Investigating the role of the carbon storage regulator A (CsrA) in Leptospira spp

Abstract

Carbon Storage Regulator A (CsrA) is a well-characterized post-transcriptional global regulator that plays a critical role in response to environmental changes in many bacteria. CsrA has been reported to regulate several metabolic pathways, motility, biofilm formation, and virulence-associated genes. The role of csrA in Leptospira spp., which are able to survive in different environmental niches and infect a wide variety of reservoir hosts, has not been characterized. To investigate the role of csrA as a gene regulator in Leptospira, we generated a L. biflexa csrA deletion mutant (ΔcsrA) and csrA overexpressing Leptospira strains. The ΔcsrA L. biflexa displayed poor growth under starvation conditions. RNA sequencing revealed that in rich medium only a few genes, including the gene encoding the flagellar filament protein FlaB3, were differentially expressed in the ΔcsrA mutant. In contrast, 575 transcripts were differentially expressed when csrA was overexpressed in L. biflexa. Electrophoretic mobility shift assay (EMSA) confirmed the RNA-seq data in the ΔcsrA mutant, showing direct binding of recombinant CsrA to flaB3 mRNA. In the pathogen L. interrogans, we were not able to generate a csrA mutant. We therefore decided to overexpress csrA in L. interrogans. In contrast to the overexpressing strain of L. biflexa, the overexpressing L. interrogans strain had poor motility on soft agar. The overexpressing strain of L. interrogans also showed significant upregulation of the flagellin flaB1, flaB2, and flaB4. The interaction of L. interrogans rCsrA and flaB4 was confirmed by EMSA. Our results demonstrated that CsrA may function as a global regulator in Leptospira spp. under certain conditions that cause csrA overexpression. Interestingly, the mechanisms of action and gene targets of CsrA may be different between non-pathogenic and pathogenic Leptospira strains.

Fig 1. csrA operon in Leptospira spp.

(A) A genetic organization of csrA in Leptospira spp. The arrangement of the genes in the csrA operon in L. interrogans, L. biflexa, and L. biflexa ΔcsrA are shown. (B) The alignment of the amino acid sequences of CsrA in L. biflexa serovar Patoc and L. interrogans serovar Manilae strains used in this study was performed in comparison with CsrA from other bacteria. (*) represents conserved amino acid and the square boxes indicate conserved residues that are important for RNA binding in E. coli [62]. Sequences highlighted in yellow indicate the conserved residues.

Fig 2. Allelic exchange of csrA in L. biflexa.

(A) Schematic representation of homologous recombination. To generate a csrA mutant, L. biflexa serovar Patoc was electroporated with a suicide vector containing the csrA locus where csrA was replaced by a kanamycin resistance cassette (Km^R). Genes and non-coding regions with their sizes (bp) are indicated. The flanking regions of csrA used for homologous recombination are indicated by the dashed line square. Arrows indicate primers used for the confirmation of double crossing-over events. (B) csrA expression in L. biflexa strains was determined by RT-qPCR. Results obtained from 3 independent cultures were presented as relative fold changes ±SEM using cysK gene for normalization. (***) indicates p-value <0.001.

Fig 3. Phenotype analysis of the ΔcsrA L. biflexa.

To investigate the effect of csrA on growth, 2×10⁶ cells of each bacterial strain were grown in 10 mL of (A) regular EMJH and (B) 5-fold diluted EMJH. OD₄₂₀ measurement for growth was performed every 24 h. Results obtained from 3 independent experiments are expressed as Mean ± SEM. (C) Soft agar assay of WT and ΔcsrA. Leptospira were inoculated onto 0.6% semisolid EMJH plate and incubated at 30°C for one week before measuring the diameter of each colony. The late exponential phase of Leptospira grown in EMJH medium were measured for (E) cell length and (D) velocity under a dark-field microscope using cellSens software (OLYMPUS).

Fig 4. RNA-sequencing.

The up- and downregulated genes in ΔcsrA or ΔcsrA+pMaORI_PcsrA_lb compared with WT are shown in the Volcano analysis. (A) Comparison between ΔcsrA and WT and (B) Comparison between ΔcsrA+pMaORI_PcsrA_lb and WT. Red dots indicated up- or downregulated genes with log2FC > ± 0.5 and adjusted p-value (padj) < 0.05. Representative genes are labeled. Blue and yellow dots indicate non-differentially expressed genes and scRNA, respectively.

Fig 5. FlaB genes expression of L. biflexa.

(A) Expression of L. biflexa flaB genes by RT-qPCR, RNAs were prepared from 3 independent cultures of each leptospiral strains. Results are presented as relative fold changes ±SEM using cysK for normalization. (*), (**), and (***) indicate p-value <0.05, <0.01 and <0.001, respectively. For statistical analysis, ΔcsrA was compared to WT; ΔcsrA+pMaORI or ΔcsrA+pMaORI_PcsrA_lb was compared to WT+pMaORI. (B) Analysis of flaB 5’ untranslated regions of L. biflexa serovar Patoc. The gene and distances to the start codon are indicated. Underlined letters represent mismatched nucleotides compared with the consensus sequence.

Fig 6. FlaB gene as a potential target of CsrA in L. biflexa.

(A) Secondary structure of 120-nucleotide 5’ untranslated region of LEPBIa1872 (flaB3) was predicted using MFOLD [64]. The putative CsrA binding site is shown and the start codon (ATG) is indicated in bold letters. The sequences of the synthesized 5’ biotinylated RNAs of LEPBIa_1872 WT and LEPBIa_1872 Mut probes for flaB3 are shown. (B) Electrophoretic mobility shift assay (EMSA), 1 nM biotinylated RNA of either LEPBIa_1872 WT or LEPBIa_1872 Mut probes were incubated with different concentrations of rCsrA of L. biflexa. The reaction solution was subjected to 10% native PAGE, transferred to a nylon membrane, probed with HRP-conjugated streptavidin, and detected for chemiluminescent signal after the detection reagent was added.

Fig 7. Overexpression of csrA in L. interrogans.

(A) Overexpression of csrA in L. interrogans. To confirm overexpression of csrA, RNA was extracted from each Leptospira strain and then subjected to RT-qPCR. Results obtained from 3 independent cultures were presented as relative fold changes ± SEM. LipL32 was used for normalization. (***) indicates p-value < 0.001. (B) Growth curve of L. interrogans. The 2×10⁶ cells of each bacterial strain were grown in 10 mL of regular EMJH. OD₄₂₀ measurement for growth was performed every 24 h. Results obtained from 3 independent experiments are expressed as Mean ± SEM. (C) Soft agar assay of L. interrogans. Leptospira at OD₄₂₀ = 0.1 were inoculated onto 0.6% semisolid EMJH plates and incubated at 30°C. (D) Measurement of cell length of L. interrogans (E) Measurement of velocity of L. interrogans. Late exponential phase of Leptospira grown in EMJH medium were measured for cell length and velocity under a dark-field microscope using cellSens software (OLYMPUS).

Fig 8. Effect of overexpressed CsrA on flaB expression.

Expression of 4 flaB genes in Leptospira strains by RT-qPCR. RNAs were prepared from 3 independent cultures of each leptospiral strain and used for RT-qPCR. Results are presented as relative fold changes ± SEM using cysK and lipL32 for normalization in L. biflexa and L. interrogans, respectively. (*), (**) and (***) indicate p-value < 0.05, < 0.01 and < 0.001, respectively.

Fig 9. L. interrogans CsrA regulated flaB expression.

(A) Analysis of flaB 5’ untranslated regions of L. interrogans serovar Manilae. The genes and distances to the start codon are indicated. Underlined letters represent mismatched nucleotides compared to the consensus sequence. (B) Secondary structure of 120-nucleotide 5’ untranslated region of LIMLP07475 (FlaB4) was predicted using MFOLD [64]. The putative CsrA binding site is shown and the start codon (ATG) is indicated in bold letters. The sequences of the synthesized 5’ biotinylated RNA of LIMLP_07475 is shown. (C) Electrophoretic mobility shift assay (EMSA), 1 nM biotinylated RNA of LIMLP_07475 was incubated with different concentrations of L. interrogans rCsrA. The reaction solution was subjected to 10% native PAGE, transferred to a nylon membrane, probed with HRP-conjugated streptavidin, and detected for chemiluminescent signal after the detection reagent was added. (D) Competitive EMSA, biotinylated RNA of LIMLP_07475 and rCsrA concentration were fixed at 1 nM and 800 nM, respectively. Unlabeled LIMLP_07475 was added in the reaction concentration range from 0.8 nM to 8 μM.