Dissection of the antimicrobial and hemolytic activity of Cap18: Generation of Cap18 derivatives with enhanced specificity

<div><p>Due to the rapid emergence of resistance to classical antibiotics, novel antimicrobial compounds are needed. It is desirable to selectively kill pathogenic bacteria without targeting other beneficial bacteria in order to prevent the negative clinical consequences caused by many broad-spectrum antibiotics as well as reducing the development of antibiotic resistance. Antimicrobial peptides (AMPs) represent an alternative to classical antibiotics and it has been previously demonstrated that Cap18 has high antimicrobial activity against a broad range of bacterial species. In this study we report the design of a positional scanning library consisting of 696 Cap18 derivatives and the subsequent screening for antimicrobial activity against <i>Y</i>. <i>ruckeri</i>, <i>A</i>. <i>salmonicida</i>, <i>S</i>. Typhimurium and <i>L</i>. <i>lactis</i> as well as for hemolytic activity measuring the hemoglobin release of horse erythrocytes. We show that the hydrophobic face of Cap18, in particular I13, L17 and I24, is essential for its antimicrobial activity against <i>S</i>. Typhimurium, <i>Y</i>. <i>ruckeri</i>, <i>A</i>. <i>salmonicida</i>, <i>E</i>. <i>coli</i>, <i>P</i>. <i>aeruginosa</i>, <i>L</i>. <i>lactis</i>, <i>L</i>. <i>monocytogenes</i> and <i>E</i>. <i>faecalis</i>. In particular, Cap18 derivatives harboring a I13D, L17D, L17P, I24D or I24N substitution lost their antimicrobial activity against any of the tested bacterial strains. In addition, we were able to generate species-specific Cap18 derivatives by particular amino acid substitutions either in the hydrophobic face at positions L6, L17, I20, and I27, or in the hydrophilic face at positions K16 and K18. Finally, our data showed the proline residue at position 29 to be essential for the inherent low hemolytic activity of Cap18 and that substitution of the residues K16, K23, or G21 by any hydrophobic residues enhances the hemolytic activity. This study demonstrates the potential of generating species-specific AMPs for the selective elimination of bacterial pathogens.</p></div

Clinical presentation of calmodulin mutations: the International Calmodulinopathy Registry

Calmodulin; Cardiomyopathies; Neurological disordersCalmodulina; Miocardiopatías; Trastornos neurológicosCalmodulina; Miocardiopaties; Trastorns neurològicsAims: Calmodulinopathy due to mutations in any of the three CALM genes (CALM1-3) causes life-threatening arrhythmia syndromes, especially in young individuals. The International Calmodulinopathy Registry (ICalmR) aims to define and link the increasing complexity of the clinical presentation to the underlying molecular mechanisms. Methods and results: The ICalmR is an international, collaborative, observational study, assembling and analysing clinical and genetic data on CALM-positive patients. The ICalmR has enrolled 140 subjects (median age 10.8 years [interquartile range 5-19]), 97 index cases and 43 family members. CALM-LQTS and CALM-CPVT are the prevalent phenotypes. Primary neurological manifestations, unrelated to post-anoxic sequelae, manifested in 20 patients. Calmodulinopathy remains associated with a high arrhythmic event rate (symptomatic patients, n = 103, 74%). However, compared with the original 2019 cohort, there was a reduced frequency and severity of all cardiac events (61% vs. 85%; P = .001) and sudden death (9% vs. 27%; P = .008). Data on therapy do not allow definitive recommendations. Cardiac structural abnormalities, either cardiomyopathy or congenital heart defects, are present in 30% of patients, mainly CALM-LQTS, and lethal cases of heart failure have occurred. The number of familial cases and of families with strikingly different phenotypes is increasing. Conclusion: Calmodulinopathy has pleiotropic presentations, from channelopathy to syndromic forms. Clinical severity ranges from the early onset of life-threatening arrhythmias to the absence of symptoms, and the percentage of milder and familial forms is increasing. There are no hard data to guide therapy, and current management includes pharmacological and surgical antiadrenergic interventions with sodium channel blockers often accompanied by an implantable cardioverter-defibrillator.The ICalmR is one of the registries supported by ERN GUARD-Heart. This research was supported by the Italian Ministry of Health Ricerca Corrente ‘Registro internazionale delle calmodulinopatie’ to L.C., F.D., P.J.S., M.C.K., and C.S.; by the 2019-ATESP-0045 Fondo di Ateneo Quota Competitiva to L.C.; and partially by the Fondation Leducq grant 18CVD05 ‘Towards Precision Medicine with Human iPSCs for Cardiac Channelopathies’ to L.C., M.-C.K., L.S., and P.J.S. J.B., L.C., and P.J.S. were partially supported by the European Joint Programme on Rare Diseases: LQTS-NEXT grant. R.H. was supported by the Canadian Institutes of Health Research, the Heart and Stroke Foundation of Canada, The Labatt Family Heart Centre, the Cartwright Family Fellowship, the Carter Heart Arrhythmia Trainee Fund and the Caitlin Elizabeth Morris fund. J.P.K. was supported by the Medical Research Council (MRC) Clinical Academic Research Partnership (CARP) Award (MR/T024062/1). G.R.W. was supported by an NIH K23HL130554 grant. A.A.M.W. was supported by the Netherlands Cardiovascular Research Initiative (CVON PREDICT-2). M.J.A. was supported in part by the Mayo Clinic Windland Smith Rice Comprehensive Sudden Cardiac Death Program. M.T.O. was supported in part by the Danish Independent Research Council (Grant no. 2032–00333B) and the Lundbeck Foundation (Grant no. R324-2019-1933). H.K.J. was supported by the Novo Nordisk Foundation (Grant NNF 18OC0031258)

Calmodulin mutations affecting Gly114 impair binding to the NaV1.5 IQ-domain

Missense variants in CALM genes encoding the Ca2+-binding protein calmodulin (CaM) cause severe cardiac arrhythmias. The disease mechanisms have been attributed to dysregulation of RyR2, for Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) and/or CaV1.2, for Long-QT Syndrome (LQTS). Recently, a novel CALM2 variant, G114R, was identified in a mother and two of her four children, all of whom died suddenly while asleep at a young age. The G114R variant impairs closure of CaV1.2 and RyR2, consistent with a CPVT and/or mild LQTS phenotype. However, the children carrying the CALM2 G114R variant displayed a phenotype commonly observed with variants in NaV1.5, i.e., Brugada Syndrome (BrS) or LQT3, where death while asleep is a common feature. We therefore hypothesized that the G114R variant specifically would interfere with NaV1.5 binding. Here, we demonstrate that CaM binding to the NaV1.5 IQ-domain is severely impaired for two CaM variants G114R and G114W. The impact was most severe at low and intermediate Ca2+ concentrations (up to 4 µM) resulting in more than a 50-fold reduction in NaV1.5 binding affinity, and a smaller 1.5 to 11-fold reduction at high Ca2+ concentrations (25–400 µM). In contrast, the arrhythmogenic CaM-N98S variant only induced a 1.5-fold reduction in NaV1.5 binding and only at 4 µM Ca2+. A non-arrhythmogenic I10T variant in CaM did not impair NaV1.5 IQ binding. These data suggest that the interaction between NaV1.5 and CaM is decreased with certain CaM variants, which may alter the cardiac sodium current, INa. Overall, these results suggest that the phenotypic spectrum of calmodulinopathies may likely expand to include BrS- and/or LQT3-like traits