Exploiting phage receptor binding proteins to enable endolysins to kill Gram-negative bacteria

Bacteriophage-encoded endolysins degrading the bacterial peptidoglycan are promising antibacterials for combating antibiotic-resistant bacteria. However, endolysins have limited use against Gram-negative bacteria, since the outer membrane prevents access to the peptidoglycan. Here, we present Innolysins, an innovative concept for engineering endolysins to exert antibacterial activity against Gram-negative bacteria. Innolysins combine the enzymatic activity of endolysins with the binding capacity of phage receptor binding proteins (RBPs). As proof-of-concept, we constructed 12 Innolysins by fusing phage T5 endolysin and RBP Pb5 in different configurations. One of these, Innolysin Ec6 displayed antibacterial activity against Escherichia coli only in the presence of Pb5 receptor FhuA, leading to 1.22 ± 0.12 log reduction in cell counts. Accordingly, other bacterial species carrying FhuA homologs such as Shigella sonnei and Pseudomonas aeruginosa were sensitive to Innolysin Ec6. To enhance the antibacterial activity, we further constructed 228 novel Innolysins by fusing 23 endolysins with Pb5. High-throughput screening allowed to select Innolysin Ec21 as the best antibacterial candidate, leading to 2.20 ± 0.09 log reduction in E. coli counts. Interestingly, Innolysin Ec21 also displayed bactericidal activity against E. coli resistant to third-generation cephalosporins, reaching a 3.31 ± 0.53 log reduction in cell counts. Overall, the Innolysin approach expands previous endolysin-engineering strategies, allowing customization of endolysins by exploiting phage RBPs to specifically target Gram-negative bacteria

In vivo testing of novel vaccine prototypes against Actinobacillus pleuropneumoniae

Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is a Gram-negative bacterium that represents the main cause of porcine pleuropneumonia in pigs, causing significant economic losses to the livestock industry worldwide. A. pleuropneumoniae, as the majority of Gram-negative bacteria, excrete vesicles from its outer membrane (OM), accordingly defined as outer membrane vesicles (OMVs). Thanks to their antigenic similarity to the OM, OMVs have emerged as a promising tool in vaccinology. In this study we describe the in vivo testing of several vaccine prototypes for the prevention of infection by all known A. pleuropneumoniae serotypes. Previously identified vaccine candidates, the recombinant proteins ApfA and VacJ, administered individually or in various combinations with the OMVs, were employed as vaccination strategies. Our data show that the addition of the OMVs in the vaccine formulations significantly increased the specific IgG titer against both ApfA and VacJ in the immunized animals, confirming the previously postulated potential of the OMVs as adjuvant. Unfortunately, the antibody response raised did not translate into an effective protection against A. pleuropneumoniae infection, as none of the immunized groups following challenge showed a significantly lower degree of lesions than the controls. Interestingly, quite the opposite was true, as the animals with the highest IgG titers were also the ones bearing the most extensive lesions in their lungs. These results shed new light on A. pleuropneumoniae pathogenicity, suggesting that antibody-mediated cytotoxicity from the host immune response may play a central role in the development of the lesions typically associated with A. pleuropneumoniae infections

Frailty, psychological well-being, and social isolation in older adults with cognitive impairment during the SARS-CoV-2 pandemic: data from the GeroCovid initiative

Background: The containment measures linked to the COVID-19 pandemic negatively affected the phyco-physical well-being of the population, especially older adults with neurocognitive disorders (NCDs). This study aims to evaluate whether the frailty of NCD patients was associated with different changes in multiple health domains, in particular in relation to loneliness and social isolation, pre- and post-lockdown. Materials and methods: Patients were recruited from 10 Italian Centers for Cognitive Disorders and Dementia. Data were collected in the pre-pandemic period (T0), during the pandemic lockdown (T1), and 6-9 months post-lockdown (T2). The UCLA Loneliness Scale-3, Activities of Daily Living (ADL), Instrumental ADL (IADL), Mini-Mental State Examination, and Neuropsychiatric Inventory (NPI) were administered. Caregivers' burden was also tested. Patients were categorized as non-frail, pre-frail, and frail according to the Fatigue, Resistance, Ambulation, Illness, and Loss of Weight scale. Results: The sample included 165 subjects (61.9% women, mean age 79.5 ± 4.9 years). In the whole sample, the ADL, IADL, and NPI scores significantly declined between T0 and T2. There were no significative variations in functional and cognitive domains between the frail groups. During lockdown we recorded higher Depression Anxiety Stress Scales and Perceived Stress Scale scores in frail people. In multivariable logistic regression, frailty was associated with an increase in social isolation, and a loss of IADL. Conclusions: We observed a global deterioration in functional and neuro-psychiatric domains irrespective of the degree of frailty. Frailty was associated with the worsening of social isolation during lockdown. Frail patients and their caregivers seemed to experience more anxiety and stress disorders during SARS-CoV-2 pandemic

Large-scale production of extracellular vesicles: Report on the “massivEVs” ISEV workshop

Extracellular vesicles (EVs) large-scale production is a crucial point for the translation of EVs from discovery to application of EV-based products. In October 2021, the International Society for Extracellular Vesicles (ISEV), along with support by the FET-OPEN projects, “The Extracellular Vesicle Foundry” (evFOUNDRY) and “Extracellular vesicles from a natural source for tailor-made nanomaterials” (VES4US), organized a workshop entitled “massivEVs” to discuss the potential challenges for translation of EV-based products. This report gives an overview of the topics discussed during “massivEVs”, the most important points raised, and the points of consensus reached after discussion among academia and industry representatives. Overall, the review of the existing EV manufacturing, upscaling challenges and directions for their resolution highlighted in the workshop painted an optimistic future for the expanding EV field

Association of kidney disease measures with risk of renal function worsening in patients with type 1 diabetes

Background: Albuminuria has been classically considered a marker of kidney damage progression in diabetic patients and it is routinely assessed to monitor kidney function. However, the role of a mild GFR reduction on the development of stage 653 CKD has been less explored in type 1 diabetes mellitus (T1DM) patients. Aim of the present study was to evaluate the prognostic role of kidney disease measures, namely albuminuria and reduced GFR, on the development of stage 653 CKD in a large cohort of patients affected by T1DM. Methods: A total of 4284 patients affected by T1DM followed-up at 76 diabetes centers participating to the Italian Association of Clinical Diabetologists (Associazione Medici Diabetologi, AMD) initiative constitutes the study population. Urinary albumin excretion (ACR) and estimated GFR (eGFR) were retrieved and analyzed. The incidence of stage 653 CKD (eGFR < 60 mL/min/1.73 m2) or eGFR reduction > 30% from baseline was evaluated. Results: The mean estimated GFR was 98 \ub1 17 mL/min/1.73m2 and the proportion of patients with albuminuria was 15.3% (n = 654) at baseline. About 8% (n = 337) of patients developed one of the two renal endpoints during the 4-year follow-up period. Age, albuminuria (micro or macro) and baseline eGFR < 90 ml/min/m2 were independent risk factors for stage 653 CKD and renal function worsening. When compared to patients with eGFR > 90 ml/min/1.73m2 and normoalbuminuria, those with albuminuria at baseline had a 1.69 greater risk of reaching stage 3 CKD, while patients with mild eGFR reduction (i.e. eGFR between 90 and 60 mL/min/1.73 m2) show a 3.81 greater risk that rose to 8.24 for those patients with albuminuria and mild eGFR reduction at baseline. Conclusions: Albuminuria and eGFR reduction represent independent risk factors for incident stage 653 CKD in T1DM patients. The simultaneous occurrence of reduced eGFR and albuminuria have a synergistic effect on renal function worsening

Design and characterization of a novel tool for the antigenic enrichment of <i>actinobacillus pleuropneumoniae</i> outer membrane

Production and isolation of recombinant proteins are costly and work-intensive processes, especially in immunology when tens or hundreds of potential immunogens need to be purified for testing. Here we propose an alternative method for fast screening of immunogen candidates, based on genetic engineering of recombinant bacterial strains able to express and expose selected antigens on their outer membrane. In Actinobacillus pleuropneumoniae, a Gram-negative porcine pathogen responsible for extensive economic losses worldwide, we identified a conserved general secretion pathway (GSP) domain in the N-terminal part of the outer membrane protein ApfA (ApfA stem: ApfAs). ApfAs was used as an outer membrane anchor, to which potential immunogens can be attached. To enable confirmation of correct positioning, ApfAs, was cloned in combination with the modified acyl carrier protein (ACP) fluorescent tag ACP mini (ACPm) and the putative immunogen VacJ. The chimeric construct was inserted in the pMK-express vector, subsequently transformed into A. pleuropneumoniae for expression. Flow cytometry, fluorescence imaging and mass spectrometry analysis were employed to demonstrate that the outer membrane of the transformed strain was enriched with the chimeric ApfAs-ACPm-VacJ antigen. Our results confirmed correct positioning of the chimeric ApfAs-ACPm-VacJ antigen and supported this system&rsquo;s potential as platform technology enabling antigenic enrichment of the outer membrane of A. pleuropneumoniae

Organocatalysis and catalyst aggregation: a study using the asymmetric synthesis of benzofuranones as a test reaction

A common problem encountered in enantioselective organocatalysis is the aggregation of the catalyst, which can result in a relevant decrease of the efficiency and selectivity of the process. In the asymmetric synthesis of chiral benzofuranones, recently reported by us, we noted a remarkable increase of the reaction yield upon the addition of one of the reagents in a portionwise manner rather than in a single addition. We investigated this phenomenon by several experimental techniques such as 1D and 2D NMR experiments, UV-Vis spectroscopy, circular dichroism and dynamic light scattering. In addition, we studied the kinetic profile of this reaction using a simple numerical model and carried out in silico investigations. All these different approaches point to the conclusion that in the reaction medium a supramolecular polymerization/aggregation phenomenon, based on weak interactions, occurs and such a process is promoted by a quinone, which is one of the reagents of the benzofuranone synthesis. The portionwise mode of addition is a known strategy which can improve the performance of many synthetic procedures and this strategy is commonly adopted on account of empirical experience. However, our results provide an explanation, based on a chemical kinetic model, of the reason why the portionwise addition affects in such a dramatic way the yield of the benzofuranone synthesis catalyzed by Cinchona alkaloids