Abstract
Non-typhoidal Salmonella includes thousands of serovars that are leading causes of foodborne diarrheal illness worldwide. In this study, we constructed three bivalent vaccines for preventing both Salmonella Typhimurium and Salmonella Newport infections by using the aspartate semialdehyde dehydrogenase (Asd)-based balanced-lethal vector-host system. The constructed Asd+ plasmid pCZ11 carrying a subset of the Salmonella Newport O-antigen gene cluster including the wzx-wbaR-wbaL-wbaQ-wzy-wbaW-wbaZ genes was introduced into three Salmonella Typhimurium mutants: SLT19 (Δasd) with a smooth LPS phenotype, SLT20 (Δasd ΔrfbN) with a rough LPS phenotype, and SLT22 (Δasd ΔrfbN ΔpagL::T araC PBAD rfbN) with a smooth LPS phenotype when grown with arabinose. Immunoblotting demonstrated that SLT19 harboring pCZ11 [termed SLT19 (pCZ11)] co-expressed the homologous and heterologous O-antigens; SLT20 (pCZ11) exclusively expressed the heterologous O-antigen; and when arabinose was available, SLT22 (pCZ11) expressed both types of O-antigens, while in the absence of arabinose, SLT22 (pCZ11) expressed only the heterologous O-antigen. Exclusive expression of the heterologous O-antigen in Salmonella Typhimurium decreased the swimming ability of the bacterium and its susceptibility to polymyxin B. Next, the crp gene was deleted from the three recombinant strains for attenuation purposes, generating the three bivalent vaccine strains SLT25 (pCZ11), SLT26 (pCZ11), and SLT27 (pCZ11), respectively. Groups of BALB/c mice (12 mice/group) were orally immunized with 109 CFU of each vaccine strain twice at an interval of 4 weeks. Compared with a mock immunization, immunization with all three vaccine strains induced significant serum IgG responses against both Salmonella Typhimurium and Salmonella Newport LPS. The bacterial loads in the mouse tissues were significantly lower in the three vaccine-strain-immunized groups than in the mock group after either Salmonella Typhimurium or Salmonella Newport lethal challenge. All of the mice in the three vaccine-immunized groups survived the lethal Salmonella Typhimurium challenge. In contrast, SLT26 (pCZ11) and SLT27 (pCZ11) conferred full protection against lethal Salmonella Newport challenge, but SLT25 (pCZ11) provided only 50% heterologous protection. Thus, we developed two novel Salmonella bivalent vaccines, SLT26 (pCZ11) and SLT27 (pCZ11), suggesting that the delivery of a heterologous O-antigen in attenuated Salmonella strains is a prospective approach for developing Salmonella vaccines with broad serovar coverage.
Highlights
Non-typhoidal Salmonella (NTS), a gram-negative and facultative intracellular bacterium, predominantly causes enteric diarrheal disease in a broad spectrum of animal hosts and humans, which represents a major public health concern, with an estimated 93.8 million cases worldwide and 155,000 deaths each year (Majowicz et al, 2010)
To maintain the Asd+ plasmid in Salmonella Typhimurium, an ATCC14028 asd mutant termed SLT19 was constructed; to block expression of the homologous Salmonella Typhimurium O-antigen, the rfbN gene, which encodes the rhamnosyl transferase that is responsible for the addition of rhamnose sugar to Und-Peyer’s patches (PPs)-Gal of the O-antigen unit (Reeves et al, 2013), was deleted from SLT19, generating SLT20 (ATCC14028 asd rfbN); and to regulate the expression of the homologous O-antigen by exogenous arabinose provided during in vitro growth, the rfbN promoter was replaced with the araC PBAD promoter, and the gene segment T araC PBAD rfbN was inserted into the pagL site, generating SLT22 (ATCC14028 asd rfbN pagL::T araC PBAD rfbN)
The recombinant strain SLT22 expressed O-antigens in an arabinose-regulated manner: both types of O-antigens were expressed when the growth medium was supplemented with arabinose (Figures 1D–F, lane 4), but only the heterologous O-antigen was expressed in the absence of arabinose (Figures 1D–F, lane 5)
Summary
Non-typhoidal Salmonella (NTS), a gram-negative and facultative intracellular bacterium, predominantly causes enteric diarrheal disease in a broad spectrum of animal hosts and humans, which represents a major public health concern, with an estimated 93.8 million cases worldwide and 155,000 deaths each year (Majowicz et al, 2010). NTS infrequently causes serious dysentery and septicemia with substantial mortality, in young infants, the elderly and immunocompromised individuals, such as HIV patients (Arshad et al, 2008; Parry et al, 2013). NTS infections often result in an asymptomatic carrier state in adult domestic animals and the establishment of persistent infections, which have serious impacts on public health due to the risks of food poisoning via consumption of contaminated products. Safe and effective NTS vaccines are urgently needed to prevent salmonellosis in both humans and domestic animals
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