Regulated programmed lysis of recombinant Salmonella in host tissues to release protective antigens and confer biological containment

Abstract

We have devised and constructed a biological containment system designed to cause programmed bacterial cell lysis with no survivors. We have validated this system, using Salmonella enterica serovar Typhimurium vaccines for antigen delivery after colonization of host lymphoid tissues. The system is composed of two parts. The first component is Salmonella typhimurium strain chi8937, with deletions of asdA and arabinose-regulated expression of murA, two genes required for peptidoglycan synthesis and additional mutations to enhance complete lysis and antigen delivery. The second component is plasmid pYA3681, which encodes arabinose-regulated murA and asdA expression and C2-regulated synthesis of antisense asdA and murA mRNA transcribed from the P22 P(R) promoter. An arabinose-regulated c2 gene is present in the chromosome. chi8937(pYA3681) exhibits arabinose-dependent growth. Upon invasion of host tissues, an arabinose-free environment, transcription of asdA, murA, and c2 ceases, and concentrations of their gene products decrease because of cell division. The drop in C2 concentration results in activation of P(R), driving synthesis of antisense mRNA to block translation of any residual asdA and murA mRNA. A highly antigenic alpha-helical domain of Streptococcus pneumoniae Rx1 PspA was cloned into pYA3681, resulting in pYA3685 to test antigen delivery. Mice orally immunized with chi8937(pYA3685) developed antibody responses to PspA and Salmonella outer membrane proteins. No viable vaccine strain cells were detected in host tissues after 21 days. This system has potential applications with other Gram-negative bacteria in which biological containment would be desirable.

Fig. 1.

The expression of Asd protein from the asd gene with ATG or GTG start codon and muramic acid-less death assay. (A) Western blot was performed with cell lysates of S. typhimurium strain χ8276 (ΔasdA16) and its derivatives cultured in LB broth with 0.2% arabinose. DAP was included in the medium for strain χ8276. Asd protein was detected by using rabbit anti-Asd serum. The 39-kDa Asd protein is indicated by an arrow. (B) Growth of Salmonella strain χ8645 (ΔP_murA7::araC P_BAD murA) with and without arabinose in the indicated media.

Fig. 2.

The regulated programmed lysis system. (A) Map of plasmid pYA3681. Plasmid sequences include the trpA, rrfG, and 5S ribosomal RNA transcriptional terminators; the P_BAD, P_trc and P22 P_R promoters; the araC gene; and the start codon-modified murA and asdA genes. (B) Diagram of model illustrating the regulatory interactions in the programmed lysis system. Details of this system are outlined in Results.

Fig. 3.

In vitro and in vivo lysis of programmed lysis system in the absence of arabinose. (A) The growth curves of strain χ8937(pYA3681) with arabinose-regulated asdA and murA expression in LB broth with or without the addition of 0.02% arabinose. (B) The ratio of released β-galactosidase versus total β-galactosidase when strain χ9380(pYA3681) with arabinose-regulated asdA and murA expression and constitutive lacZ expression was grown in LB broth with or without 0.02% arabinose; the wild-type strain χ9379 modified to express lacZ acts as a nonlysis system control. (C) Colonization of mice with S. typhimurium χ8937(pYA3681) after P.O. inoculation with 10⁹ CFU bacteria. The limits of detection for this assay were 10 CFU per Peyer's patch or gram of tissue.

Fig. 4.

Immune responses in mice after oral immunization with χ8937(pYA3685) (rPspA Rx1) and χ8937(pYA3681) (vector control) as determined by ELISA. (A) IgG antibody against S. typhimurium SOMPs and rPspA Rx1 in a 1:1,280 dilution of serum. (B) Anti-SOMP and -rPspA Rx1 IgA antibody levels in a 1:10 dilution of vaginal secretions. (C) Serum IgG2a and IgG1 responses to SOMPs and rPspA. Gray bars, IgG2a; black bars, IgG1. Serum was diluted 1:400.