Immunological characterization of a gidA mutant strain of Salmonella for potential use in a live-attenuated vaccine
Daniel C Shippy
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Amin A Fadl
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Department of Animal Sciences, University of Wisconsin-Madison
,
1675 Observatory Dr, Madison, WI 53706
,
USA
Background: Salmonella is often associated with gastrointestinal disease outbreaks in humans throughout the world due to the consumption of contaminated food. Our previous studies have shown that deletion of glucose-inhibited division gene (gidA) significantly attenuated Salmonella enterica serovar Typhimurium (STM) virulence in both in vitro and in vivo models of infection. Most importantly, immunization with the gidA mutant protected mice from a lethal dose challenge of wild-type STM. In this study, we further characterize the gidA mutant STM strain for potential use in a live-attenuated vaccine. Results: The protective efficacy of immunization with the gidA mutant was evaluated by challenging immunized mice with a lethal dose of wild-type STM. Sera levels of IgG2a and IgG1, passive transfer of sera and cells, and cytokine profiling were performed to study the induction of humoral and cellular immune responses induced by immunization with the gidA mutant strain. Additionally, a lymphocyte proliferation assay was performed to gauge the splenocyte survival in response to treatment with STM cell lysate. Mice immunized with the gidA mutant strain were fully protected from a lethal dose challenge of wild-type STM. Nave mice receiving either cells or sera from immunized mice were partially protected from a lethal dose challenge of wild-type STM. The lymphocyte proliferation assay displayed a significant response of splenocytes from immunized mice when compared to splenocytes from non-immunized control mice. Furthermore, the immunized mice displayed significantly higher levels of IgG1 and IgG2a with a marked increase in IgG1. Additionally, immunization with the gidA mutant strain evoked higher levels of IL-2, IFN-, and IL-10 cytokines in splenocytes induced with STM cell lysate. Conclusions: Together, the results demonstrate that immunization with the gidA mutant strain protects mice by inducing humoral and cellular immune responses with the humoral immune response potentially being the main mechanism of protection.
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Background
Salmonella is an enteric pathogen causing major
public health problems throughout the world due to
the consumption of contaminated food. Nontyphoidal
Salmonella species, like Salmonella enterica serovar
Typhimurium (STM), are the leading cause of
hospitalization and death among the major foodborne pathogens
[1]. Antibiotic resistance by Salmonella is dramatically
increasing, so the development of an effective vaccine
remains a global health priority [2,3].
Creating a safe and immunogenic vaccine strain is the
biggest challenge in developing an effective
liveattenuated Salmonella vaccine [4]. Several Salmonella
vaccines, including whole-cell killed and live vaccines,
have been developed with variable success [5,6]. These
vaccines either required repeated administration or
induced insufficient immune responses for long-lasting
protection against lethal challenges with virulence
Salmonella strains [7]. Many Salmonella vaccine strains
carry deletion mutations affecting metabolic functions
or virulence factors [8]. Several mutant strains of
Salmonella have been investigated in the pursuit to
develop optimal immune responses [9-11]. Our approach
in constructing a live-attenuated Salmonella vaccine
strain is to create a mutant defective in tRNA
modification [12]. This strategy enables our vaccine strain to
express multiple virulence factors at a significantly
reduced level in order to obtain a safe and
immunogenic vaccine candidate.
Glucose-inhibited division (GidA) protein (also known
as MnmG) was first described in Escherichia coli, where
deletion of gidA resulted in a filamentous morphology
when grown in a rich medium supplemented with
glucose [13]. Further studies showed GidA is a flavin
dinucleotide (FAD) binding enzyme involved in the
fruiting body development of Myxococcus xanthus [14].
Furthermore, GidA has been shown to be a tRNA
modification methylase in E. coli that forms a heterodimeric
complex with MnmE (also known as TrmE) to
catalyze the addition of a carboxymethylaminomethyl
(cmnm) group at the 5 position of the wobble uridine
(U34) of tRNAs [15-19]. Most importantly, deletion
of gidA has been shown to attenuate the pathogenesis
of some bacteria including Pseudomonas syringae,
Aeromonas hydrophila, Streptococcus pyogenes, and
Pseudomonas aeruginosa [20-23].
Our previous studies suggest a role for GidA in the
regulation of Salmonella virulence and cell division
[12,24]. In our initial study, the gidA mutant was
attenuated in vitro and showed a significant decrease in ability
to invade T84 intestinal epithelial cells as well as a
significant decrease in ability to replicate and produce
cytotoxic affects on macrophages. Furthermore, global
transcriptional and proteomic profiling indicated a
significant down-regulation in numerous ge (...truncated)