sanger and next-generation sequencing, and significantly increased fitness during colonization in adult mice. Intriguingly, instead of key genes in DNA repair systems (mutS, nfo, xthA, uvrA) or ROS and RNS scavenging systems (katG, prxA, hmpA), which were generally thought to be associated with bacterial mutagenesis, we found that deletion of the cyclin gene dps significantly increased the mutation rate (up to 53% at the fifth day post-infection) in V. cholerae. We further determined that the dpsD65A and dpsF46E point mutants showed a similar mutagenesis profile as the Δdps mutant during long-term colonization in mice, which strongly indicated that the antioxidative function of Dps directly contributes to the development of V. cholerae nonmotile mutants. Methionine metabolism pathway may be one of the mechanism for ΔflrA, ΔflrC and ΔrpoN mutant increased colonization in adult mice. Our results revealed a new phenotype in which increased fitness of V. cholerae in the host gut via spontaneous production nonmotile mutants regulated by cyclin Dps, which may represent a novel adaptation strategy for directed evolution of pathogens in the host.

Fig 1. V. cholerae produces spontaneous nonmotility-related mutations during long-term colonization in adult mice.

(A) Different morphological V. cholerae were found in the intestine of adult mice. We collected the fecal pellets of adult mice at the fifth day post-infection and plated on plates. Unexpectedly found the small, rugose, opacity and dense colony (red arrow), which was different morphology from wild-type V. cholerae large, smooth and transparent colony (black arrow). (B) Adult mice competition assay of small colony variants. We pick several small colony variants from different individual mouse and mixed as a sample, 10⁸ cells of wild-type and small colony variants were mixed in a 1:1 ratio and intragastrically administered to CD-1 adult mice. Fecal pellets were collected from each mouse at the fifth day after infection and plated on selective plates. The competitive index (CI) was calculated as the ratio of small colony variants to wild-type colonies normalized to the input ratio. Horizontal line: median CI. (C) Electron micrographs of small colony variants. Bacteria were harvested in mid-logarithmic growth and prepared for electron microscopy. Bars represent 1 μm, C6706, flagellum positive control. ΔflaA, flagellum negative control. Mutant-1-4, small colony variants. (D) Motility phenotype of small colony variants. Bacteria were inoculated into 0.3% agar LB plates and incubated at 37°C for 8 h. C6706, motility positive control. ΔflaA, motility negative control. Mutant-1-4, small colony variants. (E) Rate of nonmotile V. cholerae in vitro and in vivo culture. Wild-type C6706 were inoculated in 5 mL LB anaerobic test tubes and incubated anaerobically at 37°C for 5 days, and plated onto selective plates (in vitro culture). Bacteria were intragastrically administered to CD-1 adult mice, fecal pellets were collected from each mouse at the fifth day after infection and plated onto selective plates (in vivo culture). At the indicated time points, one hundred V. cholerae colonies per sample were picked and the motility was detected in 0.3% agar LB plates. Rate of nonmotile colonies were calculated as the ratio of nonmotile mutant colonies to all colonies per sample. Horizontal line: median. The illustration was created with BioRender.com.

Fig 2. High impact variants in nonmotile mutants are located on the flagellum regulator genes flrA, flrC and rpoN.

(A) Schematic experimental overview for next-generation sequencing. Wild-type C6706 were intragastrically administered to 16 CD-1 adult mice. A single-cage single-mouse infection experiment were performed to avoid a cage effect of mutants’ collection. Fecal pellets were collected from each mouse at the fifth day post-infection, one hundred V. cholerae colonies from one mouse were randomly selected for motility screening in 0.3% agar LB plates. To avoid picking siblings of the same bacterium, we picked only 3–4 nonmotile colonies per mouse for next-generation sequencing. The illustration was created with BioRender.com. (B) High impact variants of V. cholerae from intestine of adult mice. Fifty-three nonmotile mutants from 16 mice were chosen for DNA purification and next-generation sequencing, the genes of 20 High impact variants were showed. (C) Motility phenotype of five High impact knockout mutants. Bacteria were inoculated into 0.3% agar LB plates and incubated at 37°C for 8 h. C6706, motility positive control. ΔflaA, motility negative control. (D) Adult mice competition assay of ΔflrA, ΔflrC, ΔrpoN. 10⁸ cells of wild-type and knockout or complemented strain were mixed in a 1:1 ratio and intragastrically administered to CD-1 adult mice, the competitive index (CI) of the fifth day after infection was calculated as the ratio of mutant to wild-type colonies normalized to the input ratio. Horizontal line: median CI. Significance was determined by Mann Whitney test, p-value: *, < 0.05, **, < 0.01. (E) Reparable motility phenotype of nonmotile mutants derived from C6706 by flrA, flrC, rpoN genes. The pBBR-P_bad-flrA, pBBR-P_bad-flrC and pBBR-P_bad-rpoN plasmids were constructed to complement the motility phenotype of fifty-three nonmotile mutants derived from C6706. Detection of reparable motility by 0.3% agar LB plates.

Fig 3. Nonmotility-related mutation frequency is dps-dependent in V. cholerae.

(A) Schematic experimental overview. The illustration was created with BioRender.com. (B) Effect of V. cholerae defective in ROS or RNS detoxification genes on the rate of nonmotile colonies in vivo. V. cholerae defective in ROS (Δdps, ΔkatG, ΔprxA) or RNS (ΔhmpA) detoxification genes were individually intragastrically inoculated into CD-1 mice treated with antibiotics cocktail. Fecal pellets were collected at the fifth day post-infection, and plated on selective plates. One hundred V. cholerae colonies from one mouse were randomly selected for motility screening in 0.3% agar LB plates. Rate of nonmotile colonies were calculated as the ratio of nonmotile mutant colonies to all colonies per sample. Horizontal line: median. Significance was determined by Kruskal-Wallis test, p-value: ns, not significant, *, < 0.05. (C) Effect of V. cholerae overexpression in ROS or RNS detoxification genes on the rate of nonmotile colonies in vivo. Wild-type V. cholerae containing pACYC-P_bad-dps, pACYC-P_bad-katG or pACYC-P_bad-prxA plasmid for overexpression in ROS detoxification, and pACYC-P_bad-hmpA plasmid for overexpression in RNS detoxification were individually intragastrically inoculated into CD-1 mice. Fecal pellets were collected at the fifth day post-infection, and plated on selective plates. One hundred V. cholerae colonies from one mouse were randomly selected for motility screening in 0.3% agar LB plates. Rate of nonmotile colonies were calculated as the ratio of nonmotile mutant colonies to all colonies per sample. Horizontal line: median. Significance was determined by Kruskal-Wallis test, p-value: ns, not significant. Effect of V. cholerae defective (D) and overexpression (E) in bacterial gene repair system on the rate of nonmotile colonies in vivo. Horizontal line: median. Significance was determined by Kruskal-Wallis test, p-value: ns, not significant.

Fig 4. ROS detoxification function of Dps contributes to the nonmotility-related mutation frequency in V. cholerae.

(A) Dps protein structure of ROS detoxification. Structural model of Dps generated from the crystal structure of the V. cholerae N16961 (PDB entry 3IQ1) was showed. The monomer of the Dps-like 12-mer assemblies was showed by green (top left), which the ferroxidase center amino acid residues (H38, H50, D65, E69) were highlighted in red. Dps protects DNA with ferroxidase center by greatly ameliorating the potentially lethal combination of Fe²⁺ and H₂O₂. F46 was colored red, residues of AB loop were colored green (bottom left). A mutation at F46 might generate a 24-mer from Dps, the conformations of ferroxidation site residues were altered and no iron was bound due to disruption of stacking interactions with F46, which may result in Dps ROS detoxification function deficiency and consequent cell damage. (B) Growth of wild-type and dps point mutations under ROS stress. Exponentially growing cultures of wild-type C6706 (blue) and Δdps (red), dps^D65A (green) and dps^F46E (purple) mutants were grown in LB with 300 μM H₂O₂. The recovery and growth of each strains were monitored over time. The averages of 4 experiments were showed for each strains. (C) Binding of Dps to supercoiled plasmid pUC19. Different concentration wild-type Dps or D65A, F46E Dps mutants protein were individually incubated with 0.6 pM of supercoiled plasmid pUC19 (in 50 mM MOPS buffer pH 7.0, containing 50 mM NaCl). BSA, negative control. (D) Rate of nonmotile mutants in dps^D65A and dps^F46E mutations in adult mice intestine. Δdps, dps^D65A and dps^F46E mutants were individually intragastrically inoculated into CD-1 mice treated with antibiotics cocktail. Fecal pellets were collected at the fifth day post-infection, and plated on selective plates. One hundred V. cholerae colonies from one mouse were randomly selected for motility screening in 0.3% agar LB plates. Rate of nonmotile colonies were calculated as the ratio of nonmotile mutant colonies to all colonies per sample. Horizontal line: median. Significance was determined by Kruskal-Wallis test, p-value: *, < 0.05, **, < 0.01. (E) Rate of nonmotile mutants in wild-type C6706 and Δdps in adult mice intestine with or without ROS. Bacteria were intragastrically administered to CD-1 adult mice treated with antibiotics cocktail (ROS+) or NAC (ROS-), fecal pellets were collected from each mouse at the fifth day post-infection, and plated onto selective plates. One hundred V. cholerae colonies per mouse were randomly selected for motility screen in 0.3% agar LB plates. Rate of nonmotile colonies were calculated as the ratio of nonmotile mutant colonies to all colonies per sample. Horizontal line: median. Significance was determined by Mann Whitney test, p-value: *, < 0.05.

Fig 5. Similar mutation characteristics between nonmotile mutants derived from Δdps mutant or wild-type V. cholerae.

(A) Distribution of flrA, flrC, rpoN mutation in nonmotile mutants derived from Δdps mutant or wild-type V. cholerae in vivo. The arrangement of the domains of FlrA, FlrC, RpoN were generated by Pfam database. The FlrA protein has three domains, an N-terminal flagellar regulatory protein FleQ domain, a central sigma-54 interaction domain and a C-terminal DNA binding helix turn helix (HTH) domain. The FlrC protein has three domains, an N-terminal response regulator receiver domain, a central sigma-54 interaction domain and a C-terminal DNA binding HTH domain. The RpoN protein has three domains, an N-terminal sigma-54 factor Activator interacting domain (AID), a central sigma-54 factor core binding domain (CBD) and a C-terminal DNA binding domain (DBD). Positions of the mutations in the domain were indicated with amino acid or nucleotide. The positions of mutations in nonmotile mutants derived from C6706 were showed by black, and derived from Δdps were showed by blue. Red box represents identical mutation sites in nonmotile mutants derived from C6706 and Δdps, * and number represents the number of mutants. (B) Adult mice competition assay of flrA, flrC, rpoN point mutants. We constructed point mutants of flrA, flrC, and rpoN, 10⁸ cells of wild-type and mutant were mixed in a 1:1 ratio and intragastrically administered to CD-1 adult mice. Fecal pellets were collected from each mouse at the fifth day post-infection, and plated onto selective plates. The competitive index (CI) was calculated as the ratio of mutant to wild-type colonies normalized to the input ratio. Horizontal line: median CI.

Fig 6. Nonmotility-related mutations increase V. cholerae transmission between hosts.

(A) Schematic experimental overview. The illustration is created with BioRender.com. (B) Rate of nonmotile mutants from second colonization in adult mice intestine. Collection of V. cholerae from the feces of mice gavaged with Δdps or wild-type alone on the fifth day of gavage (as Δdps-Mix, wt-Mix), and then performed the competition colonization assay (second colonization) using wild-type C6706 and Δdps-Mix or wt-Mix. Fecal pellets were collected at the fifth day post-infection, and plated on selective plates. One hundred V. cholerae colonies from one mouse were randomly selected for motility screening in 0.3% agar LB plates. Rate of nonmotile colonies were calculated as the ratio of nonmotile mutant colonies to all colonies per sample. Horizontal line: median. Adult mice competition assay of methionine addition and Δ4ΔflrA (C), Δ4ΔflrC (D), Δ4ΔrpoN (E). 10⁸ cells of wild-type and mutant were mixed in a 1:1 ratio and intragastrically administered to CD-1 adult mice. Methionine addition indicates adult mice supplemented with 25 mM L-methionine (Met) in drinking water. Fecal pellets were collected from each mouse at the fifth day post-infection, and plated onto selective plates. The competitive index (CI) was calculated as the ratio of mutant to wild-type colonies normalized to the input ratio. Δ4: ΔmetRΔmetIΔmetTΔmsrC. Horizontal line: median CI. Significance was determined by Mann Whitney test, p-value: *, < 0.05, **, < 0.01.

Fig 7. Proposed Model of generating nonmotility-related mutants.

V. cholerae entering the mouse intestine are exposed to ROS, and the flagella of V. cholerae itself can also trigger inflammation. Dps is an important oxidative stress resistance protein, and its expression is significantly induced by ROS exposure. Increased Dps expression can confer resistance to ROS, but overexpression of Dps can also lead to sequestration of iron, inactivation of iron ion-dependent enzymes and inhibition bacterial growth. When the amount of Dps decreases, bacteria have decreased ability to resist ROS and DNA damage. V. cholerae produced nonmotility-related mutations, which had a stronger ability to resist ROS and increased fitness in the host by the methionine metabolism pathway. The illustration is created with BioRender.com.