Guanylate-binding protein 1 inhibits inflammatory factors produced by H5N1 virus through Its GTPase activity.

The combination of inflammatory factors resulting from an influenza A virus infection is one of the main causes of death in host animals. Studies have shown that guinea pig guanosine monophosphate binding protein 1 (guanylate-binding protein 1, gGBP1) can downregulate cytokine production induced by the influenza virus. Therefore, exploring the innate immune defense mechanism of GBP1 in the process of H5N1 influenza virus infection has important implications for understanding the pathogenic mechanism, disease prevention, and the control of influenza A virus infections. We found that, in addition to inhibiting the early replication of influenza virus, gGBP1 also inhibited the production of CCL2 and CXCL10 cytokines induced by the influenza virus as well as the proliferation of mononuclear macrophages induced by these cytokines. These findings further confirmed that gGBP1 inhibited the production of cytokines through its GTPase activity and cell proliferation through its C-terminal α-helix structure. This study revealed the effect of gGBP1 on the production of cellular inflammatory factors during influenza virus infection and determined the key amino acid residues that assist in the inhibitory processes mediated by gGBP1.

Microencapsulated Limosilactobacillus reuteri Encoding Lactoferricin-Lactoferrampin Targeted Intestine against Salmonella typhimurium Infection.

Salmonella enterica serovar Typhimurium (S. typhimurium) is an important foodborne pathogen that infects both humans and animals and develops acute gastroenteritis. As porcine intestines are relatively similar to the human ones due to their relatively similar sizes and structural similarity, S. typhimurium causes analogous symptoms in both. Novel strategies for controlling S. typhimurium infection are also desired, such as mucosal-targeted delivery of probiotics and antimicrobial peptides. The bovine lactoferricin-lactoferrampin-encoding Limosilactobacillus reuteri (LR-LFCA) strain improves intestinal barrier function by strengthening the intestinal barrier. Weaned piglets were selected for oral administration of microencapsulated LR-LFCA (microcapsules entrap LR-LFCA into gastro-resistant polymers) and then infected with S. typhimurium for 3 days. We found that orally administering microencapsulated LR-LFCA to weaned piglets attenuated S. typhimurium-induced production of inflammatory factors in the intestinal mucosa by inhibiting the nuclear factor-kappa B (NF-κB) and P38 mitogen-activated protein kinases (MAPK) signaling pathway. Moreover, microencapsulated LR-LFCA administration significantly suppressed the oxidative stress that may correlate with gut microbiota (reduced Salmonella population and increased α-diversity and Lactobacillus abundance) and intestinal function (membrane transport and metabolism). Our work demonstrated that microencapsulated LR-LFCA effectively targeted intestine delivery of Lactobacillus and antimicrobial peptides and modulated gut microbiota and mucosal immunity. This study reveals a novel targeting mucosal strategy against S. typhimurium infection.

Optimum Fermentation Conditions for Bovine Lactoferricin-Lactoferrampin-Encoding LimosiLactobacillus reuteri and Regulation of Intestinal Inflammation.

The multifunctional antibacterial peptide lactoferricin-lactoferrampin (LFCA) is derived from bovine lactoferrin. Optimization of the fermentation process should be studied since different microorganisms have their own favorable conditions and processes for growth and the production of metabolites. In this study, the culture conditions of a recombinant strain, pPG-LFCA-E/LR-CO21 (LR-LFCA), expressing LFCA was optimized, utilizing the high-density fermentation process to augment the biomass of LimosiLactobacillus reuteri and the expression of LFCA. Furthermore, an assessment of the protective effect of LR-LFCA on intestinal inflammation induced by lipopolysaccharide (LPS) was conducted to evaluate the impact of LR-LFCA on the disease resistance of piglets. The findings of this study indicate that LR-LFCA fermentation conditions optimally include 2% inoculation volume, 36.5 °C fermentation temperature, 9% dissolved oxygen concentration, 200 revolutions/minute stirring speed, pH 6, 10 mL/h glucose flow, and 50% glucose concentration. The inclusion of fermented LR-LFCA in the diet resulted in an elevation of immunoglobulin levels, significant upregulation of tight junction proteins ZO-1 and occludin, reinforcement of the intestinal barrier function, and significant amelioration of the aberrant alterations in blood physiological parameters induced by LPS. These results offer a theoretical framework for the implementation of this micro-ecological preparation in the field of piglet production to enhance intestinal well-being.

Effect of Microencapsulation Techniques on the Stress Resistance and Biological Activity of Bovine Lactoferricin-Lactoferrampin-Encoding Lactobacillus reuteri.

Bovine lactoferricin-lactoferrampin-encoding Lactobacillus reuteri (LR-LFCA) has been found to benefit its host by strengthening its intestinal barrier. However, several questions remain open concerning genetically engineered strains maintaining long-term biological activity at room temperature. In addition, probiotics are vulnerable to harsh conditions in the gut, such as acidity and alkalinity, and bile salts. Microencapsulation is a technique to entrap probiotic bacteria into gastro-resistant polymers to carry them directly to the intestine. We selected nine kinds of wall material combinations to encapsulate LR-LFCA by spray drying microencapsulation. The storage stability, microstructural morphology, biological activity, and simulated digestion in vivo or in vitro of the microencapsulated LR-LFCA were further evaluated. The results showed that LR-LFCA had the highest survival rate when microcapsules were prepared using a wall material mixture (skim milk, sodium glutamate, polyvinylpyrrolidone, maltodextrin, and gelatin). Microencapsulated LR-LFCA increased the stress resistance capacity and colonization abilities. In the present study, we have identified a suitable wall material formulation for spray-dried microencapsulation of genetically engineered probiotic products, which would facilitate their storage and transport.

A bovine lactoferricin-lactoferrampin-encoding Lactobacillus reuteri CO21 regulates the intestinal mucosal immunity and enhances the protection of piglets against enterotoxigenic Escherichia coli K88 challenge.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea in human and animal. To determine the mechanism of a bovine lactoferricin-lactoferrampin (LFCA)-encoding Lactobacillus reuteri CO21 (LR-LFCA) to enhance the intestinal mucosal immunity, we used a newborn piglet intestine model to study the intestinal response to ETEC. Pigs were chosen due to the anatomical similarity between the porcine and the human intestine.4-day-old piglets were orally administered with LR-LFCA, LR-con (L. reuteri CO21 transformed with pPG612 plasmid) or phosphate buffered saline (PBS) for three consecutive days, within 21 days after these treatments, we found that LR-LFCA can colonize the intestines of piglets, improve the growth performance, enhance immune response and is beneficial for intestinal health of piglets by improving intestinal barrier function and modulating the composition of gut microbiota. Twenty-one days after, piglets were infected with ETEC K88 for 5 days, we found that oral administration of LR-LFCA to neonatal piglets attenuated ETEC-induced the weight loss of piglets and diarrhea incidence. LR-LFCA decreased the production of inflammatory factors and oxidative stress in intestinal mucosa of ETEC-infected piglets. Additionally, LR-LFCA increased the expression of tight junction proteins in the ileum of ETEC-infected piglets. Using LPS-induced porcine intestinal epithelial cells (IPEC-J2) in vitro, we demonstrated that LR-LFCA-mediated increases in the tight junction proteins might depend on the MLCK pathway; LR-LFCA might increase the anti-inflammatory ability by inhibiting the NF-κB pathway. We also found that LR-LFCA may enhance the antioxidant capacity of piglets by activating the Nrf2/HO-1 pathway. This study demonstrates that LR-LFCA is effective at maintaining intestinal epithelial integrity and host homeostasis as well as at repairing intestinal damage after ETEC infection and is thus a promising alternative therapeutic method for intestinal inflammation.

Oral delivery of a Lactococcus lactis strain secreting bovine lactoferricin-lactoferrampin alleviates the development of acute colitis in mice.

Ulcerative colitis (UC) is a chronic relapsing disease. Treatment of UC would benefit from specific targeting of therapeutics to the intestine. Previous studies have demonstrated that bovine lactoferricin and lactoferrampin have bactericidal, anti-inflammatory, and immunomodulatory effects. Here, we investigated whether oral administration of a bovine lactoferricin-lactoferrampin (LFCA)-encoding Lactococcus lactis (LL-LFCA) strain could alleviate experimental colitis. LFCA derived from LL-LFCA inhibited the growth of Escherichia coli and Staphylococcus aureus in vitro. In mice, administration of LL-LFCA decreased the disease activity index and attenuated dextran sulfate sodium (DSS)-induced body weight loss and colon shortening. LL-LFCA treatment also ameliorated DSS-induced colon damage, inhibited inflammatory cell infiltration, significantly decreased myeloperoxidase activity, and ameliorated DSS-induced disruption of intestinal permeability and tight junctions. In addition, 16S rDNA sequencing showed that LL-LFCA reversed DSS-induced gut dysbiosis. The production of proinflammatory mediators in serum and the colon was also reduced by administration of LL-LFCA. In vitro, LFCA derived from LL-LFCA decreased the messenger RNA expression of proinflammatory factors. The underlying mechanisms may involve inhibition of the nuclear factor kappa B (NF-κB) pathway. The results demonstrate that LL-LFCA ameliorates DSS-induced intestinal injury in mice, suggesting that LL-LFCA might be an effective drug for the treatment of inflammatory bowel diseases.