The Effect of Tryptophan-to-Tyrosine Mutation at Position 61 of the Nonstructural Protein of Severe Fever with Thrombocytopenia Syndrome Virus on Viral Replication through Autophagosome Modulation.

In our prior investigations, we elucidated the role of the tryptophan-to-tyrosine substitution at the 61st position in the nonstructural protein NSsW61Y in diminishing the interaction between nonstructural proteins (NSs) and nucleoprotein (NP), impeding viral replication. In this study, we focused on the involvement of NSs in replication via the modulation of autophagosomes. Initially, we examined the impact of NP expression levels, a marker for replication, upon the infection of HeLa cells with severe fever thrombocytopenia syndrome virus (SFTSV), with or without the inhibition of NP binding. Western blot analysis revealed a reduction in NP levels in NSsW61Y-expressing conditions. Furthermore, the expression levels of the canonical autophagosome markers p62 and LC3 decreased in HeLa cells expressing NSsW61Y, revealing the involvement of individual viral proteins on autophagy. Subsequent experiments confirmed that NSsW61Y perturbs autophagy flux, as evidenced by reduced levels of LC3B and p62 upon treatment with chloroquine, an inhibitor of autophagosome-lysosome fusion. LysoTracker staining demonstrated a decrease in lysosomes in cells expressing the NS mutant compared to those expressing wild-type NS. We further explored the mTOR-associated regulatory pathway, a key regulator affected by NS mutant expression. The observed inhibition of replication could be linked to conformational changes in the NSs, impairing their binding to NP and altering mTOR regulation, a crucial upstream signaling component in autophagy. These findings illuminate the intricate interplay between NSsW61Y and the suppression of host autophagy machinery, which is crucial for the generation of autophagosomes to facilitate viral replication.

Intracellular Survival and Pathogenicity Modulation of Salmonella Lon, CpxR, and RfaL Mutants Used as Live Bacterial Vectors under Abiotic Stress, Unveiling the Link between Stress Response and Virulence in Epithelial Cells.

In the current study, two Salmonella Typhimurium strains, JOL 912 and JOL 1800, were engineered from the wild-type JOL 401 strain through in-frame deletions of the lon and cpxR genes, with JOL 1800 also lacking rfaL. These deletions significantly attenuated the strains, impairing their intracellular survival and creating unique immunological profiles. This study investigates the response of these strains to various abiotic stress conditions commonly experienced in vivo, including temperature, acidity, osmotic, and oxidative stress. Notably, cold stress induced a non-significant trend towards increased invasion by Salmonella compared to other stressors. Despite the observed attenuation, no significant alterations in entry mechanisms (trigger vs. zipper) were noted between these strains, although variations were evident depending on the host cell type. Both strains effectively localized within the cytoplasm, demonstrating their ability to invade and interact with the intracellular environment. Immunologically, JOL 912 elicited a robust response, marked by substantial activation of nuclear factor kappa B (NF-kB), and chemokines, interleukin 8 (CXCL 8) and interleukin 10 (CXCL 10), comparable to the wild-type JOL 401 (over a fourfold increase compared to JOL 1800). In contrast, JOL 1800 exhibited a minimal immune response. Additionally, these attenuations influenced the expression of cyclins D1 and B1 and caspases 3 and 7, indicating cell cycle arrest at the G2/M phase and promotion of the G0/G1 to S phase transition, alongside apoptosis in infected cells. These findings provide valuable insights into the mechanisms governing the association, internalization, and survival of Salmonella mutants, enhancing our understanding of their regulatory effects on host cell physiology.

Probiotic effects of Pseudoalteromonas ruthenica: Antibacterial, immune stimulation and modulation of gut microbiota composition.

This study aimed to characterise and evaluate the probiotic properties of a newly isolated marine bacterium, strain S6031. The isolated strain was identified as Pseudoalteromonas ruthenica. In vivo experiments were conducted with P. ruthenica-immersed larvae and P. ruthenica-enriched Artemia fed to adult zebrafish. Disease tolerance of larval zebrafish against Edwardsiella piscicida was demonstrated by 66.34% cumulative per cent survival (CPS) in the P. ruthenica-exposed group, which was higher than the CPS of the control (46.67%) at 72 h post challenge (hpc). Heat-stressed larvae had 55% CPS in the P. ruthenica-immersed group, while the control had 30% CPS at 60 hpc. Immune-stress response gene transcripts (muc5.1, muc5.2, muc5.3, alpi2, alpi3, hsp70, and hsp90a) were induced, while pro-inflammatory genes (tnfα, il1b, and il6) were downregulated in P. ruthenica-immersed larvae compared to the control. This trend was confirmed by low pro-inflammatory and high stress-responsive protein expression levels in P. ruthenica-exposed larvae. Adult zebrafish had higher CPS (27.2%) in the P. ruthenica-fed group than the control (9.52%) upon E. piscicida challenge, suggesting increased disease tolerance. Histological analysis demonstrated modulation of goblet cell density and average villus height in the P. ruthenica-supplemented group. Metagenomics analysis clearly indicated modulation of alpha diversity indices and the relative abundance of Proteobacteria in the P. ruthenica-supplemented zebrafish gut. Furthermore, increased Firmicutes colonisation and reduced Bacteroidetes abundance in the gut were observed upon P. ruthenica supplementation. Additionally, this study confirmed the concentration-dependent increase of colony dispersion and macrophage uptake upon mucin treatment. In summary, P. ruthenica possesses remarkable functional properties as a probiotic that enhances host defence against diseases and thermal stress.

Complete genome sequence analysis and phylogenetic classification of the novel Aeromonas phage AHP-1, a potential member of the genus Tequatrovirus.

Aeromonas phage AHP-1 was originally isolated from crucian carp (Carassius carassius) tissue. It was able to infect Aeromonas hydrophila and A. salmonicida. Genome sequence analysis revealed a 218,317-bp-long linear genome with an overall G + C content of 47.9%, 315 open reading frames (ORFs), and 25 tRNA sequences. Its genome was found to contain 67 unique ORFs (21.26%) that did not show any homology to previously characterized proteins. A comparative genome analysis suggested that its closest neighbors are unclassified phages belonging to the genus Tequatrovirus of the subfamily Tevenvirinae.

Genetic interference exerted by Salmonella-delivered CRISPR/Cas9 significantly reduces the pathological burden caused by Marek's disease virus in chickens.

Efficient in vivo delivery of a CRISPR/Cas9 plasmid is of paramount importance for effective therapy. Here, we investigated the usability of Salmonella as a plasmid carrier for in vivo therapy against virus-induced cancer using Marek's disease virus (MDV) as a model for study in chickens. A green fluorescent protein-expressing CRISPR/Cas9 plasmid encoding the virulence gene pp38 was constructed against Marek's disease virus. Therapeutic plasmids were transformed into Salmonella carrying lon and sifA gene deletions. The animals in 5 groups were intraperitoneally inoculated with phosphate-buffered saline, vector control, or Salmonella before or after MDV infection, or left uninfected as a naïve control. Therapeutic effectiveness was evaluated by observing disease outcomes and the viral copy number in peripheral blood mononuclear cells. The efficacy of plasmid delivery by Salmonella was 13 ± 1.7% in the spleen and 8.0 ± 1.8% in the liver on the 6th day post-infection. The Salmonella-treated groups showed significant resistance to MDV infection. The maximum effect was observed in the group treated with Salmonella before MDV infection. None of the chickens fully recovered; however, the results suggested that timely delivery of Salmonella could be effective for in vivo CRISPR/Cas9-mediated genetic interference against highly pathogenic MDV. The use of Salmonella in CRISPR systems provides a simpler and more efficient platform for in vivo therapy with CRISPR than the use of conventional in vivo gene delivery methods and warrants further development.

Copper-impregnated three-layer mask efficiently inactivates SARS-CoV2.

The present study investigates the potential of SARS-CoV-2 inactivation by a copper sulfide (CuS) incorporated three-layer mask design. The mask consisted of the outer, middle, and inner layers to give comfort, strength, shape, and safety. The outer layer contained a total of 4.4% CuS (w/w) (2.2% CuS coated & 2.2% CuS impregnated) nylon fibers and the middle entrapment area contain a total of 17.6% CuS (w/w) impregnated nylon. No CuS was present in the inner layer. The antiviral efficacy assessment revealed, CuS incorporated mask is highly effective in inactivating SARS-CoV-2 within 30 min exposure. After, 1h and 2 h exposure, near-complete elimination of virus were observed by cytopathy, fluorescence, and viral copy number. The antiviral activity of the mask material was derived by incorporated solid-state CuS. Noticeably, the antiviral activity of CuS against SARS-CoV-2 was in the form of solid-state CuS, but not as Cu2+ ionic form derived by dissolved CuSO4. The kinetics of droplet entrapment revealed, that the three-layered mask almost completely block virus-containing droplet pass-through for short exposure periods of 1-2 min, and 80% efficacy for longer exposure times of 5-10 min. We also demonstrated the incorporated CuS is evenly distributed all over the fibers assuring the uniformity of potential antiviral activity and proves, CuS particles are not easily shed out of the fabric fibers. The inactivation efficacy demonstrated against SARS-CoV-2 proves that the CuS incorporated three-layer mask will be a lifesaver during the present intense global pandemic.

A Novel Pseudoalteromonas xiamenensis Marine Isolate as a Potential Probiotic: Anti-Inflammatory and Innate Immune Modulatory Effects against Thermal and Pathogenic Stresses.

A marine bacterial strain was isolated from seawater and characterized for it beneficial probiotic effects using zebrafish as a model system. The strain was identified by morphological, physiological, biochemical, and phylogenetic analyses. The strain was most closely related to Pseudoalteromonas xiamenensis Y2, with 99.66% similarity; thus, we named it Pseudoalteromonas xiamenensis S1131. Improvement of host disease tolerance for the P. xiamenensis isolate was adapted in a zebrafish model using Edwardsiella piscicida challenge. The larvae were pre-exposed to P. xiamenensis prior to E. piscicida challenge, resulting in a 73.3% survival rate compared to a 46.6% survival for the control. The treated larvae tolerated elevated temperatures at 38 °C, with 85% survival, compared to 60% survival for the control. Assessment of immunomodulatory responses at the mRNA level demonstrated the suppression of pro-inflammatory markers tnfα and il6, and upregulation of heat shock protein hsp90 and mucin genes. The same effect was corroborated by immunoblot analysis, revealing significant inhibition of Tnfα and an enhanced expression of the Hsp90 protein. The antibacterial activity of P. xiamenensis may be related to mucin overexpression, which can suppress bacterial biofilm formation and enhance macrophage uptake. This phenomenon was evaluated using nonstimulated macrophage RAW264.7 cells. Further studies may be warranted to elucidate a complete profile of the probiotic effects, to expand the potential applications of the present P. xiamenensis isolate.

Enhancement of host infectivity, immunity, and protective efficacy by addition of sodium bicarbonate antacid to oral vaccine formulation of live attenuated Salmonella secreting Brucella antigens.

In the present study, the importance of sodium bicarbonate antacid as an agent for an orally delivered attenuated Salmonella strain secreting Brucella antigens Cu-Zn superoxide dismutase (SodC) and outer membrane protein 19 (Omp19) as a live vaccine candidate against Brucella infection was investigated. First, Brucella antigens SodC and Omp19 were cloned into a prokaryotic constitutive expression vector, pJHL65. Then secretion of proteins was verified after transformation into an attenuated Salmonella typhimurium (ST) strain, JOL1800 (Δlon, ΔcpxR, Δasd, ΔrfaL), using western blot analysis. Mice were orally inoculated with phosphate-buffered saline (PBS) or with a co-mixture Salmonella secreting each antigens at a 1:1 ratio, each containing 1 × 108 CFU/mouse with and without sodium bicarbonate treatment. For antacid treatment, 1.3% w/v sodium bicarbonate was orally administered 30 min before and immediately after immunization with the Salmonella formulation. Humoral and cell-mediated immune responses were evaluated to investigate the efficacy of sodium bicarbonate in an oral formulation. The results indicated that addition of sodium bicarbonate to the vaccine significantly increased (P < 0.05) levels of anti-Brucella-specific systemic IgG responses, lymphocyte proliferation, and CD4+ T cell responses, indicating induction of a mixed Th1-Th2 response. Immunohistochemical assays and bacterial enumeration in intestinal samples also indicated that administration of sodium bicarbonate enhanced colonization of Salmonella. These results indicate that ingestion of the Salmonella formulation with sodium bicarbonate can enhance colonization of Salmonella and induce a significant protective immune response against Brucella compared with a formulation without sodium bicarbonate. Thus, incorporation of sodium bicarbonate as an antacid buffer is highly recommended for this oral live vaccine.

Parenteral immunization of Salmonella Typhimurium ghosts with surface-displayed Escherichia coli flagellin enhancesTLR-5 mediated activation of immune responses that protect the chicken against Salmonella infection.

The present study investigates the enhancement of immunogenicity and protection efficacy of Salmonella Typhimurium ghosts surface-displayed with FliC against chicken salmonellosis. The membrane-anchored FliC is a potential TLR-5 agonist, delivers an essential adjuvant effect for the ghost vaccine candidate. The present ghost plasmid pJHL184 construct carries a convergent dual promoter system that has the temperature-dependent induction of the phage lysis gene E and the target antigen FliC at the same time. Under permissible conditions of temperatures, less than 30 °C at the presence of 20 mM l-arabinose effectively suppresses expression of the lysis gene. Once the temperature is up-lifted to 42 °C without arabinose, cause the generation of ST ghosts expelling the cytoplasmic content. The addition of FliC adjuvant significantly enhanced the IgY response, cell-mediated immune responses, regulatory cytokine induction and subsequently enhanced protection against Salmonella challenge. Further, intramuscular immunization with ST ghosts displaying FliC induced particularly high CD8+ response demarcating its proficiency to elicit Type I immune responses. Further, ST ghosts displaying FliC caused an increase in both CD4+ and CD8+ response compared to the PBS control suggesting its capability to engage both cell-mediated and humoral immune responses essential for the elimination of Salmonella. Upon the virulent challenge, we could observe a significant reduction in challenged bacterial load on spleen, liver and cecum tissues in the ST ghosts surface-displaying FliC adjuvant. Our study suggests the biological incorporation of FliC on ST ghosts enhances vaccine immunogenic potency and acts as a safe and effective prevention strategy against chicken salmonellosis.

Live vaccine consisting of attenuated Salmonella secreting and delivering Brucella ribosomal protein L7/L12 induces humoral and cellular immune responses and protects mice against virulent Brucella abortus 544 challenge.

The present study employs the Brucella abortus L7/L12 antigen in a Salmonella secretion platform and investigates its ability to induce protective immune responses against wild type challenge in BALB/c mice. The highly conserved L7/L12 open reading frame was PCR amplified from B. abortus and cloned into a prokaryotic expression vector, pJHL65, directly under the beta-lactamase secretory signal. The plasmid constructs pJHL65::L7/L12 was then transformed into an attenuated Salmonella Typhimurium strain, JOL1800 (∆lon, ∆cpxR, ∆asd, and ∆rfaL), and protein secretion was verified by Western blot. Three mice groups were inoculated with either phosphate-buffered saline (PBS), vector-only control, or the vaccine strain secreting L7/L12 antigen. Assessment of humoral and cell-mediated immune responses revealed successful elicitation of Brucella antigen-specific Th1 and Th2 immune responses that were significantly higher than PBS and vector control groups. The immune responses were confirmed by splenocyte proliferation assay, flow cytometry analysis for CD4+ and CD8+ markers, and RT-PCR based cytokine profiling upon restimulation with L7/L12 purified antigen. Results indicate that immunization with Salmonella secreting L7/L12 antigen demonstrated significant enhancement of cell-mediated immune (CMI) responses in immunized mice. The overall effectiveness of the immunization was evaluated by challenging with virulent B. abortus that revealed significant reduction in Brucella colonization in spleen and liver tissues in Salmonella L7/L12 immunized mice. Delivery of Brucella protective antigen L7/L12 using the Salmonella secretion system can effectively accomplish immunogenic advantages of both Salmonella and L7/L12 to derive robust CMI responses and induce humoral immunity to protect against Brucella infection in the mouse model.

Intranasally administered protein coated chitosan nanoparticles encapsulating influenza H9N2 HA2 and M2e mRNA molecules elicit protective immunity against avian influenza viruses in chickens.

Chitosan nanoparticles (CNPs) represent an efficient vaccination tool to deliver immunogenic antigens to the antigen-presenting cells (APCs), which subsequently stimulate protective immune responses against infectious diseases. Herein, we prepared CNPs encapsulating mRNA molecules followed by surface coating with conserved H9N2 HA2 and M2e influenza proteins. We demonstrated that CNPs efficiently delivered mRNA molecules into APCs and had effectively penetrated the mucosal barrier to reach to the immune initiation sites. To investigate the potential of CNPs delivering influenza antigens to stimulate protective immunity, we intranasally vaccinated chickens with empty CNPs, CNPs delivering HA2 and M2e in both mRNA and protein formats (CNPs + RNA + Pr) or CNPs delivering antigens in protein format only (CNPs + Pr). Our results demonstrated that chickens vaccinated with CNPs + RNA + Pr elicited significantly (p < 0.05) higher systemic IgG, mucosal IgA antibody responses and cellular immune responses compared to the CNPs + Pr vaccinated group. Consequently, upon challenge with either H7N9 or H9N2 avian influenza viruses (AIVs), efficient protection, in the context of viral load and lung pathology, was observed in chickens vaccinated with CNPs + RNA + Pr than CNPs + Pr vaccinated group. In conclusion, we show that HA2 and M2e antigens elicited a broad spectrum of protection against AIVs and incorporation of mRNAs in vaccine formulation is an effective strategy to induce superior immune responses.

Oral immunization with an attenuated Salmonella Gallinarum encoding the H9N2 haemagglutinin and M2 ectodomain induces protective immune responses against H9N2 infection in chickens.

H9N2, a low pathogenic avian influenza virus, causes significant economic losses in the poultry industry worldwide. Herein, we describe the construction of an attenuated Salmonella Gallinarum (SG) strain for expression and delivery of H9N2 haemagglutinin (HA) 1 (SG-HA1), HA2 (SG-HA2) and/or the conserved matrix protein 2 ectodomain (SG-M2e). We demonstrated that recombinant SG strains expressing HA1, HA2 and M2e antigens were immunogenic and safe in a chicken model. Chickens (n = 8) were vaccinated once orally with SG alone, SG-HA1, SG-HA2, SG-M2e, or mixture of SG-HA1, SG-HA2 and SG-M2e, or vaccinated once intramuscularly with an oil-adjuvant inactivated H9N2 vaccine. Our results demonstrated that vaccination with SG mutants encoding influenza antigens, administered individually or as a mixture, elicited significantly (P < 0.05) greater antigen-specific humoral and cell-mediated immune responses in chickens compared with those vaccinated with SG alone. A conventional H9N2 vaccine induced significantly (P < 0.05) greater HA1 and HA2 antibody responses than SG-based H9N2 vaccine strains, but significantly (P < 0.05) less robust M2e-specific responses. Upon challenge with the virulent H9N2 virus on day 28 post-vaccination, chickens vaccinated with either the SG-based H9N2 or conventional H9N2 vaccines exhibited comparable lung inflammation and viral loads, although both were significantly lower (P < 0.05) than in the group vaccinated with SG alone. In conclusion, our results showed that SG-based vaccination stimulated efficient immune responses against virulent H9N2. Further studies are needed to fully develop this approach as a preventive strategy for low pathogenic avian influenza viruses affecting poultry. RESEARCH HIGHLIGHTS S. gallinarum expressing HA1, HA2 and M2e antigens are immunogenic and safe. Salmonella has dual function of acting as a delivery system and as a natural adjuvant. Vaccine constructs elicit specific humoral and cell-mediated immune responses.

Isolation and Characterization of Multidrug Resistance Aeromonas salmonicida subsp. salmonicida and Its Infecting Novel Phage ASP-1 from Goldfish (Carassius auratus).

In this study, Aeromonas salmonicida subsp. salmonicida was isolated, identified by 16S RNA sequencing and its potential lytic phage (ASP-1) was isolated and characterized. The bacterium was positive for virulence genes (ascV, fla, ahyB, gcaT, lip, alt and act) and phenotypic parameters (haemolysis, slime production, lipase activity, DNase test, gelatinase activity and protease activity) were tested. The bacterium was resistant to 27%, intermediate resistant to 14% and susceptible to 59% of tested common antibiotics. Transmission electron microscopy analysis revealed that lytic ASP-1 belongs to the Myoviridae family. The isolated phage was more specific against A. salmonicida subsp. salmonicida (efficiency of plating index = 1), but also had infectivity to A. hydrophila lab strain 1. The bacteriolytic effect of ASP-1 was tested at early exponential phase culture of A. salmonicida subsp. salmonicida, and bacteria growth was apparently decreased with time and MOI dependent manner. One-step growth of ASP-1 showed approximately 30 min of latent period, 16 PFU/infected cells of burst size and 40 min of rise period. The adsorption rate was determined as 3.61 × 108 PFU mL-1 min-1 for 3 min, and rate decreased with time. The ASP-1 genome size was estimated to be approximately 55-60 kD. The phage was stable over wide-range of temperatures, pH and salinity, thus could withstand at severe environmental conditions, indicating that ASP-1 has a potential to develop as an alternative antibiotic to use in ornamental and aquaculture industry.

Complete genome sequence analysis of a novel Staphylococcus phage StAP1 and proposal of a new species in the genus Silviavirus.

Bacteriophage StAP1 was isolated from a soil sample infecting Staphylococcus aureus and S. xylosus. Its genome was found to be 135,502 base pairs (bp) long with 30.00 mol% G+C content and 192 open reading frames. While no tRNA encoding genes were identified, 7 mobile elements were found to interrupt five StAP1 open reading frames. Comparative genomic and proteomic analysis consistently supports the establishment of a new species in the genus Silviavirus.

Putative Inv is essential for basolateral invasion of Caco-2 cells and acts synergistically with OmpA to affect in vitro and in vivo virulence of Cronobacter sakazakii ATCC 29544.

Cronobacter sakazakii is an opportunistic pathogen that causes neonatal meningitis and necrotizing enterocolitis. Its interaction with intestinal epithelium is important in the pathogenesis of enteric infections. In this study, we investigated the involvement of the inv gene in the virulence of C. sakazakii ATCC 29544 in vitro and in vivo. Sequence analysis of C. sakazakii ATCC 29544 inv revealed that it is different from other C. sakazakii isolates. In various cell culture models, an Δinv deletion mutant showed significantly lowered invasion efficiency, which was restored upon genetic complementation. Studying invasion potentials using tight-junction-disrupted Caco-2 cells suggested that the inv gene product mediates basolateral invasion of C. sakazakii ATCC 29544. In addition, comparison of invasion potentials of double mutant (ΔompA Δinv) and single mutants (ΔompA and Δinv) provided evidence for an additive effect of the two putative outer membrane proteins. Finally, the importance of inv and the additive effect of putative Inv and OmpA were also proven in an in vivo rat pup model. This report is the first to demonstrate two proteins working synergistically in vitro, as well as in vivo in C. sakazakii pathogenesis.

Assessment of environmental safety and protective efficacy of O-antigen deficient DIVA capable Salmonella Enteritidis against chicken salmonellosis.

In this study, we incorporated deletion of the O-antigen ligase gene to an attenuated Salmonella Enteritidis (SE) strain, JOL919 (SE PS; Δlon ΔcpxR), using the Lambda-Red recombination method and evaluated the safety and immunological aspects of the novel genotype, JOL2381 (SE VS: Δlon, ΔcpxR, ΔrfaL). Assessment of fecal shedding and organ persistence following administration via oral and IM routes revealed that the SE VS was safer than its parent strain, SE PS. Immunological assays confirmed that immunization via the oral route with SE PS was superior to the SE VS. However, chickens immunized with SE PS and SE VS strains via the IM route showed higher humoral and cell-mediated immune responses. Compared to PBS control, the IM route of immunization with SE VS resulted in a higher IgY antibody titer and expansion of CD4+ and CD8+ T-cell populations, which resulted in the clearance of Salmonella from the liver and splenic tissues. Furthermore, deletion of the O-antigen ligase gene caused lower production of LPS-specific antibodies in the host, promoting DIVA functionality and making it a plausible candidate for field utilization. Due to significant protection, high attenuation, and environmental safety concerns, the present SE VS strain is an ideal choice to prevent chicken salmonellosis and ensure public health.

Salmonella vector induces protective immunity against Lawsonia and Salmonella in murine model using prokaryotic expression system.

BACKGROUND: Lawsonia intracellularis is the causative agent of proliferative enteropathy and is associated with several outbreaks, causing substantial economic loss to the porcine industry. OBJECTIVES: In this study, we focused on demonstrating the protective effect in the mouse model through the immunological bases of two vaccine strains against porcine proliferative enteritis. METHODS: We used live-attenuated Salmonella Typhimurium (ST) secreting two selected immunogenic LI antigens (Lawsonia autotransporter A epitopes and flagellin [FliC]-peptidoglycan-associated lipoprotein-FliC) as the vaccine carrier. The constructs were cloned into a Salmonella expression vector (pJHL65) and transformed into the ST strain (JOL912). The expression of immunogenic proteins within Salmonella was evaluated via immunoblotting. RESULTS: Immunizing BALB/c mice orally and subcutaneously induced high levels of LI-specific systemic immunoglobulin G and mucosal secretory immunoglobulin A. In immunized mice, there was significant upregulation of interferon-γ and interleukin-4 cytokine mRNA and an increase in the subpopulations of cluster of differentiation (CD) 4+ and CD 8+ T lymphocytes upon splenocytes re-stimulation with LI antigens. We observed significant protection in C57BL/6 mice against challenge with 106.9 times the median tissue culture infectious dose of LI or 2 × 109 colony-forming units of the virulent ST strain. Immunizing mice with either individual vaccine strains or co-mixture inhibited bacterial proliferation, with a marked reduction in the percentage of mice shedding Lawsonia in their feces. CONCLUSIONS: Salmonella-mediated LI gene delivery induces robust humoral and cellular immune reactions, leading to significant protection against LI and salmonellosis.

Genome Characterization of Bacteriophage KPP-1, a Novel Member in the Subfamily Vequintavirinae, and Use of Its Endolysin for the Lysis of Multidrug-Resistant Klebsiella variicola In Vitro.

Multidrug-resistant members of the Klebsiella pneumoniae complex have become a threat to human lives and animals, including aquatic animals, owing to the limited choice of antimicrobial treatments. Bacteriophages are effective natural tools available to fight against multidrug-resistant bacteria. The bacteriophage KPP-1 was found to be strictly lytic against K. variicola, a multidrug-resistant isolate, producing clear plaques. The genome sequence analysis of KPP-1 revealed that it comprised 143,369 base pairs with 47% overall GC content. A total of 272 genes (forward 161, complementary 111) encode for 17 tRNAs and 255 open reading frames (ORFs). Among them, 32 ORFs could be functionally annotated using the National Center for Biotechnology Information (NCBI) Protein Basic Local Alignment Search Tool (BLASTp) algorithm while 223 were found to code for hypothetical proteins. Comparative genomic analysis revealed that the closest neighbor of KPP-1 can be found in the genus Mydovirus of the subfamily Vequintavirinae. KPP-1 not only markedly suppressed the growth of the host but also worked synergistically with ampicillin. Useful genes for pathogen control such as endolysin (locus tag: KPP_11591) were found to have activity against multidrug-resistant isolate of K. variicola. Further studies are necessary to develop a strategy to control the emerging pathogen K. variicola using bacteriophages such as KPP-1.

The impact of lipid A modification on biofilm and related pathophysiological phenotypes, endotoxicity, immunogenicity, and protection of Salmonella Typhimurium.

This study presents the engineering of a less endotoxic Salmonella Typhimurium strain by manipulating the lipid-A structure of the lipopolysaccharide (LPS) component. Salmonella lipid A was dephosphorylated by using lpxE from Francisella tularensis. The 1-phosphate group from lipid-A was removed selectively, resulting in a close analog of monophosphoryl lipid A. We observed a significant impact of ∆pagL on major virulence factors such as biofilm formation, motility, persistency, and immune evasion. In correlation with biofilm and motility retardation, adhesion and invasion were elevated but with reduced intracellular survival, a favorable phenotype prospect of a vaccine strain. Western blotting and silver staining confirmed the absence of the O-antigen and truncated lipid-A core in the detoxified Salmonella mutant. In vitro and in vivo studies demonstrated that the dephosphorylated Salmonella mutant mediated lower pro-inflammatory cytokine secretion than the wild-type strain. The vaccine strains were present in the spleen and liver for five days and were cleared from the organs by day seven. However, the wild-type strain persisted in the spleen, liver, and brain, leading to sepsis-induced death. Histological evaluations of tissue samples further confirmed the reduced endotoxic activity of the detoxified Salmonella mutant. The detoxification strategy did not compromise the level of protective immunity, as the vaccine strain could enhance humoral and cellular immune responses and protect against the wild-type challenge in immunized mice.

Inflammation-Related Immune-Modulatory SLURP1 Prevents the Proliferation of Human Colon Cancer Cells, and Its Delivery by Salmonella Demonstrates Cross-Species Efficacy against Murine Colon Cancer.

This study investigates the anticancer properties of the α7-nAChR antagonist SLURP1 with a specific focus on its effect as an inflammation modulator on human colorectal cancer cell lines Caco2, Colo320DM, and H508 cells. The investigation includes the evaluation of cell cycle arrest, cell migration arrest, endogenous expression of SLURP1 and related proteins, calcium influx, and inflammatory responses. The results demonstrate that SLURP1 not only inhibits cell proliferation but also has the potential to arrest the cell cycle at the G1/S interface. The impact of SLURP1 on cell cycle regulation varied among cell lines, with H508 cells displaying the strongest response to exogenous SLURP1. Additionally, SLURP1 affects the nuclear factor kappa B expression and effectively reverses inflammatory responses elicited by purified lipopolysaccharides in H508 and Caco2 cells. This study further confirmed the expression of human SLURP1 by Salmonella, under Ptrc promoter, through Western blot analysis. Moreover, Salmonella secreting SLURP1 revealed a significant tumor regression in a mouse CT26 tumor model, suggesting the cross-species anticancer potential of human SLURP1. However, further investigations are required to fully understand the mechanisms underlying SLURP1's ability to prevent cancer proliferation and its protective function in humans.