A soluble DR5-Fc chimeric protein attenuates inflammatory responses induced by coronavirus MHV-A59 and SARS-CoV-2.

Mortality in coronavirus disease 2019 (COVID-19) patients has been linked to the presence of a "cytokine storm" induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, which involves elevated levels of circulating cytokines and immune-cell hyperactivation. Targeting cytokines during the management of COVID-19 patients has the potential to improve survival rates and reduce mortality. Although cytokine blockers and immune-host modulators are currently being tested in severely ill COVID-19 patients to cope with the overwhelming systemic inflammation, there is not too many successful cases, thus finding new cytokine blockers to attenuate the cytokine storm syndrome is meaningful. In this paper, we significantly attenuated the inflammatory responses induced by mouse hepatitis viruses A59 and SARS-CoV-2 through a soluble DR5-Fc (sDR5-Fc) chimeric protein that blocked the TNF-related apoptosis-inducing ligand-death receptor 5 (TRAIL-DR5) interaction. Our findings indicates that blocking the TRAIL-DR5 pathway through the sDR5-Fc chimeric protein is a promising strategy to treat COVID-19 severe patients requiring intensive care unit  admission or with chronic metabolic diseases.

Comparison of model-specific histopathology in mouse models of COVID-19.

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has been identified as the causative agent of the current coronavirus disease 2019 pandemic. Development of animal models that parallel the clinical and pathologic features of disease are highly essential to understanding the pathogenesis of SARS-CoV-2 infection and the development of therapeutics and prophylactics. Several mouse models that express the human angiotensin converting enzyme 2 (hACE2) have been created, including transgenic and knock-in strains, and viral vector-mediated delivery of hACE2. However, the comparative pathology of these mouse models infected with SARS-CoV-2 are unknown. Here, we perform systematic comparisons of the mouse models including K18-hACE2 mice, KI-hACE2 mice, Ad5-hACE2 mice and CAG-hACE2 mice, which revealed differences in the distribution of lesions and the characteristics of pneumonia induced. Based on these observations, the hACE2 mouse models meet different needs of SARS-CoV-2 researches. The similarities or differences among the model-specific pathologies may help in better understanding the pathogenic process of SARS-CoV-2 infection and aiding in the development of effective medications and prophylactic treatments for SARS-CoV-2.

Salmonella effector SpvB interferes with intracellular iron homeostasis via regulation of transcription factor NRF2.

Iron is a necessary nutrient for humans and nearly all bacterial species. During Salmonella infection, macrophages limit the availability of iron to intracellular pathogens in one of the central components of nutritional immunity. However, Salmonella also have mechanisms to interfere with the antimicrobial effect of host iron withdrawal and meet their own nutrient requirements by scavenging iron from the host. Here, we provide what is, to our knowledge, the first report that SpvB, a pSLT-encoded cytotoxic protein whose function is associated with the intracellular stage of salmonellosis, perturbs macrophage iron metabolism, thereby facilitating Salmonella survival and intracellular replication. In investigating the underlying mechanism, we observed that the Salmonella effector SpvB down-regulated nuclear factor erythroid-derived 2-related factor 2 (NRF2), and its C-terminal domain was necessary and sufficient for NRF2 degradation via the proteasome pathway. Decreased NRF2 expression in the nucleus resulted in a decrease in its transcriptional target ferroportin, encoding the sole macrophage iron exporter, thus ultimately decreasing iron efflux and increasing the intracellular iron content. Additionally, SpvB contributes to the pathogenesis of Salmonella including severe serum hypoferremia, increased splenic and hepatic bacterial burden, and inflammatory injury in vivo. Together, our observations uncovered a novel contribution of SpvB to Salmonella pathology via interference with host intracellular iron metabolism.-Yang, S., Deng, Q., Sun, L., Dong, K., Li, Y., Wu, S., Huang, R. Salmonella effector SpvB interferes with intracellular iron homeostasis via regulation of transcription factor NRF2.

spv locus aggravates Salmonella infection of zebrafish adult by inducing Th1/Th2 shift to Th2 polarization.

Salmonella enterica serovar typhimurium (S. typhimurium) are facultative intracellular enteric pathogens causing disease with a broad range of hosts. It was known that Th1-type cytokines such as IFN-γ, IL-12, and TNF-α etc. could induce protective immunity against intracellular pathogens, while Th2-type cytokines such as IL-4, IL-10, and IL-13 etc. are proved to help pathogens survive inside hosts and cause severe infection. One of the critical virulence factor attributes to the pathogenesis of S. typhimurium is Salmonella plasmid virulence genes (spv). Until now, the interaction between spv locus and the predictable generation of Th1 or Th2 immune responses to Salmonella has not been identified. In this study, zebrafish adults were employed to explore the effect of spv locus on Salmonella pathogenesis as well as host adaptive immune responses especially shift of Th1/Th2 balance. The pathological changes of intestines and livers in zebrafish were observed by hematoxylin-eosin (HE) staining and electron microscopy. Levels of the transcription factors of Th1 (Tbx21) and Th2 (GATA3) were measured by real-time quantitative PCR (RT-qPCR). Expression of cytokines were determined by using RT-qPCR and ELISA, respectively. Results showed that spv operon aggravates damage of zebrafish. Furthermore, it demonstrated that spv locus could inhibit the transcription of tbx21 gene and suppress the expression of cytokines IFN-γ, IL-12 and TNF-α. On the contrary, the transcription of gata3 gene could be promoted and the expression of cytokines IL-4, IL-10 and IL-13 were enhanced by spv locus. Taken together, our data revealed that spv locus could aggravate Salmonella infection of zebrafish adult by inducing an imbalance of Th1/Th2 immune response and resulting in a detrimental Th2 bias of host.

Protein/peptide-based entry/fusion inhibitors as anti-HIV therapies: challenges and future direction.

The failures of several first-generation and second-generation small molecule drug-based anti-HIV therapies in various stages of clinical trials are an indication that there is a need for a paradigm shift in the future designs of anti-HIV therapeutics. Over the past several decades, various anti-HIV drugs have been developed, among them, protein/peptide-based therapies. From the first peptide discovered (SJ2176) to the first peptide approved by the Food and Drug Administration (DP178/T20/enfuvirtide/Fuzeon®), anti-HIV proteins/peptides as fusion/entry inhibitors have been shown to provide potent effects and benefits. This review summarizes the past and current endeavors in this area, discusses the potential mechanisms of action for various anti-HIV proteins/peptides, compares the advantages and disadvantages between the different proteins/peptides, and finally, examines the future direction of the field, specifically, strategies that will enhance the therapeutic efficacy of fusion/entry inhibitor-based anti-HIV proteins/peptides. Although there are numerous reviews highlighting the general field of entry/fusion inhibitors, there is a lack of literature focused on protein/peptide-based entry/fusion inhibitors for HIV therapy, and as a result, this review is intended to fill this void by summarizing the past, current, and future development of these macromolecules. Copyright © 2015 John Wiley & Sons, Ltd.

Biodegradable Film for the Targeted Delivery of siRNA-Loaded Nanoparticles to Vaginal Immune Cells.

The goal of this study was to develop and characterize a novel intravaginal film platform for targeted delivery of small interfering RNA (siRNA)-loaded nanoparticles (NP) to dendritic cells as a potential gene therapy for the prevention of sexually transmitted human immunodeficiency virus (HIV) infection. Poly(ethylene glycol) (PEG)-functionalized poly(D, L-lactic-co-glycolic acid) (PLGA)/polyethylenimine (PEI)/siRNA NP (siRNA-NP) were fabricated using a modified emulsion-solvent evaporation method and characterized for particle size, zeta potential, encapsulation efficiency (EE), and siRNA release. siRNA-NP were decorated with anti-HLA-DR antibody (siRNA-NP-Ab) for targeting delivery to HLA-DR+ dendritic cells (DCs) and homogeneously dispersed in a biodegradable film consisting of poly vinyl alcohol (PVA) and λ-carrageenan. The siRNA-NP-Ab-loaded film (siRNA-NP-Ab-film) was transparent, displayed suitable physicomechanical properties, and was noncytotoxic. Targeting activity was evaluated in a mucosal coculture model consisting of a vaginal epithelial monolayer (VK2/E6E7 cells) and differentiated KG-1 cells (HLA-DR+ DCs). siRNA-NP-Ab were rapidly released from the film and were able to penetrate the epithelial layer to be taken up by differentiated KG-1 cells. siRNA-NP-Ab demonstrated higher targeting activity and significantly higher knockdown of synaptosome-associated 23-kDa protein (SNAP-23) mRNA and protein when compared to siRNA-NP without antibody conjugation. Overall, these data suggest that our novel siRNA-NP-Ab-film may be a promising platform for preventing HIV infection within the female genital tract.

Development of an Analytical Method for the Rapid Quantitation of Peptides Used in Microbicide Formulations.

Recently, a growing number of macromolecules such as peptides and proteins have been formulated into various microbicide formulations for the prevention of sexually transmitted infections. However, a fast and reliable high-throughput method for quantitating peptide/protein in polymer-based microbicide formulations is still lacking. As a result, we developed and validated a reversed-phase high-performance liquid chromatography method for the quantitation of gp120 fragment and LL-37 simultaneously in various microbicide gel formulations. This method was capable of detecting a limit of linearity (regression coefficient of 0.999) for gp120 fragment and LL-37 within a range of 0.625-80 and 1.25-80 µg mL-1, respectively. The lower limit of quantification for gp120 fragment and LL-37 was 1.14 and 0.31 µg mL-1, respectively. Method validation demonstrated acceptable intra- and inter-day RSD % (<5 %) and accuracy (95.67-100.5 %). Formulating both peptides into polymeric pharmaceutical gel formulations showed high extraction efficiency (in the range of 95.90 ± 3.03 to 111.45 ± 2.51 %). Using this method, we were able to separate and identify the forced degraded products from both peptides simultaneously without affecting the quantitation of both peptides in the polymeric dosage forms. Furthermore, this method was able to detect and separate degradants that were unable to be revealed using gel eletrophoresis.

pH-sensitive dual-preventive siRNA-based nanomicrobicide reactivates autophagy and inhibits HIV infection in vaginal CD4+ cells.

Women are more susceptible to HIV transmission through unprotected heterosexual intercourse due to biological and social vulnerabilities. Intravaginal delivery of siRNAs targeting viral genes, host genes, or in combination has shown promising outcomes against HSV, HPV and HIV. Therefore, in this study, we designed, developed and evaluated a pH-sensitive RNAi-based combination nanomicrobide for the prevention/reduction of vaginal transmission of HIV. The nanomicrobide was composed of siRNA-PEI encapsulated PLGA-PEG nanoparticles (siRNA NP) loaded in a HEC gel dosage form with siRNA targeting host gene CCR5 and the viral gene Nef as a dual preventive strategy. Knocking down CCR5, a co-receptor for HIV could prevent HIV from attaching to and entering host cells and knocking down Nef could reactivate autophagy that was inhibited by Nef to improve the elimination of intracellular virus that escaped the first line of defense. The siRNA NP showed a desirable particle size and zeta potential for intravaginal delivery and a pH-dependent release profile whereby low amounts of siRNA was released under acidic vaginal conditions (vaginal fluid simulant; VFS, pH 4.2) (6.0 ± 0.4% released over 15 days) but significantly higher amounts of siRNA was released under neutral pH conditions (phosphate buffered saline; PBS, pH 7.4) (22.9 ± 0.4% released over 15 days). The CCR5-Nef-specific siRNA NP efficiently knocked down CCR5 and Nef protein expression by 43% and 63%, respectively, reactivated Nef-blocked autophagy and inhibited the replication of HIV in vitro (71.8% reduction in p24 expression). After being formulated into a gel dosage form, siRNA NP could be readily released from the gel, penetrate the vaginal epithelial layer, get taken up into the target cells and knockdown Nef and CCR5 without causing cytotoxicity in a vaginal mucosal co-culture model. Functionalization of siRNA NP with anti-CD4 antibody and loaded into a 0.5% HEC gel improved vaginal distribution and uptake of siRNA in a mouse model with distribution of siRNA restricted to the reproductive tract without any unwanted systemic uptake. The 0.5% HEC gel loaded with siRNA NP-(m)CD4 significantly downregulated approximately 40% of CCR5 protein in the lower vagina and 36% of CCR5 protein in the upper vaginal and cervical region. In contrast, 0.5% HEC gel loaded with siRNA NP-IgG did not result in significant gene knockdown.

REV-ERBα negatively regulates NLRP6 transcription and reduces the severity of Salmonella infection in mice.

Non-typhoidal Salmonella infection is among the most frequent foodborne diseases threatening human health worldwide. The host circadian clock orchestrates daily rhythms to adapt to environmental changes, including coordinating immune function in response to potential infections. However, the molecular mechanisms underlying the interplay between the circadian clock and the immune system in modulating infection processes are incompletely understood. Here, we demonstrate that NLRP6, a novel nucleotide-oligomerization domain (NOD)-like receptor (NLR) family member highly expressed in the intestine, is closely associated with the differential day-night response to Salmonella infection. The core clock component REV-ERBα negatively regulates NLRP6 transcription, leading to the rhythmic expression of NLRP6 and the secretion of IL-18 in intestinal epithelial cells, playing a crucial role in mediating the differential day-night response to Salmonella infection. Activating REV-ERBα with agonist SR9009 in wild-type mice attenuated the severity of infection by decreasing the NLRP6 level in intestinal epithelial cells. Our findings provide new insights into the association between the host circadian clock and the immune response to enteric infections by revealing the regulation of Salmonella infection via the inhibitory effect of REV-ERBα on NLRP6 transcription. Targeting REV-ERBα to modulate NLRP6 activation may be a potential therapeutic strategy for bacterial infections.

NLRP6 induces RIP1 kinase-dependent necroptosis via TAK1-mediated p38MAPK/MK2 phosphorylation in S. typhimurium infection.

Programmed cell death (PCD) is tightly orchestrated by molecularly defined executors and signaling pathways. NLRP6, a member of cytoplasmic pattern recognition receptors, has a multifaceted role in host resistance to bacterial infection. However, whether and how NLRP6 may contribute to regulate host PCD during Gram-negative bacterial infection remain to be illuminated. Here, we report that NLRP6 promotes RIP1 kinase-mediated necroptosis, a form of lytic PCD, in both an in vitro and in vivo model of Salmonella typhimurium infection. By downregulating TAK1-mediated p38MAPK/MK2 phosphorylation, NLRP6 decreased RIP1 phosphorylation at residue S321 and subsequently increased RIP1 kinase-dependent MLKL phosphorylation. Suppression of p38MAPK/MK2 cascade not only reduced the number of dead cells caused by NLRP6 but also decreased the systemic dissemination of S. typhimurium resulting from NLRP6. Taken together, our findings provide new insights into the role and regulatory mechanism of NLRP6-associated antimicrobial responses by revealing a function for NLRP6 in regulating necroptosis.

SARS-CoV-2 ORF3a sensitizes cells to ferroptosis via Keap1-NRF2 axis.

Viral infection-induced cell death has long been considered as a double-edged sword in the inhibition or exacerbation of viral infections. Patients with severe Coronavirus Disease 2019 (COVID-19) are characterized by multiple organ dysfunction syndrome and cytokine storm, which may result from SARS-CoV-2-induced cell death. Previous studies have observed enhanced ROS level and signs of ferroptosis in SARS-CoV-2 infected cells or specimens of patients with COVID-19, but the exact mechanism is not clear yet. Here, we find SARS-CoV-2 ORF3a sensitizes cells to ferroptosis via Keap1-NRF2 axis. SARS-CoV-2 ORF3a promotes the degradation of NRF2 through recruiting Keap1, thereby attenuating cellular resistance to oxidative stress and facilitated cells to ferroptotic cell death. Our study uncovers that SARS-CoV-2 ORF3a functions as a positive regulator of ferroptosis, which might explain SARS-CoV-2-induced damage in multiple organs in COVID-19 patients and imply the potential of ferroptosis inhibition in COVID-19 treatment.

Synthesis of deuterated S-217622 (Ensitrelvir) with antiviral activity against coronaviruses including SARS-CoV-2.

S-217622 (Ensitrelvir) is a reversible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3-chymotrypsin-like protease (3CLpro) inhibitor which obtained emergency regulatory approval in Japan for the treatment of SARS-CoV-2 infection on Nov 22, 2022. Herein, analogs of S-271622 with deuterium-for-hydrogen replacement were synthesized for comparison of the antiviral activities and pharmacokinetic (PK) profiles. Compared to the parent compound, C11-d2-S-217622 compound YY-278 retained in vitro activity against 3CLpro and SARS-CoV-2. X-ray crystal structural studies showed similar interactions of SARS-CoV-2 3CLpro with YY-278 and S-271622. The PK profiling revealed the relatively favorable bioavailability and plasma exposure of YY-278. In addition, YY-278, as well as S-217622, displayed broadly anti-coronaviral activities against 6 other coronaviruses that infect humans and animals. These results laid the foundation for further research on the therapeutic potential of YY-278 against COVID-19 and other coronaviral diseases.

Fused deposition modeling three-dimensional printing of flexible polyurethane intravaginal rings with controlled tunable release profiles for multiple active drugs.

We designed and engineered novel intravaginal ring (IVR) medical devices via fused deposition modeling (FDM) three-dimensional (3D) printing for controlled delivery of hydroxychloroquine, IgG, gp120 fragment (encompassing the CD4 binding site), and coumarin 6 PLGA-PEG nanoparticles (C6NP). The hydrophilic polyurethanes were utilized to 3D-print reservoir-type IVRs containing a tunable release controlling membrane (RCM) with varying thickness and adaptable micro porous structures (by altering the printing patterns and interior fill densities) for controlled sustained drug delivery over 14 days. FDM 3D printing of IVRs were optimized and implemented using a lab-developed Cartesian 3D printer. The structures were investigated by scanning electron microscopy (SEM) imaging and in vitro release was performed using 5 mL of daily-replenished vaginal fluid simulant (pH 4.2). The release kinetics of the IVR segments were tunable with various RCM (outer diameter to inner diameter ratio ranging from 1.12 to 2.61) produced from FDM 3D printing by controlling the printing perimeter to provide daily zero-order release of HCQ ranging from 23.54 ± 3.54 to 261.09 ± 32.49 µg/mL/day. IgG, gp120 fragment, and C6NP release rates demonstrated pattern and in-fill density-dependent characteristics. The current study demonstrated the utility of FDM 3D printing to rapidly fabricate complex micro-structures for tunable and sustained delivery of a variety of compounds including HCQ, IgG, gp120 fragment, and C6NP from IVRs in a controlled manner.

Salmonella pSLT-encoded effector SpvB promotes RIPK3-dependent necroptosis in intestinal epithelial cells.

Salmonella is one of the most important worldwide zoonotic pathogens. After invading a host orally, the bacteria break through the intestinal epithelial barrier for further invasion. Intestinal epithelial cells (IECs) play a crucial role in maintaining the integrity of the intestinal epithelial barrier. Necroptosis is considered one of the virulence strategies utilized by invasive Salmonella. Our previous work has shown that SpvB, an effector encoded by S. Typhimurium virulence plasmid (pSLT), promotes bacterial translocation via the paracellular route. However, it is still unknown whether SpvB could promote bacterial invasion through disrupting the integrity of IECs. Here, we demonstrated that SpvB promoted necroptosis of IECs and contributed to the destruction of the intestinal barrier during Salmonella infection. We found that SpvB enhanced the protein level of receptor-interacting protein kinase 3 (RIPK3) through inhibiting K48-linked poly-ubiquitylation of RIPK3 and the degradation of the protein in an autophagy-dependent manner. The abundant accumulation of RIPK3 upregulated the phosphorylation of MLKL, which contributed to necroptosis. The damage to IECs ultimately led to the disruption of the intestinal barrier and aggravated infection. In vivo, SpvB promoted the pathogenesis of Salmonella, favoring intestinal injury and colonic necroptosis. Our findings reveal a novel function of Salmonella effector SpvB, which could facilitate salmonellosis by promoting necroptosis, and broaden our understanding of the molecular mechanisms of bacterial invasion.

A detrimental role of NLRP6 in host iron metabolism during Salmonella infection.

Maintaining host iron homeostasis is an essential component of nutritional immunity responsible for sequestrating iron from pathogens and controlling infection. Nucleotide-oligomerization domain-like receptors (NLRs) contribute to cytoplasmic sensing and antimicrobial response orchestration. However, it remains unknown whether and how NLRs may regulate host iron metabolism, an important component of nutritional immunity. Here, we demonstrated that NLRP6, a member of the NLR family, has an unconventional role in regulating host iron metabolism that perturbs host resistance to bacterial infection. NLRP6 deficiency is advantageous for maintaining cellular iron homeostasis in both macrophages and enterocytes through increasing the unique iron exporter ferroportin-mediated iron efflux in a nuclear factor erythroid-derived 2-related factor 2 (NRF2)-dependent manner. Additional studies uncovered a novel mechanism underlying NRF2 regulation and operating through NLRP6/AKT interaction and that causes a decrease in AKT phosphorylation, which in turn reduces NRF2 nuclear translocation. In the absence of NLRP6, increased AKT activation promotes NRF2/KEAP1 dissociation via increasing mTOR-mediated p62 phosphorylation and downregulates KEAP1 transcription by promoting FOXO3A phosphorylation. Together, our observations provide new insights into the mechanism of nutritional immunity by revealing a novel function of NLRP6 in regulating iron metabolism, and suggest NLRP6 as a therapeutic target for limiting bacterial iron acquisition.

Evidence of Infection of Human Embryonic Stem Cells by SARS-CoV-2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was initially described to target the respiratory system and now has been reported to infect a variety of cell types, including cardiomyocytes, neurons, hepatocytes, and gut enterocytes. However, it remains unclear whether the virus can directly infect human embryonic stem cells (hESCs) or early embryos. Herein, we sought to investigate this question in a cell-culture system of hESCs. Both the RNA and S protein of SARS-CoV-2 were detected in the infected hESCs and the formation of syncytium was observed. The increased level of subgenomic viral RNA and the presence of dsRNA indicate active replication of SARS-CoV-2 in hESCs. The increase of viral titers in the supernatants revealed virion release, further indicating the successful life cycle of SARS-CoV-2 in hESCs. Remarkably, immunofluorescence microscopy showed that only a small portion of hESCs were infected, which may reflect low expression of SARS-CoV-2 receptors. By setting |log2 (fold change)| > 0.5 as the threshold, a total of 1,566 genes were differentially expressed in SARS-CoV-2-infected hESCs, among which 17 interferon-stimulated genes (ISGs) were significantly upregulated. Altogether, our results provide novel evidence to support the ability of SARS-CoV-2 to infect and replicate in hESCs.

The adenosine analog prodrug ATV006 is orally bioavailable and has preclinical efficacy against parental SARS-CoV-2 and variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus driving the ongoing coronavirus disease 2019 (COVID-19) pandemic, continues to rapidly evolve. Because of the limited efficacy of vaccination in prevention of SARS-CoV-2 transmission and continuous emergence of variants of concern (VOCs), orally bioavailable and broadly efficacious antiviral drugs are urgently needed. Previously, we showed that the parent nucleoside of remdesivir, GS-441524, has potent anti-SARS-CoV-2 activity. Here, we report that esterification of the 5'-hydroxyl moieties of GS-441524 markedly improved antiviral potency. This 5'-hydroxyl-isobutyryl prodrug, ATV006, demonstrated excellent oral bioavailability in rats and cynomolgus monkeys and exhibited potent antiviral efficacy against different SARS-CoV-2 VOCs in vitro and in three mouse models. Oral administration of ATV006 reduced viral loads and alleviated lung damage when administered prophylactically and therapeutically to K18-hACE2 mice challenged with the Delta variant of SARS-CoV-2. These data indicate that ATV006 represents a promising oral antiviral drug candidate for SARS-CoV-2.

Salmonella effector SpvB aggravates dysregulation of systemic iron metabolism via modulating the hepcidin-ferroportin axis.

Iron withholding, an essential component of nutritional immunity, plays a fundamental role in host resistance to Salmonella infection. Our previous study showed that SpvB, an important pSLT-encoded cytotoxic effector, facilitated Salmonella pathogenesis within macrophages via perturbing cellular iron metabolism. However, the underlying mechanisms of SpvB in Salmonella-relevant disorders of systemic iron metabolism have not yet been identified. Here, we demonstrated that SpvB facilitated Salmonella to scavenge iron from the host by modulating the hepcidin-ferroportin axis, a key regulator of systemic iron metabolism. We observed that SpvB enhanced hepatic hepcidin synthesis in a STAT3-dependent manner, but not the BMP/SMAD pathway. This subsequently resulted in a reduction of the unique cellular iron exporter ferroportin, which facilitated hypoferremia and hepatic iron accumulation and ultimately countered the limitation of iron availability, thereby improving the chances of Salmonella survival and replication. Moreover, SpvB promoted the production of proinflammatory molecules associated with the infiltration of inflammatory cells via highly upregulating TREM-1 signaling. Our data supported a role of TREM-1 in SpvB-related dysregulation of host iron metabolism and suggested that targeting TREM-1 might provide a potential therapeutic strategy to prevent or alleviate Salmonella pathogenesis.

Salmonella Effector SpvB Inhibits NF-κB Activity via KEAP1-Mediated Downregulation of IKKß.

Bacterial pathogens have a broad arsenal of genes that are tightly regulated and coordinated to facilitate adaptation to alter host inflammatory response and prolong intracellular bacterial survival. Salmonella enterica serovar Typhimurium utilizes a type III secretion system (T3SS) to deliver effector molecules into host cells and regulate signal transduction pathways such as NF-κB, thereby resulting in salmonellosis. SpvB, a pSLT-encoded cytotoxic protein secreted by Salmonella pathogenicity island-2 T3SS, is associated with enhanced Salmonella survival and intracellular replication. In this report, we characterized the effects of SpvB on NF-κB signaling pathway. We showed that SpvB has a potent and specific ability to prevent NF-κB activation by targeting IκB kinase ß (IKKß). Previous studies from our laboratory showed that SpvB decreases Nrf2 through its C-terminal domain. Here we further demonstrated that KEAP1, a cytoplasmic protein that interacts with Nrf2 and mediates its proteasomal degradation, is involved in SpvB-induced downregulation of IKKß expression and phosphorylation. Reduction of KEAP1 by small-interfering RNA prevented the suppression of IKKß and its phosphorylation mediated by SpvB. These findings revealed a novel mechanism by which Salmonella modulates NF-κB activity to ultimately facilitate intracellular bacterial survival and proliferation and delay host immune response to establish infection.

Salmonella Effector SpvB Disrupts Intestinal Epithelial Barrier Integrity for Bacterial Translocation.

Salmonella are common enteric bacterial pathogens that infect both humans and animals. Intestinal epithelial barrier, formed by a single layer of epithelial cells and apical junctional complex (AJC), plays a crucial role in host defense against enteric pathogens to prevent bacterial translocation. However, the underlying mechanisms of intestinal epithelial barrier dysfunction caused by Salmonella are poorly understood. It is found that a locus termed Salmonella plasmid virulence (spv) gene exists extensively in clinically important Salmonella serovars. SpvB is a key effector encoded within this locus, and closely related to Salmonella pathogenicity such as interfering with autophagy and iron homeostasis. To investigate the interaction between SpvB and intestinal epithelial barrier and elucidate the underlying molecular mechanism, we used the typical foodborne disease agent Salmonella enterica serovar Typhimurium (Salmonella typhimurium) carrying spvB or not to construct infection models in vivo and in vitro. C57BL/6 mice were orally challenged with S. typhimurium wild-type strain SL1344 or spvB-deficient mutant strain SL1344-ΔspvB. Caco-2 cell monolayer model, as a widely used model to mimic the human intestinal epithelium in vitro, was infected with SL1344, SL1344-ΔspvB, or spvB complementary strain SL1344-c-ΔspvB, respectively. The results showed that SpvB enhanced bacterial pathogenicity during S. typhimurium infection in vivo, and contributed to intestinal epithelial barrier dysfunction in both infection systems. This SpvB-mediated barrier dysfunction was attributed to the cellular redistribution of Claudin-1, Occludin, and E-cadherin junctional proteins. Moreover, by using pharmacological inhibitors, we found that F-actin rearrangement and suppression of protein kinase C (PKC) signaling pathway were involved in SpvB-mediated barrier dysfunction. In conclusion, the study reveals the contribution of Salmonella effector SpvB to the dysfunction of intestinal epithelial barrier integrity, which facilitates bacterial translocation via the paracellular route to promote Salmonella systemic dissemination. Our findings broaden the understanding of host-pathogen interactions in salmonellosis, and provide new strategies for the therapy in limiting bacterial dissemination during infection.