Search

Introduction: The limitations of conventional cancer therapies necessitate target-oriented, highly invasive, and safe treatment approaches. Hence, the intrinsic anti-tumor activity of Salmonella can offer better options to combat cancers. Objectives: This study aims to utilize attenuated Salmonella and deliver cytolytic protein cytolysin A (ClyA) under quorum sensing (QS) signaling for precise localized expression in tumors but not in healthy organs. Methods: The therapeutic delivery strain was imposed with tryptophan auxotroph for selective colonization in tumors by trpA and trpE deletion, and lipid-A and O-antigen were altered by pagL and rfaL deletions using lambda red recombination method. The strain was transformed with the designed QS-controlled ClyA expression vector which was validated by western blot. The in vivo passaged therapeutic strain was used for treatment four times at a weekly interval, with a dose of 5 × 106 CFU/mouse for cancer therapy. Results: The attenuated strain induced minimal endotoxicity-related cytokines TNF-α, IL-1β, and IFN-γ and exhibited adequate colonization despite earlier exposure in mice. The QS-controlled ClyA expression was confirmed by western blot from bacterial cultures grown at different cell densities. The results demonstrated that the in vivo passaged strain preferentially colonized the tumor after vacating the spleen, liver, and lung, leaving no outward histological scars. The anti-cancer effect of the designed construct was evaluated in the murine CT26 colon cancer model. The expression of ClyA increased tumoricidal activity by 67 % compared to vector control. Conclusion: Hence, the anti-tumor effect of the engineered Salmonella strain was improved by ClyA expression via QS activation after achieving the threshold bacterial cell density. Further, immunohistochemical staining of the tumor and other organs corroborated the QS-controlled tumor-specific expression of ClyA. Overall, the results imply that the developed anti-cancer Salmonella has low endotoxicity and QS-controlled expression of ClyA as beneficial safety elements and supports recurrent Salmonella inoculation by O-antigen deficiency.

A universal vaccine is in high demand to address the uncertainties of antigenic drift and the reduced effectiveness of current influenza vaccines. In this study, a strategy called computationally optimized broadly reactive antigen (COBRA) was used to generate a consensus sequence of the hemagglutinin globular head portion (HA1) of influenza virus samples collected from 1918 to 2021 to trace evolutionary changes and incorporate them into the designed constructs. Constructs carrying different HA1 regions were delivered into eukaryotic cells by Salmonella-mediated bactofection using a Semliki Forest virus RNA-dependent RNA polymerase (RdRp)-based eukaryotic expression system, pJHL204. Recombinant protein expression was confirmed by Western blot and immunofluorescence assays. Mice immunized with the designed constructs produced a humoral response, with a significant increase in immunoglobulin G (IgG) levels, and a cell-mediated immune response, including a 1.5-fold increase in CD4+ and CD8+ T cells. Specifically, constructs #1 and #5 increased the production of interferon-γ (IFN-γ) producing CD4+ and CD8+ T cells, skewing the response toward the T helper type 1 cell (Th1) pathway. Additionally, interleukin-4 (IL-4)-producing T cells were upregulated 4-fold. Protective efficacy was demonstrated, with up to 4-fold higher production of neutralizing antibodies and a hemagglutination inhibition titer > 40 against the selected viral strains. The designed constructs conferred a broadly protective immune response, resulting in a notable reduction in viral titer and minimal inflammation in the lungs of mice challenged with the influenza A/PR8/34, A/Brisbane/59/2007, A/California/07/2009, KBPV VR-92, and NCCP 43021 strains. This discovery revolutionizes influenza vaccine design and delivery; Salmonella-mediated COBRA-HA1 is a highly effective in vivo antigen presentation strategy. This approach can effectively combat seasonal H1N1 influenza strains and potential pandemic outbreaks.

Emerging chemo- and radiotherapy resistance exacerbated the cancer risk and necessitated novel treatment strategies. Although RNA therapeutics against pro-oncogenic genes are highly effective, tumor-specific delivery remains a barrier to the implementation of this valuable tool. In this study, we report a tryptophan-auxotrophic Salmonella typhimurium strain as an onco-therapeutic delivery system with tumor-targeting ability using 4T1 mice breast-cancer model. The receptor-binding cancer antigen expressed on SiSo cell (RCAS1) is a cancer-specific protein that induces the apoptosis of peripheral lymphocytes and confers tumor immune evasion. We designed a long non-coding antisense-RNA against RCAS1 (asRCAS1) and delivered by Salmonella using a non-antibiotic, auxotrophic-selective, eukaryotic expression plasmid, pJHL204. After in vivo tumor-to-tumor passaging, the JOL2888 (ΔtrpA, ΔtrpE, Δasd + asRCAS1) strain exhibited high sustainability in tumors, but did not last in healthy organs, thereby demonstrating tumor specificity and safety. RCAS1 inhibition in the tumor was confirmed by western blotting and qPCR. In mice, JOL2888 treatment reduced tumor-associated macrophages, improved the T cell population, elicited cell-mediated immunity, and suppressed cancer-promoting genes. Consequently, the JOL2888 treatment significantly decreased the tumor volume by 80%, decreased splenomegaly by 30%, and completely arrested lung metastasis. These findings highlight the intrinsic tumor-targeting ability of tryptophan-auxotrophic Salmonella for delivering onco-therapeutic macromolecules.

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-1β 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.

Abstract In the present study, two prospective Salmonella delivery strains, JOL2782 and JOL2837, were developed by gene deletions of lon and cpxR, which are related to cellular adhesion and intracellular survival. Additionally, sifA deletion was introduced for JOL2782, which confers immune susceptibility and improves antigen delivery. Similarly, the rfaL deletion and lpxE substitution for pagL were accomplished in JOL2837 to reduce virulence and endotoxicity. Thus, enhanced adhesion and invasion and reduced intracellular survival were attained. Furthermore, aspartic acid auxotrophic (asd) was deleted to impose Darwinian selection on retention of the foreign antigen-expressing plasmid. Both delivery strains induced sufficient cytokine expression, but the level was significantly lower than that of the wild-type strain; the lowest cytokine expression was induced by the JOL2837 strain, indicating reduced endotoxicity. In parallel, IgG production was significantly enhanced by both delivery strains. Thus, the innate and adaptive immunogenicity of the strains was ensured. The environmental safety of these strains was ascertained through faecal dissemination assays. The nonpathogenicity of these strains to the host was confirmed by body weight monitoring, survival assays, and morphological and histological assessments of the vital organs. The in vitro assay in murine and human cell lines and in vivo safety assessments in mice suggest that these novel strains possess safety, invasiveness, and immunogenicity, making them ideal delivery strains. Overall, the results clearly showed that strain JOL2782 with sifA deletion had higher invasiveness, demonstrating superior vaccine deliverability, while JOL2837 with lpxE substitution for pagL and rfaL deletion had outstanding safety potential with drastically abridged endotoxicity.

The non-structural protein (NSs) and nucleoprotein (NP) of the severe fever with thrombocytopenia syndrome virus (SFTSV) encoded by the S segment are crucial for viral pathogenesis. They reside in viroplasm-like structures (VLS), but their interaction and their significance in viral propagation remain unclear. Here, we investigated the significance of the association between NSs and NP during viral infection through in-silico and in-vitro analyses. Through in-silico analysis, three possible binding sites were predicted, at positions C6S (Cystein at 6th position to Serine), W61Y (Tryptophan 61st to Tyrosine), and S207T (Serine 207th to Threonine), three mutants of NSs were developed by site-directed mutagenesis and tested for NP interaction by co-immunoprecipitation. NSsW61Y failed to interact with the nucleoprotein, which was substantiated by the conformational changes observed in the structural analyses. Additionally, molecular docking analysis corroborated that the NSW61Y mutant protein does not interact well compared to wild-type NSs. Over-expression of wild-type NSs in HeLa cells increased the SFTSV replication by five folds, but NSsW61Y exhibited 1.9-folds less viral replication than wild-type. We demonstrated that the W61Y alteration was implicated in the reduction of NSs-NP interaction and viral replication. Thus, the present study identified a critical NSs site, which could be targeted for development of therapeutic regimens against SFTSV.

Currently, there are no commercial vaccines or therapeutics against severe fever with thrombocytopenia syndrome (SFTS) virus. This study explored an engineered Salmonella as a vaccine carrier to deliver a eukaryotic self-mRNA replicating vector, pJHL204. This vector expresses multiple SFTS virus antigenic genes for the nucleocapsid protein (NP), glycoprotein precursor (Gn/Gc), and nonstructural protein (NS) to induce host immune responses. The engineered constructs were designed and validated through 3D structure modeling. Western blot and qRT-PCR analyses of transformed HEK293T cells confirmed the delivery and expression of the vaccine antigens. Significantly, mice immunized with these constructs demonstrated a cell-mediated and humoral response as balanced Th1/Th2 immunity. The JOL2424 and JOL2425 delivering NP and Gn/Gc generated strong immunoglobulin IgG and IgM antibodies and high neutralizing titers. To further examine the immunogenicity and protection, we utilized a human DC-SIGN receptor transduced mouse model for SFTS virus infection by an adeno-associated viral vector system. Among the SFTSV antigen constructs, the construct with full-length NP and Gn/Gc and the construct with NP and selected Gn/Gc epitopes induced robust cellular and humoral immune responses. These were followed by adequate protection based on viral titer reduction and reduced histopathological lesions in the spleen and liver. In conclusion, these data indicate that recombinant attenuated Salmonella JOL2424 and JOL2425 delivering NP and Gn/Gc antigens of SFTSV are promising vaccine candidates that induce strong humoral and cellular immune responses and protection against SFTSV. Moreover, the data proved that the hDC-SIGN transduced mice as a worthy tool for immunogenicity study for SFTSV.

Abstract Acinetobacter baumannii (AB) is rising as a human pathogen of critical priority worldwide as it is the leading cause of chronic opportunistic infections in healthcare settings and the condition is ineradicable with antibiotic therapy. AB possesses the ability to form biofilm on abiotic as well as biotic surfaces which plays a major role in its pathogenesis and resistance in clinical settings. Hence, the demand for an alternative therapy to combat the biofilm-associated infections is increasing. The present study explored the antibiofilm potential of myrtenol, a bicyclic monoterpene present in various plants against reference and clinical strains of AB. Myrtenol (200 μg/mL) exhibited a strong antibiofilm activity without exerting any harmful effect on growth and metabolic viability of AB strains. Microscopic analyses confirmed the reduction in the biofilm thickness and surface coverage upon myrtenol treatment. Especially, myrtenol was found to be effective in disrupting the mature biofilms of tested AB strains. Furthermore, myrtenol inhibited the biofilm-associated virulence factors of AB strains such as extracellular polysaccharide, cell surface hydrophobicity, oxidant resistance, swarming and twitching motility. Transcriptional analysis unveiled the suppression of the biofilm-associated genes such as bfmR, csuA/B, bap, ompA, pgaA, pgaC, and katE by myrtenol. Notably, myrtenol improved the susceptibility of AB strains towards conventional antibiotics such as amikacin, ciprofloxacin, gentamicin and trimethoprim. Thus, the present study demonstrates the therapeutic potential of myrtenol against biofilm-associated infections of AB.

Bacteria-mediated cancer therapy has become a topic of interest under the broad umbrella of oncotherapy. Among many bacterial species, Salmonella remains at the forefront due to its ability to localize and proliferate inside tumor microenvironments and often suppress tumor growth. Salmonella Typhimurium is one of the most promising mediators, with engineering plasticity and cancer specificity. It can be used to deliver toxins that induce cell death in cancer cells specifically, and also as a cancer-specific instrument for immunotherapy by delivering tumor antigens and exposing the tumor environment to the host immune system. Salmonella can be used to deliver prodrug converting enzymes unambiguously against cancer. Though positive responses in Salmonella-mediated cancer treatments are still at a preliminary level, they have paved the way for developing combinatorial therapy with conventional chemotherapy, radiotherapy, and surgery, and can be used synergistically to combat multi-drug resistant and higher-stage cancers. With this background, Salmonella-mediated cancer therapy was approved for clinical trials by U.S. Food and Drug Administration, but the results were not satisfactory and more pre-clinical investigation is needed. This review summarizes the recent advancements in Salmonella-mediated oncotherapy in the fight against cancer. The present article emphasizes the demand for Salmonella mutants with high stringency toward cancer and with amenable elements of safety by virulence deletions.

Curcumin, a yellow-colored molecule derived from the rhizome of Curcuma longa, has been identified as the bioactive compound responsible for numerous pharmacological activities of turmeric, including anticancer, antimicrobial, anti-inflammatory, antioxidant, antidiabetic, etc. Nevertheless, the clinical application of curcumin is inadequate due to its low solubility, poor absorption, rapid metabolism and elimination. Advancements in recent research have shown several components and techniques to increase the bioavailability of curcumin. Combining with adjuvants, encapsulating in carriers and formulating in nanoforms, in combination with other bioactive agents, synthetic derivatives and structural analogs of curcumin, have shown increased efficiency and bioavailability, thereby augmenting the range of applications of curcumin. The scope for incorporating biotechnology and nanotechnology in amending the current drawbacks would help in expanding the biomedical applications and clinical efficacy of curcumin. Therefore, in this review, we provide a comprehensive overview of the plethora of therapeutic potentials of curcumin, their drawbacks in efficient clinical applications and the recent advancements in improving curcumin’s bioavailability for effective use in various biomedical applications.