O-antigen-deficient, live, attenuated Salmonella typhimurium confers efficient uptake, reduced cytotoxicity, and rapid clearance in chicken macrophages and lymphoid organs and induces significantly high protective immune responses that protect chickens against Salmonella infection

https://doi.org/10.1016/j.dci.2020.103745Get rights and content

Highlights

  • Attenuated Salmonella Typhimurium strain was tested against chicken salmonellosis.

  • The strain was safe, did not secrete in feces, and rapidly cleared from host tissues.

  • Cytokine response elicited by the mutant was higher than the wild type strain.

  • The mutant strain was completely safe in chicken macrophages.

  • Immunization resulted in significant protection against wild type challenge.

Abstract

In the present study, we developed an O-antigen-deficient, live, attenuated Salmonella Typhimurium (ST) strain (JOL2377) and assessed its safety, macrophage toxicity, invasion into lymphoid tissues, immunogenicity, and protection against Salmonella infection in chickens. The JOL2377 induced significantly lower cytotoxicity and higher level of cytokine response in IL-2, IL-10, IL-4, and IFN- γ than the WT strain upon macrophage uptake. It did not persist in macrophages or in chicken organs and rapidly cleared without systemic infection. None of the chicken were found to secrete Salmonella in feces into the environment exacerbating its attenuation. Interestingly JOL2377 successfully arrived in immunological hot-spots such as spleen, liver and bursa of Fabricius for an efficient antigen presentation and immune stimulation. Mucosal and parenteral immunization with JOL2377 significantly elicit antigen-specific humoral (IgY) and cell mediated responses marked by peripheral blood mononuclear cell proliferation, cytokine induction, increase in T-cell responses than non-immunized control. JOL2377 did not generate significant levels of LPS specific antibodies as compared to the WT strain due to the lack of immunogenic O-antigen component from its LPS structure. Upon virulent challenge, route dependent efficacy differences were leaving the intramuscular route is superior to the oral route on reducing splenic and liver colonization of the challenge ST. The least cytotoxicity, virulence, and superior immunogenicity of JL2377 that effectively engage both humoral and IFN- γ mediated CMI responses present an ideal scenario in host immune modulation to fight against intracellular pathogen Salmonella.

Introduction

Salmonella enterica serovar Typhimurium infects different hosts and are among the most prevalent causes of food poisoning among humans and a wide variety of farm animals. The predominant source of human acquisition of Salmonella is through contaminated farm products such as poultry. It is estimated that about 17% of Salmonella-associated foodborne diseases are derived due to the poultry industry in the United States (USA, Tsolis et al., 1999). Between 2009 and 2015, 30% of all food-borne diseases in the USA were caused by Salmonella (Dewey-Mattia et al., 2018). Due to its significant prevalence within human society, Salmonella causes a huge economic loss in terms of human productivity and public health expenses. Infected animals, especially layer hens, act as a reservoir for both vertical and horizontal disease transfer; however, infected chicken usually remain asymptomatic (Shah et al., 2012), and contamination can occur unnoticed. Horizontal contamination occurs when an egg contacts a contaminated environment, allowing bacteria to adhere to the surface of the shell and subsequently pass through the egg pores or form biofilms. To reduce the risk of Salmonella contamination, many farms adopt multiple layers of biosecurity, including routine chemical decontamination (De Cort et al., 2016), the addition of organic acids to feed and drinking water, use of probiotics, and animal vaccination (Desin et al., 2013). In many countries, vaccination of layer hens has resulted in the reduction in human Salmonella infection, indicating the importance of vaccination in disease prevention (Cogan and Humphrey, 2003). However, the generation of an ideal vaccine candidate with key safety and immunological factors is a daunting task. In ideal circumstances, a candidate strain would be highly attenuated, not secreted into the environment via fecal matter, would not cause cytotoxicity/apoptosis, and would induce significant protective immune responses that eliminate the invading pathogen.

Due to advances in immunology and molecular biology, the field of vaccine development has undergone considerable changes over the past few decades. Novel molecular techniques that allow precise gene deletion, strain attenuation, identification of novel antigenic molecules, and adjuvant development have contributed to the uplift in vaccine development against important diseases such as salmonellosis (André, 2003; McCullers and Dunn, 2008). Over the past century, successful vaccines have caused almost complete elimination of certain diseases from the globe; however, adaptable pathogens such as Salmonella continue to cause epidemic outbreaks. Further, vast antigenic variations within the genus (Elias et al., 1974) and acquisition of novel antigenic variations cause existing vaccines to become obsolete. Hence, the development of more effective vaccines is a continuous and relentless process. Currently, killed vaccines, subunit vaccines, and live attenuated vaccines are available to prevent salmonellosis. Although each of them has pros and cons, live attenuated vaccines hold a reputation for robust induction of humoral and cell-mediated protective immune responses in immunized hosts (Clark-Curtiss and Curtiss, 2018). For Salmonella to be used as an immunization agent, it would need to undergo significant attenuation to ensure that no disease could be caused in the immunized host and that there would be no persistence in host immune cells such as macrophages.

In the present study, a Salmonella Typhimurium (ST) strain was attenuated by deleting the three major virulence genes lon, cpxR, and rfaL. The Lon protease of Salmonella participates in the regulation of several virulence-related genes (SPI-1 genes), and its removal confers impaired intracellular survival (Takaya et al., 2002). The CpxA/CpxR two-component system of Gram-negative bacteria regulates a vast number of genetic loci responsible for stress adaptation and fimbriae expression, and deletion of the cpxR gene not only attenuates the ST strain but also enhances fimbriae expression on the cell surface (Natasha Weatherspoon-Griffin et al., 2014). The novel mutant strain could not produce systemic infection yet retained efficient adhesion and uptake by antigen presentation cells such as dendritic cells and macrophages. The vaccine ST strain was further attenuated by deletion of the rfaL gene, which is responsible for O-antigen expression. Research studies have reported that removal of the rfaL gene causes increased sensitivity to complement-mediated killing (Kong et al., 2011). Also, O-antigen-deficient Salmonella strains demonstrate increased uptake by dendritic cells (DC), altered intracellular processing, and increased degradation (Zenk et al., 2009). Collectively, the deletion of lon, cpxR, and rfaL confers significant attenuation, reducing the risk of intracellular proliferation while maintaining efficient antigen presentation essential for sufficient elicitation of protective immune responses (Cheminay et al., 2005).

Several reports have demonstrated that rfaL deletion causes a deficiency in Payer's patches in small intestine infection by mutant strains (Kong et al., 2011), leading to a deficiency in systemic infection and inefficient antigen presentation. Our observations of chicken lymphoid organ bursa of Fabricius revealed significant attenuation of the present attenuated mutant upon inoculation via both mucosal and parenteral routes. Interestingly, the present mutant demonstrated remarkable safety in cultured bone marrow-derived macrophage cells without significant cytotoxicity compared to the wild type strain (Chen et al., 1996), while proinflammatory responses were significantly higher than those of the wild type counterpart. Such actions are ideal for a vaccine candidate, and more experiments are needed to understand the underlying mechanisms of the mutant strain. Further, removal of the O-antigen component of the vaccine strain confers a significant reduction in O-antigen-specific antibody development. Thus, such removal might enable the bacterium to avoid O-antigen-mediated complement activity while allowing DIVA (Differentiation of Infected and Vaccinated Animals) capability for serodiagnostic approaches based on O-antigen-specific serodiagnostic antibodies (Liang et al., 2013).

Considering these advantages, we constructed an attenuated and rough S. Typhimurium strain by deleting the three major virulence genes lon, cpxR, and rfaL and investigated efficacy against virulent ST challenge in a chicken model. We also extensively investigated the safety, environmental shedding, macrophage uptake and persistence, organ persistence, immunogenicity, DIVA capability, and protection against wild-type challenge using mucosal and systemic routes. The resulting significantly high safety profile and protection derived by the novel ST strain open a novel platform for vaccination against Salmonella with improved safety and protection efficacy that is highly appealing for environmental safety and prevention of human salmonellosis.

Section snippets

Bacterial strains, plasmids, primers, and genetic manipulation

All chosen bacterial strains, plasmids, and primers are listed in Table 1. All bacterial strains were grown in Lauria Bertani (LB; BD, Sparks, USA) medium or Brilliant Green Agar (BGA; BD, Sparks, USA) at 37 °C with or without vigorous shaking. An allelic exchange procedure was implemented to delete lon and cpxR genes from the host chromosome (Nandre et al., 2012). In-frame deletion of the rfaL gene was achieved by Lambda Red recombination as per the original description by Datsanko et al. (

Characterization of Salmonella Typhimurium vaccine strain

The deletion of the rfaL gene from the ST JOL911 (ΔlonΔcpxR) double mutant was achieved by the Lambda Red recombination method. The resultant mutations were confirmed by PCR for the three genes using the flanking primers listed in Table 1 (Fig. 1 A). Further, knockout of the rfaL gene resulted in the truncation of LPS by removal of the O-antigen component. Truncation of O-antigen was confirmed by SDS PAGE of purified LPS of ST mutant (Fig. 1 B) and silver staining.

Safety of the attenuated Salmonella Typhimurium strain

The safety of the ST mutant

Discussion

The present attenuated ST mutant demonstrated significant attenuation compared to its wild type counterpart due to specific deletion of three major virulence-related genes, lon, cpxR, and rfaL, from its genome. Attributes of the respective attenuations resulted in no environmental shedding in chicken feces during the inoculation study conducted via mucosal (oral) and parenteral (intramuscular) routes (Gibani et al., 2019). Upon inoculation, bacterial recovery from the spleen and liver

Declaration of competing interest

The authors declare that they have no competing interests.

Acknowledgment

This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, and Forestry (IPET) through Animal Disease Management Technology Development Program, funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (grant number. 119058-02).

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