Deciphering the Interrelationship of arnT Involved in Lipid-A Alteration with the Virulence of Salmonella Typhimurium.

The lipopolysaccharide (LPS) that resides on the outermost surface and protects Gram-negative bacteria from host defenses is one of the key components leading to Salmonella infection, particularly the endotoxic lipid A domain of LPS. Lipid A modifications have been associated with several genes such as the arnT that encodes 4-amino-4-deoxy-L-arabinose transferase, which can be critical for bacteria to resist cationic antimicrobial peptides and interfere with host immune recognition. However, the association of arnT with virulence is not completely understood. Thus, this study aimed to elucidate the interrelationship of the major lipid A modification gene arnT with Salmonella Typhimurium virulence. We observed that the arnT-deficient S. Typhimurium (JOL2943), compared to the wild type (JOL401), displayed a significant decrease in several virulence phenotypes such as polymyxin B resistance, intracellular survival, swarming, and biofilm and extracellular polymeric substance (EPS) production. Interestingly, the cell-surface hydrophobicity, adhesion, and invasion characteristics remained unaffected. Additionally, LPS isolated from the mutant induced notably lower levels of endotoxicity-related cytokines in RAW and Hela cells and mice, particularly IL-1ß with a nine-fold decrease, than WT. In terms of in vivo colonization, JOL2943 showed diminished presence in internal organs such as the spleen and liver by more than 60%, while ileal infectivity remained similar to JOL401. Overall, the arnT deletion rendered the strain less virulent, with low endotoxicity, maintained gut infectivity, and reduced colonization in internal organs. With these ideal characteristics, it can be further explored as a potential attenuated Salmonella strain for therapeutics or vaccine delivery systems.

Live-Attenuated Salmonella-Based Oral Vaccine Candidates Expressing PCV2d Cap and Rep by Novel Expression Plasmids as a Vaccination Strategy for Mucosal and Systemic Immune Responses against PCV2d.

Oral vaccines are highly envisaged for veterinary applications due to their convenience and ability to induce protective mucosal immunity as the first line of defense. The present investigation harnessed live-attenuated Salmonella Typhimurium to orally deliver novel expression vector systems containing the Cap and Rep genes from porcine circovirus type 2 (PCV2), a significant swine pathogen. The antigen expression by the vaccine candidates JOL2885 and JOL2886, comprising eukaryotic pJHL204 and pro-eukaryotic expression pJHL270 plasmids, respectively, was confirmed by Western blot and IFA. We evaluated their immunogenicity and protective efficacy through oral vaccination in a mouse model. This approach elicited both mucosal and systemic immunity against PCV2d. Oral administration of the candidates induced PCV2-specific sIgA, serum IgG antibodies, and neutralizing antibodies, resulting in reduced viral loads in the livers and lungs of PCV2d-challenged mice. T-lymphocyte proliferation and flow-cytometry assays confirmed enhanced cellular immune responses after oral inoculation. The synchronized elicitation of both Th1 and Th2 responses was also confirmed by enhanced expression of TNF-α, IFN-γ, IL-4, MHC-I, and MHC-II. Our findings highlight the effectiveness and safety of the constructs with an engineered-attenuated S. Typhimurium, suggesting its potential application as an oral PCV2 vaccine candidate.

Structure, Substrate Specificity and Role of Lon Protease in Bacterial Pathogenesis and Survival.

Proteases are the group of enzymes that carry out proteolysis in all forms of life and play an essential role in cell survival. By acting on specific functional proteins, proteases affect the transcriptional and post-translational pathways in a cell. Lon, FtsH, HslVU and the Clp family are among the ATP-dependent proteases responsible for intracellular proteolysis in bacteria. In bacteria, Lon protease acts as a global regulator, governs an array of important functions such as DNA replication and repair, virulence factors, stress response and biofilm formation, among others. Moreover, Lon is involved in the regulation of bacterial metabolism and toxin-antitoxin systems. Hence, understanding the contribution and mechanisms of Lon as a global regulator in bacterial pathogenesis is crucial. In this review, we discuss the structure and substrate specificity of the bacterial Lon protease, as well as its ability to regulate bacterial pathogenesis.

Salmonella-mediated oral delivery of multiple-target vaccine constructs with conserved and variable regions of SARS-CoV-2 protect against the Delta and Omicron variants in hamster.

Since the emergence of the pandemic COVID19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the development of vaccines has been the prime strategy to control the disease transmission. Most of the developed vaccines target the spike protein, however, the emerging variants have alterations, particularly at the same region which may pose resistance to neutralizing antibodies. In this study, we explored the variable and conserved regions of SARS-CoV-2 as a potential inclusion in a multiple-target vaccine with the exploitation of Salmonella-based vector for oral mRNA vaccine against Delta and Omicron variants. Increased IgG and IgA levels imply the induction of humoral response and the CD4+, CD8+ and IFN-γ+ sub-population level exhibits cell-mediated immune responses. The degree of CD44+ cells indicates the induction of memory cells corresponding to long-term immune responses. Furthermore, we assessed the protective efficacy of the vaccines against the Delta and Omicron variants in the hamster model. The vaccine constructs induced neutralizing antibodies and protected the viral-challenged hamsters with significant decrease in lung viral load and reduced histopathological lesions. These results reinforce the use of the conserved and variable regions as potential antigen targets of SARS-CoV-2 as well as the exploitation of bacteria-mediated delivery for oral mRNA vaccine development.