A conditionally lethal mutant of Salmonella Typhimurium induces a protective response in mice

https://doi.org/10.1016/j.bbrc.2016.01.058Get rights and content

Highlights

  • The tetracycline-resistant cassette tetRA was used to control expression of genes.

  • The mutant Salmonella Typhimurium yabB::tetRA presented tetracycline-dependent growth.

  • Immunization with S. Typhimurium yabB::tetRA induced antibodies against Salmonella.

  • IP immunization of mice with the yabB mutant protects against S. Typhimurium WT.

Abstract

Here we present the design of a conditionally lethal mutant of Salmonella enterica serovar Typhimurium (S. Typhimurium) which growth depends on tetracycline (Tet). Four mutants of S. Typhimurium, with Tet-conditional growth, were created by inserting the tetRA cassette. Three of the mutants presented a conditional-lethal phenotype in vitro. One mutant in the yabB gene remained conditional inside cells and did not persisted after 24 h in cell cultures. The capacity of S. Typhimurium yabB::tetRA to invade deep organs was investigated in intraperitoneally (IP) infected mice fed with or without chlortetracycline (CTet), a Tet analog with lower antibiotic activity. The yabB::tetRA mutant was undetectable in liver or spleen of animals under normal diet, while in mice under diet including CTet, yabB::tetRA invaded at a level comparable to the WT in mice under normal diet. Moreover, yabB::tetRA produced a strong humoral-immunoresponse after one IP immunization with 106 bacteria, measured as serum reactivity against S. Typhimurium whole cell extract. By contrast, oral immunization with 106 bacteria was weaker and variable on inducing antibodies. Consistently, IP infected mice were fully protected in a challenge with 104 oral S. Typhimurium, while protection was partial in orally immunized mice. Our data indicate that S. Typhimurium yabB::tetRA is a conditionally attenuated strain capable of inducing a protective response in mice in non-permissive conditions.

Introduction

The ideal genetically engineered bacterial vaccine should achieve immunogenicity while attenuated enough to avoid disease of animals or humans, with not shedding of genetically modified live vaccines [1]. These qualities have been typically addressed by introducing mutations in metabolic or virulence genes [1]. A good example of these type of bacterial vaccines are the S. Typhimurium mutants in the genes cya (adenylate cyclase) and crp (cAMP receptor protein) [2]. Recently, delayed attenuation has been proposed in Salmonella as a mean to produce attenuated yet invasive strains that, after few replication cycles into the host, became attenuated. To achieve this, genes involved in iron regulation (fur), catabolite repression (crp), regulation of virulence factors (phoPQ) and stationary-phase protein expression (rpoS) were subjected to transcriptional control of the arabinose inducible PBAD promoter [1], [3]. Here we present the design of a conditionally lethal mutant of S. Typhimurium which growth depends on Tet. Previously, in the search for essential genes, we screened 1700 mutants of S. Typhi with insertions of the T-POP transposon which provides downstream Tet-dependent transcription from the tetRA cassette [4]. The tetRA cassette present in T-POP is defective on the intrinsic transcriptional terminators located at the ends of tetR and tetA genes, thus it can support transcription of genes adjacent to the tetRA insertion by a positive polar effect [4]. Replica plating with and without Tet revealed four insertions of the transposon T-POP (in yabB, in yabB promoter, in tyrS promoter, and in yqgE) that determined a Tet-dependent growth [5]. We created S. Typhimurium strains with insertions of the tetRA cassette equivalent to the insertions described for S. Typhi. All mutants in S. Typhimurium repeated the conditional-lethal phenotype previously described in vitro [5]; except the yqgE mutant, in which the absence of Tet-induction produced only slower growth. To advance in creating a vaccine strain, we investigated whether S. Typhimurium mutants remained conditional inside cells; we found that after 24 h, S. Typhimurium yabB::tetRA was undetectable from infected cells in culture. The yabB::tetRA mutant contains an insertion of the tetRA (Tet resistant) cassette in the yabB gene, the first gene of the conserved mra operon. Sequence analyses indicated that at least the first 16 genes of mra operon may be transcribed as a polycistron that may stop transcribing at a Rho-independent terminator at the lpxC gene (also known as envA) [6]. This operon includes 6 genes involved in cell division (fts genes), seven genes involved in biosynthesis of murein precursors (including mur, mra and ddlB) and one gene involved in lipopolysaccharide synthesis (lpxC) [7], [8]. The only function associated to yabB is as an inhibitor of the yabC encoded methyltransferase, but this information is only supported by one disputed publication [9]. In this work, we constructed a S. Typhimurium yabB::tetRA Tet-conditional mutant by inserting the T-POP-encoded tetRA cassette at the equivalent locus where S. Typhi yabB::T-POP, previously described, exhibits the T-POP transposon [5] (Supplementary Figures 1 and 2). The insertion of the tetRA cassette provides Tet-dependent transcription downstream of the gene tetA [10], [11] to transcribe genes of the mra operon in S. Typhimurium yabB::tetRA. To further characterize this strain, we studied its capacity to invade deep organs in mice, after an intraperitoneal infection. In mice under a normal diet, S. Typhimurium yabB::tetRA was unable to reach the liver or spleen. In mice fed with a diet comprising CTet (an analog of Tet with lower antibiotic activity), S. Typhimurium yabB::tetRA invaded deep organs at a level comparable to the WT strain. Furthermore, S. Typhimurium yabB::tetRA induced strong humoral-immunoresponse after both IP and oral immunization measured as serum reactivity against S. Typhimurium whole cell extract, albeit the response was weaker with the oral immunization. Consistently with the strong antibody production of IP infected mice, these IP immunized mice were fully protected against a challenge with S. Typhimurium WT; while protection was partial in orally immunized mice.

The data presented indicate that S. Typhimurium yabB::tetRA is a conditionally attenuated strain capable of inducing anti-Salmonella antibodies and a protective response, against S. Typhimurium, in mice.

Section snippets

Construction of mutants and phenotype assessment

Tet-dependent conditional mutants were obtained by allelic exchange using a modification of Red-Swap method [12]. Briefly, 60 bp primers, whose 40 bp of the 5′ end exhibited 100% identity to target genes (yabB or yqgE) or to target promoters (PyabB or PtyrS) (Supplementary Table 1), were used to PCR amplify the tetRA cassette. Amplicons were electroporated into S. Typhimurium ATCC14028s/pKD46 [12] and recombinants selected on LB-agar + Tet 10 μg/mL incubated overnight at 37 °C. Tet-dependency

Tet-dependent control of genes encoding essential functions produced Tet-conditional growth

The tetRA cassette was introduced by allelic interchange in yabB and yqgE genes, and in yabB and tyrS promoters to produce Tet-dependent transcription of downstream genes. To first test Tet-dependent conditional phenotype, disk diffusion assays were performed. Tet-dependent growth was observed for all mutants, except for yqgE::tetRA, which only presented slower growth in the absence of Tet (Supplementary Figure 3). The same four equivalent mutants of S. Typhi produced Tet-conditional growth [5]

Discussion

The first approach to create Salmonella vaccines targeted serovar Typhi and consisted of inactivated whole-cell administered parentally [15], [16], [17]. Due to its high reactogenicity, this vaccine was replaced by parental purified components and by live attenuated oral bacteria [18]. Ty21a, a live attenuated strain generated by chemical mutagenesis of the virulent S. Typhi Ty21 [19], achieved protection over 60% of population for at least 7 years [15], [20], [21]. Besides Typhi, it is

Conflict of interest

The authors have nothing to declare.

Acknowledgment

We thank Mr. Víctor Ahumada and David Pezoa DVM, Ph.D. for assisting with animal procedures. Work supported by FONDECYT grants 3130523 (to A.H.), 1151393 (to G.M.) and 11121506 (to J.F.).

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