Pasteurella multocida activates apoptosis via the FAK-AKT-FOXO1 axis to cause pulmonary integrity loss, bacteremia, and eventually a cytokine storm.

Pasteurella multocida is an important zoonotic respiratory pathogen capable of infecting a diverse range of hosts, including humans, farm animals, and wild animals. However, the precise mechanisms by which P. multocida compromises the pulmonary integrity of mammals and subsequently induces systemic infection remain largely unexplored. In this study, based on mouse and rabbit models, we found that P. multocida causes not only lung damage but also bacteremia due to the loss of lung integrity. Furthermore, we demonstrated that bacteremia is an important aspect of P. multocida pathogenesis, as evidenced by the observed multiorgan damage and systemic inflammation, and ultimately found that this systemic infection leads to a cytokine storm that can be mitigated by IL-6-neutralizing antibodies. As a result, we divided the pathogenesis of P. multocida into two phases: the pulmonary infection phase and the systemic infection phase. Based on unbiased RNA-seq data, we discovered that P. multocida-induced apoptosis leads to the loss of pulmonary epithelial integrity. These findings have been validated in both TC-1 murine lung epithelial cells and the lungs of model mice. Conversely, the administration of Ac-DEVD-CHO, an apoptosis inhibitor, effectively restored pulmonary epithelial integrity, significantly mitigated lung damage, inhibited bacteremia, attenuated the cytokine storm, and reduced mortality in mouse models. At the molecular level, we demonstrated that the FAK-AKT-FOXO1 axis is involved in P. multocida-induced lung epithelial cell apoptosis in both cells and animals. Thus, our research provides crucial information with regard to the pathogenesis of P. multocida as well as potential treatment options for this and other respiratory bacterial diseases.

Pasteurella multocida activates Rassf1-Hippo-Yap pathway to induce pulmonary epithelial apoptosis.

Pasteurella multocida is an opportunistic zoonotic pathogen that primarily causes fatal respiratory diseases, such as pneumonia and respiratory syndromes. However, the precise mechanistic understanding of how P. multocida disrupts the epithelial barrier in mammalian lung remains largely unknown. In this study, using unbiased RNA-seq analysis, we found that the evolutionarily conserved Hippo-Yap pathway was dysregulated after P. multocida infection. Given the complexity of P. multocida infection associated with lung injury and systemic inflammatory processes, we employed a combination of cell culture models, mouse models, and rabbit models to investigate the dynamics of the Hippo-Yap pathway during P. multocida infection. Our findings reveal that P. multocida infection activates the Hippo-Yap pathway both in vitro and in vivo, by upregulating the upstream factors p-Mst1/2, p-Lats1, and p-Yap, and downregulating the downstream effectors Birc5, Cyr61, and Slug. Conversely, pharmacological inhibition of the Hippo pathway by XMU-MP-1 significantly rescued pulmonary epithelial cell apoptosis in vitro and reduced lung injury, systemic inflammation, and mouse mortality in vivo. Mechanistic studies revealed that P. multocida induced up-regulation of Rassf1 expression, and Rassf1 enhanced Hippo-Yap pathway through phosphorylation. Accordingly, in vitro knockdown of Rassf1 significantly enhanced Yap activity and expression of Yap downstream factors and reduced apoptosis during P. multocida infection. P. multocida-infected rabbit samples also showed overexpression of Rassf1, p-Lats1, and p-Yap, suggesting that P. multocida activates the Rassf1-Hippo-Yap pathway. These results elucidate the pathogenic role of the Rassf1-Hippo-Yap pathway in P. multocida infection and suggest that this pathway has the potential to be a drug target for the treatment of pasteurellosis.

Attenuated vaccine PmCQ2Δ4555-4580 effectively protects mice against Pasteurella multocida infection.

Pasteurella multocida type A (PmA) mainly causes respiratory diseases such as pneumonia in bovines, leading to great economic losses to the breeding industry. At present, there is still no effective commercial vaccine against PmA infection. In this study, a mutant strain (PmCQ2Δ4555-4580) with brand-new phenotypes was obtained after serially passaging at 42 °C. Whole genome resequencing and PCR analysis showed that PmCQ2Δ4555-4580 missed six genes, including PmCQ2_004555, PmCQ2_004560, PmCQ2_004565, PmCQ2_004570, PmCQ2_004575, and PmCQ2_004580. Importantly, the virulence of PmCQ2Δ4555-4580 was reduced by approximately 2.8 × 109 times in mice. Notably, live PmCQ2Δ4555-4580 could provide 100%, 100% and 40% protection against PmA, PmB and PmF, respectively; and inactivated PmCQ2Δ4555-4580 could provide 100% and 87.5% protection against PmA and PmB. Interestingly, immune protection-related proteins were significantly upregulated in PmCQ2Δ4555-4580 based on RNA-seq and bioinformatics analysis. Meaningfully, by in vitro expression, purification and in vivo immunization, 12 proteins had different degrees of immune protective effects. Among them, PmCQ2_008205, PmCQ2_010435, PmCQ2_008190, and PmCQ2_004170 had the best protective effect, the protection rates against PmA were 50%, 40%, 30%, and 30%, respectively, and the protective rates against PmB were 62.5%, 42.9%, 37.5%, and 28.6%, respectively. Collectively, PmCQ2Δ4555-4580 is a potential vaccine candidate for the prevention of Pasteurellosis involving in high expression of immune protective related proteins.

Corrigendum: L-ascorbic acid shapes bovine Pasteurella multocida serogroup A infection.

[This corrects the article DOI: 10.3389/fvets.2021.687922.].

Plant green pigment of chlorophyllin attenuates inflammatory bowel diseases by suppressing autophagy activation in mice.

Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are intestinal complications characterized by chronic inflammation, autophagy abnormality, and lysosomal stress, which are derived from genetic predisposition and environmental risk factors. It is generally precepted that dietary green vegetable is beneficial for physiological homeostasis. In this study, we found that dextran sulfate sodium (DSS)-induced colitis and altered intestinal epithelia in mice were attenuated by oral administration of chlorophyllin (CHL), a water-soluble derivate of chlorophyll. In DSS-treated mice, autophagy was persistently activated in intestinal tissues and associated with bowel disorders. Conversely, supplement of CHL in diet or gavage suppressed intestinal inflammation, downregulated autophagy flux in intestinal tissue, and relieved endoplasmic reticulum stress. In vitro studies show that CHL could activate Akt and mTOR pathways, leading to downregulation of autophagic and lysosomal flux. Thus, consumption of green vegetables and chlorophyllin may be beneficial for IBD recovery in part through alleviation of inflammation and autolysosomal flux.NEW & NOTEWORTHY Inflammatory bowel disease (IBD) is a chronic and recurrent gastrointestinal disease, while the etiology remains poorly understood. Dietary composition and lifestyle are crucial for pathogenesis and progression of IBD. In this study, we observed that autophagy in the intestinal tissue was persistently activated in IBD mice. Chlorophyllin (CHL), a water-soluble derivate of chlorophyll, can attenuate colitis by regulating autophagy and inflammation. Thus, consumption of green vegetables and chlorophyllin may be beneficial for IBD recovery.

Sequestration of Intestinal Acidic Toxins by Cationic Resin Attenuates Pancreatic Cancer Progression through Promoting Autophagic Flux for YAP Degradation.

Pancreatic cancer is driven by risk factors such as diabetes and chronic pancreatic injury, which are further associated with gut dysbiosis. Intestinal toxins such as bile acids and bacterial endotoxin (LPS), in excess and persistence, can provoke chronic inflammation and tumorigenesis. Of interest is that many intestinal toxins are negatively charged acidic components in essence, which prompted us to test whether oral administration of cationic resin can deplete intestinal toxins and ameliorate pancreatic cancer. Here, we found that increased plasma levels of endotoxin and bile acids in Pdx1-Cre: LSL-KrasG12D/+ mice were associated with the transformation of the pancreatic ductal carcinoma (PDAC) state. Common bile-duct-ligation or LPS injection impeded autolysosomal flux, leading to Yap accumulation and malignant transformation. Conversely, oral administration of cholestyramine to sequestrate intestinal endotoxin and bile acids resumed autolysosomal flux for Yap degradation and attenuated metastatic incidence. Conversely, chloroquine treatment impaired autolysosomal flux and exacerbated malignance, showing jeopardization of p62/ Sqxtm1 turnover, leading to Yap accumulation, which is also consistent with overexpression of cystatin A (CSTA) in situ with pancreatic cancer cells and metastatic tumor. At cellular levels, chenodeoxycholic acid or LPS treatment activated the ligand-receptor-mediated AKT-mTOR pathway, resulting in autophagy-lysosomal stress for YAP accumulation and cellular dissemination. Thus, this work indicates a potential new strategy for intervention of pancreatic metastasis through sequestration of intestinal acidic toxins by oral administration of cationic resins.

L-Ascorbic Acid Shapes Bovine Pasteurella multocida Serogroup A Infection.

Bovine Pasteurella multocida serogroup A (bovine PmA) is one of the most important pathogens causing fatal pneumonia in cattle. However, it is largely unknown how nutrition shapes bovine PmA infection. Here, we discovered that the infected lung held the highest bacterial density than other tissues during infection. By screening the different metabolites between high (lung)- and low (liver)-bacterial density tissues, the present work revealed that L-ascorbic acid and L-aspartic acid directly influenced bovine P. multocida growth. Interestingly, L-ascorbic acid, which is expressed at higher levels in the infected livers, inhibited bovine PmA growth as well as virulence factor expression and promoted macrophage bactericidal activity in vitro. In addition, ascorbic acid synthesis was repressed upon bovine PmA infection, and supplementation with exogenous L-ascorbic acid significantly reduced the bacterial burden of the infected lungs and mouse mortality. Collectively, our study has profiled the metabolite difference of the murine lung and liver during bovine PmA infection. The screened L-ascorbic acid showed repression of bovine PmA growth and virulence expression in vitro and supplementation could significantly increase the survival rate of mice and reduce the bacterial load in vivo, which implied that L-ascorbic acid could serve as a potential protective agent for bovine PmA infection in clinic.

Unexpected Function of a Heptapeptide-Conjugated Zwitterionic Polymer that Coassembles into ß-Amyloid Fibrils and Eliminates the Amyloid Cytotoxicity.

Fibrillogenesis of amyloid ß-protein (Aß) is pathologically associated with Alzheimer's disease (AD), so modulating Aß aggregation is crucial for AD prevention and treatment. Herein, a zwitterionic polymer with short dimethyl side chains (pID) is synthesized and conjugated with a heptapeptide inhibitor (Ac-LVFFARK-NH2, LK7) to construct zwitterionic polymer-inhibitor conjugates for enhanced inhibition of Aß aggregation. However, it is unexpectedly found that the LK7@pID conjugates remarkably promote Aß fibrillization to form more fibrils than the free Aß system but effectively eliminate Aß-induced cytotoxicity. Such an unusual behavior of the LK7@pID conjugates is unraveled by extensive mechanistic studies. First, the hydrophobic environment within the assembled micelles of LK7@pID promotes the hydrophobic interaction between Aß molecules and LK7@pID, which triggers Aß aggregation at the very beginning, making fibrillization occur at an earlier stage. Second, in the aggregation process, the LK7@pID micelles disassemble by the intensive interactions with Aß, and LK7@pID participates in the fibrillization by being embedded in the Aß fibrils, leading to the formation of hybrid and heterogeneous fibrillar aggregates with a different structure than normal Aß fibrils. This unique Trojan horse-like feature of LK7@pID conjugates has not been observed for any other inhibitors reported previously and may shed light on the design of new modulators against ß-amyloid cytotoxicity.

Hepatitis B Virus X Protein (HBx) Suppresses Transcription Factor EB (TFEB) Resulting in Stabilization of Integrin Beta 1 (ITGB1) in Hepatocellular Carcinoma Cells.

Hepatitis B virus (HBV) infection is a major etiological risk for the incidence of hepatocellular carcinoma (HCC), and HBV X protein (HBx) is essential for oncogenic transformation. It is not known that if HBx can sabotage the lysosomal system for transformation and tumorigenesis, or its mechanism if it does have an effect. Examining clinical data, we observed that the downregulation of lysosomal components and transcription factor EB (TFEB) was associated with a poor prognosis of HCC patients. In HCC cells, we found that expression of HBx suppressed TFEB, impaired biogenesis of autophagic-lysosome, and promoted cellular dissemination. HBx mediated downregulation of TFEB led to impairment of autophagic/lysosomal biogenesis and flux, and consequently, accumulation of integrin beta 1 (ITGB1) for motility of HCC cells. Conversely, TFEB, in a steady-state condition, through induction of lysosomal biogenesis restrained ITGB1 levels and limited mobility of HCC cells. Specifically, overexpression of TFEB upregulated and activated the cysteine proteases including cathepsin L (CTSL) to degrade ITGB1. Conversely, expression of cystatin A (CSTA) or cystatin B (CSTB), the cellular inhibitors of lysosomal cysteine proteinases, spared ITGB1 from degradation and promoted dissemination of HCC cells. Taken together, this study suggests a potential mechanism for HBV-mediated malignancy, showing that HBx mediated downregulation of TFEB leads to accumulation of ITGB1 for HCC cell migration.

Transcriptomic Analysis of Chicken Lungs Infected With Avian and Bovine Pasteurella multocida Serotype A.

Pasteurella multocida (P. multocida) is a common animal pathogen responsible for many animal diseases. Strains from different hosts exhibit disparate degrees of effect in other species. Here, we characterize an avian P. multocida serogroup A strain (PmQ) showing high lethality to chickens and a bovine P. multocida serogroup A strain (PmCQ2) with no lethality to chickens. We used RNA-seq to profile the transcriptomes of chicken lungs infected with PmQ and PmCQ2. A total of 1,649 differentially expressed genes (DEGs) due to PmQ infection (831 upregulated genes and 818 downregulated genes) and 1427 DEGs (633 upregulated genes and 794 downregulated genes) due to PmCQ2 infection were identified. Functional analysis of these DEGs demonstrated that the TNF signaling pathway, the toll-like receptor signaling pathway, complement and coagulation cascades, and cytokine-cytokine receptor interaction were both enriched in PmQ and PmCQ2 infection. STAT and apoptosis signaling pathways were uniquely enriched by PmQ infection, and the NOD-like receptor signaling pathway was enriched only by PmCQ2 infection. Cell-type enrichment analysis of the transcriptomes showed that immune cells, including macrophages and granulocytes, were enriched in both infection groups. Collectively, our study profiled the transcriptomic response of chicken lungs infected with P. multocida and provided valuable information to understand the chicken responses to P. multocida infection.

Zwitterionic Polymer Micelles with Dual Conjugation of Doxorubicin and Curcumin: Synergistically Enhanced Efficacy against Multidrug-Resistant Tumor Cells.

This paper reports a novel redox-sensitive micellar system for the co-delivery of doxorubicin (Dox) and a chemosensitizer (curcumin, Cur) to overcome the multidrug resistance (MDR) in cancer cells. Dox and Cur were co-conjugated onto a zwitterionic polymer, poly(carboxybetaine) (pCB), to form Cur-pCB-Dox that self-assembled into stable micelles (164.2 ± 4.8 nm). Single-drug conjugates (pCB-Dox and pCB-Cur) were prepared for comparisons. Compared to the high half-maximal inhibitory concentration (IC50) of Dox (437.2 µg/mL), the IC50 value of pCB-Dox (14.1 µg/mL) was only 1/33 that of Dox. Confocal laser scanning microscopy and flow cytometry revealed the greatly enhanced cell uptake of the conjugate due to the enhanced permeability and retention effect of tumor cells on the micellar conjugate. Co-delivery of pCB-Dox with pCB-Cur further reduced the IC50 value by 37% (8.9 µg/mL). More importantly, Cur-pCB-Dox exhibited the strongest cytotoxicity against MCF-7/Adr cells (IC50, 5.87 µg/mL) because the co-delivered Dox and Cur on one carrier specifically transported into the same cells, which inhibited the efflux of Dox by Cur, led to a higher intracellular Dox concentration and made the drugs exert synergistic effects at the targeting regions. The results proved the zwitterionic micelles as promising drug co-delivery vehicles for fighting against MDR.

Self-Assembled Curcumin-Poly(carboxybetaine methacrylate) Conjugates: Potent Nano-Inhibitors against Amyloid ß-Protein Fibrillogenesis and Cytotoxicity.

Fibrillogenesis of amyloid ß-protein (Aß) is a pathological hallmark of Alzheimer's disease, so inhibition of Aß aggregation is considered as an important strategy for the precaution and treatment of AD. Curcumin (Cur) has been recognized as an effective inhibitor of Aß fibrillogenesis, but its potential application is limited by its poor bioavailability. Herein, we proposed to conjugate Cur to a zwitterionic polymer, poly(carboxybetaine methacrylate) (pCB), and synthesized three Cur@pCB conjugates of different degrees of substitution (DS, 1.9-2.9). Cur@pCB conjugates self-assembled into nanogels of 120-190 nm. The inhibition effects of Cur@pCB conjugates on the fibrillation and cytotoxicity of Aß42 was investigated by extensive biophysical and biological analyses. Thioflavin T fluorescence assays and atomic force microscopic observations revealed that the Cur@pCB conjugates were much more efficient than molecular curcumin on inhibiting Aß42 fibrillation, and cytotoxicity assays also indicated the same tendency. Of the three conjugates, Cur1@pCB of the lowest DS (1.97) exhibited the best performance; 5 µM Cur1@pCB functioned similarly with 25 µM free curcumin. Moreover, 5 µM Cur1@pCB increased the cell viability by 43% but free curcumin at the same concentration showed little effect. It is considered that the highly hydrated state of the zwitterionic polymers resulted in the superiority of Cur@pCB over free curcumin. Namely, the dense hydration layer on the conjugates strongly stabilized the bound Aß on curcumin anchored on the polymer, suppressing the conformational transition of the protein to ß-sheet-rich structures. This was demonstrated by circular dichroism spectroscopy, in which Cur1@pCB was proven to be the strongest in the three conjugates. The research has thus revealed a new function of zwitterionic polymer pCBMA and provided new insights into the development of more potent nanoinhibitors for suppressing Aß fibrillogenesis and cytotoxicity.

Betaine in Inflammation: Mechanistic Aspects and Applications.

Betaine is known as trimethylglycine and is widely distributed in animals, plants, and microorganisms. Betaine is known to function physiologically as an important osmoprotectant and methyl group donor. Accumulating evidence has shown that betaine has anti-inflammatory functions in numerous diseases. Mechanistically, betaine ameliorates sulfur amino acid metabolism against oxidative stress, inhibits nuclear factor-κB activity and NLRP3 inflammasome activation, regulates energy metabolism, and mitigates endoplasmic reticulum stress and apoptosis. Consequently, betaine has beneficial actions in several human diseases, such as obesity, diabetes, cancer, and Alzheimer's disease.