Induced protein expression in Leptospira spp. and its application to CRISPR/Cas9 mutant generation

Abstract

Expanding the genetic toolkit for Leptospira spp. is a crucial step toward advancing our understanding of the biology and virulence of these atypical bacteria. Pathogenic Leptospira are responsible for over 1 million human leptospirosis cases annually and significantly impact domestic animals. Bovine leptospirosis causes substantial financial losses due to abortion, stillbirths, and suboptimal reproductive performance. The advent of the CRISPR/Cas9 system has marked a turning point in genetic manipulation, with applications across multiple Leptospira species. However, incorporating controlled protein expression into existing genetic tools could further expand their utility. We developed and demonstrated the functionality of IPTG-inducible heterologous protein expression in Leptospira spp. This system was applied for regulated expression of dead Cas9 (dCas9) to generate knockdown mutants, and Cas9 to produce knockout mutants by inducing double-strand breaks (DSB) into desired targets. IPTG-induced dCas9 expression enabled validation of essential genes and non-coding RNAs. Additionally, IPTG-controlled Cas9 expression combined with a constitutive non-homologous end-joining (NHEJ) system allowed for successful recovery of knockout mutants, even in the absence of IPTG. These newly controlled protein expression systems will advance studies on the basic biology and virulence of Leptospira, as well as facilitate knockout mutant generation for improved veterinary vaccines.

Keywords: Leptospira; Cas9; IPTG; Knockdown; Knockout; Mutagenesis; dCas9.

Fig. 1

Validation of an IPTG-inducible cassette in L. biflexa.(A) An inducible cassette developed for Leptospira spp. comprising the lipL21 promoter directing the transcription of codon optimized lac repressor LacI, followed by a B. burgdorferi transcription terminator. The constitutive lipL41 promoter was used to transcribe the reporter gene (either lipL41 or lipL32), with inclusion of the lacO sequence (red) after TSS. Shine-Dalgarno sequence (SD, blue) is responsible for ribosome binding. L. biflexa cells containing the plasmid for IPTG-controlled expression of LipL41 (pMaOri.Inducible:LipL41) (B) or LipL32 (pMaOri.Inducible:LipL32) (C) were grown in liquid media in the absence (-) or presence ( +) of IPTG and protein expression evaluated by immunoblot. Controlled LipL32 expression was also evaluated in different concentrations of IPTG (D) and times of induction (E). Cell lysates from L. interrogans serovar Copenhageni strain Fiocruz L1-130 were used as a control.

Fig. 2

Evaluation of IPTG-induced expression of dCas9 and Cas9 in L. biflexa. Total protein from L. biflexa cells containing the plasmids pMaOri.Inducible:dCas9 (A) and pMaOriNHEJ.Inducible:Cas9 (B) and grown without (-) or with ( +) IPTG were evaluated by Sypro Ruby staining. Upper panel highlights the appearance of dCas9 or Cas9 protein in the presence of IPTG. Wild-type (WT) and L. biflexa containing the plasmid pMaOri.dCas9 were used as controls. dCas9 and Cas9 controlled expression was validated by immunoblotting with anti-Cas9 antibodies (C).

Fig. 3

Evaluation of conditional dnaK knockdown mutants inL. biflexa. (A) L. biflexa cells containing the plasmids pMaOri.Inducible dCas9 alone or with a sgRNA targeting the dnaK gene were seeded onto EMJH media containing spectinomycin without (-) or with ( +) 1 mM IPTG. Colony formation was monitored and recorded at different time points. (B) Colonies observed across four independent experiments. Number of colonies recovered in the absence or presence of IPTG was compared by t-test (*p < 0.05). Colonies from plates without IPTG (n = 3) were picked, cells released from the agar and inoculated in liquid HAN media containing spectinomycin at 10⁵ cells/mL, in the absence or presence of IPTG, and growth was monitored daily at both 29 (C) and 37 °C (D).

Fig. 4

Silencing of the ncRNA RNase P in L. biflexa. (A) L. biflexa cells containing plasmids pMaOri.dCas9 alone, or with sgRNA cassettes for pairing to the sense (complete silencing) or antisense (incomplete) strand of the RNaseP sequence, were plated onto EMJH media containing spectinomycin. Colonies obtained from two independent experiments were counted. (B)Validation of essentiality of ncRNA RNaseP was demonstrated by inducible silencing. Cells harboring the pMaOri.Inducible:dCas9 alone or with the sgRNasePsense were seeded onto EMJH plates with spectinomycin without (-) or with ( +) IPTG. Two independent experiments were performed and at each, two clones of conjugative E. coli were used. Number of colonies recovered in the absence or presence of IPTG was compared by t-test (*p < 0.05).

Fig. 5

OmpL1 porin silencing in L. biflexa and L. interrogans. (A) L. biflexa cells containing plasmid pMaOri.Inducible:dCas9 alone, or with a sgRNA cassette targeting the ompL1 gene, were seeded onto EMJH media containing spectinomycin without (-) or with ( +) 1 mM IPTG. Colony formation was monitored and recorded at different time points. (B) Three independent experiments were performed, and numbers of colonies recovered in the absence or presence of IPTG were recorded. (C) Cell viability was also demonstrated in liquid media in the absence (-) or presence ( +) of IPTG. (D-F) Silencing was also performed in the ompL1 ortholog in L. interrogans strain Fiocruz L1-130. (D) Three independent conjugation experiments were performed and for each, two clones of conjugative E. coli containing the plasmid for ompL1 silencing were used. (E) Colonies from plates without IPTG (n = 2) were picked, cells released from the agar and inoculated in liquid HAN media containing spectinomycin in the absence (-) or presence ( +) of IPTG. Cells at mid-log phase were recovered and cell lysates evaluated by immunoblotting. (F) Viability was also confirmed by growth curves in liquid EMJH containing spectinomycin, in the presence or absence of IPTG.

Fig. 6

lipL21 knockout by inducible Cas9 in different strains of L. interrogans. Conjugation reactions for L. interrogans serogroup Canicola strains LAD-1 (A) and serovar Copenhageni strain Fiocruz L1-130 (B) were seeded onto HAN plates with spectinomycin, without (-) or with ( +) 1 mM IPTG. Numbers of recovered colonies after 3 independent experiments were recorded, utilizing two clones of E. coli with the plasmid containing the sgRNA targeting lipL21 in each experiment. Control plasmid with no sgRNA, therefore no DSB, was also employed. Distinct colonies from the plates without IPTG were selected, grown in liquid media without or with IPTG, and whole cell lysates were by SDS-PAGE followed by Sypro Ruby staining (C, LAD-1; D, L1-130) and immunoblotting (E, LAD-1; F, L1-130). Cultures (a-c for LAD-1; a and b for L1-130) were plated for individual colonies, which were selected and lipL21 mutations were assessed by sequencing. (G) Mutation in distinct colonies (mutation/total colonies) from original colonies “a-c” in LAD-1, and (H) from original colonies “a and b” in L1-130. Sequence in green denotes the protospacer contained in the sgRNA, including a mismatch (underlined) for LAD-1, followed by the PAM (purple, not contained in the sgRNA sequence). Cas9 cleavage sites are indicated by the red triangles.