Engineered probiotics represent a cutting-edge therapy in intestinal inflammatory disease (IBD). Genetically modified bacteria have provided a new strategy to release therapeutically operative molecules in the intestine and have grown into promising new therapies for IBD. Current IBD treatments, such as corticosteroids and immunosuppressants, are associated with relevant side effects and a significant proportion of patients are dependent on these therapies, thus exposing them to the risk of relevant long-term side effects. Discovering new and effective therapeutic strategies is a worldwide goal in this research field and engineered probiotics could potentially provide a viable solution. This review aims at describing the proceeding of bacterial engineering and how genetically modified probiotics may represent a promising new biotechnological approach in IBD treatment.

Similar to canine inflammatory enteropathy, inflammatory bowel disease (IBD) is a chronic idiopathic condition characterized by remission periods and recurrent flares in which diarrhea, visceral pain, rectal bleeding/bloody stools, and weight loss are the main clinical symptoms. Intestinal barrier function alterations often persist in the remission phase of the disease without ongoing inflammatory processes. However, current therapies include mainly anti-inflammatory compounds that fail to promote functional symptoms-free disease remission, urging new drug discoveries to handle patients during this step of the disease. ALIAmides (ALIA, autacoid local injury antagonism) are bioactive fatty acid amides that recently gained attention because of their involvement in the control of inflammatory response, prompting the use of these molecules as plausible therapeutic strategies in the treatment of several chronic inflammatory conditions. N-palmitoyl-D-glucosamine (PGA), an under-researched ALIAmide, resulted in being safe and effective in preclinical models of inflammation and pain, suggesting its potential engagement in the treatment of IBD. In our study, we demonstrated that micronized PGA significantly and dose-dependently reduces colitis severity, improves intestinal mucosa integrity by increasing the tight junction proteins expression, and downregulates the TLR-4/NLRP3/iNOS pathway via PPAR-α receptors signaling in DNBS-treated mice. The possibility of clinically exploiting micronized PGA as support for the treatment and prevention of inflammation-related changes in IBD patients would represent an innovative, effective, and safe strategy.

As of October 2022, the COVID-19 pandemic continues to pose a major public health conundrum, with increased rates of symptomatic infections in vaccinated individuals. An ideal vaccine candidate for the prevention of outbreaks should be rapidly scalable, easy to administer, and able to elicit a potent mucosal immunity. Towards this aim, we proposed an engineered Escherichia coli (E. coli) Nissle 1917 (EcN) strain with SARS-CoV-2 spike protein (SP)-coding plasmid, which was able to expose SP on its cellular surface by a hybridization with the adhesin involved in diffuse adherence 1 (AIDA1). In this study, we presented the effectiveness of a 16-week intragastrically administered, engineered EcN in producing specific systemic and mucosal immunoglobulins against SARS-CoV-2 SP in mice. We observed a time-dependent increase in anti-SARS-CoV-2 SP IgG antibodies in the sera at week 4, with a titre that more than doubled by week 12 and a stable circulating titre by week 16 (+309% and +325% vs. control; both p < 0.001). A parallel rise in mucosal IgA antibody titre in stools, measured via intestinal and bronchoalveolar lavage fluids of the treated mice, reached a plateau by week 12 and until the end of the immunization protocol (+300, +47, and +150%, at week 16; all p < 0.001 vs. controls). If confirmed in animal models of infection, our data indicated that the engineered EcN may be a potential candidate as an oral vaccine against COVID-19. It is safe, inexpensive, and, most importantly, able to stimulate the production of both systemic and mucosal anti-SARS-CoV-2 spike-protein antibodies.

Metabolic and mood disorders elicited by chronic exposure of high-fat diet (HFD) are often associated with intestinal dysbiosis and persistent low-grade inflammation in the small intestine. This leads to remodeling of the epithelial barrier with disruption of the neuroepithelial circuits that control energy homeostasis by the gut-brain axis. Therefore, therapies that restoreintestinal microbial niche and barrier function are promising candidates to counter peripheral metabolic challenges that affect behaviors controlled by the brain. The endogenous oleoylethanolamine (OEA) was found to shape the intestinal microbiota profile towards a "lean-like phenotype", ameliorating pathological profiles of metabolic diseases. Further, OEA displays beneficial effects in several cognitive paradigms and preserves the epithelial barrier integrity, acting as an intestinal "gate-keeper". Here, we developed an "intestinal OEA factory" for the in-situ and controlled release of OEA by using a probiotic-based delivery system. We engineered the Lactobacillus paracasei F19 (LP) to express the human N-acylphosphatidylethanolamine-preferring phospholipase D (NAPEpld) gene and to produce OEA in response to dietary ultra-low oleate supply. We treated 12-week HFD male mice with oleate-probiotic formulations and assessed their impact on metabolic and behavioral dysfunctions, and microbiota-gut-brain signaling after 8 weeks of treatment. NAPE-expressing LP (pNAPE-LP) led to significant reduced weight loss and improved metabolic dysfunction in HFD-treated mice. Further, a parallel improvement in depressive- and anxiety-like phenotypes was associated with the duodenal barrier function retrieval, the restoration of the Firmicutes/Bacteroidetes ratio, and an increase in beneficial bacteria, such as Lactobacillus, Prevotella, and Parabacteroides. The HFD-driven changes both in the enteric and central nervous system were prevented by pNAPE-LP/oleate treatment. Collectively, our data suggest that these effects were mediated by the oleate-dependent release of OEA by pNAPE-LP since no significant effects were observed in HFD mice treated with the native probiotic alone (pLP). This oleate-regulated delivery system of OEA is a safe and efficient probiotic-based strategy for the treatment of metabolic syndrome and related behavioral disorders.

Developing drugs for brain infection by Naegleria fowleri is an unmet medical need. We used a combination of cheminformatics, target-, and phenotypic-based drug discovery methods to identify inhibitors that target an essential N. fowleri enzyme, sterol 14-demethylase (NfCYP51). A total of 124 compounds preselected in silico were tested against N. fowleri. Nine primary hits with EC₅₀ ≤ 10 μM were phenotypically identified. Cocrystallization with NfCYP51 focused attention on one primary hit, miconazole-like compound 2a. The S-enantiomer of 2a produced a 1.74 Å cocrystal structure. A set of analogues was then synthesized and evaluated to confirm the superiority of the S-configuration over the R-configuration and the advantage of an ether linkage over an ester linkage. The two compounds, S-8b and S-9b, had an improved EC₅₀ and K_D compared to 2a. Importantly, both were readily taken up into the brain. The brain-to-plasma distribution coefficient of S-9b was 1.02 ± 0.12, suggesting further evaluation as a lead for primary amoebic meningoencephalitis.