Broad spectrum capture of clinical pathogens using engineered Fc-Mannose-Binding Lectin (FcMBL) enhanced by antibiotic treatment

Abstract

25FcMBL, an engineered version of the blood opsonin mannose-binding 26 lectin (MBL) that contains the carbohydrate recognition domain (CRD) and27 flexible neck regions of MBL fused to the Fc portion of human IgG1, has been 28 shown to bind various microbes and pathogen-associated molecular patterns 29 (PAMPs). FcMBL also has been used to create an enzyme-linked lectin sorbent 30 assay (ELLecSA) for use as a rapid ( 1 hr) diagnostic of bloodstream infections.31 Here we extended this work by using the ELLecSA to test FcMBL's ability to bind 32 to more than 200 different isolates from over 100 different pathogen species. 33 FcMBL bound to 86% of the isolates and 110 of the 122 (90%) different pathogen 34 species tested, including bacteria, fungi, viruses, and parasites. It also bound to 35 PAMPs including, lipopolysaccharide endotoxin (LPS) and lipoteichoic acid (LTA) 36 from Gram-negative and Gram-positive bacteria, as well as lipoarabinomannan 37 (LAM) and phosphatidylinositol mannoside 6 (PIM 6 ) from Mycobacterium 38 tuberculosis. The efficiency of pathogen detection and variation between binding 39 of different strains of the same species also could be improved by treating the 40 bacteria with antibiotics prior to FcMBL capture to reveal previously concealed 41 binding sites within the bacterial cell wall. As FcMBL can bind to pathogens and 42 PAMPs in urine as well as blood, its broad-binding capability could be leveraged 43 to develop a variety of clinically relevant technologies, including infectious 44 disease diagnostics, therapeutics, and vaccines. 45 46 3 47 Introduction 48 49 Mannose-binding lectin (MBL) is a key host-defense protein associated 50 with the lectin pathway of the innate immune system [1], and deficiency of MBL 51 can lead to increased susceptibility to a wide-spectrum of infectious diseases [2-52 4]. MBL functions as a calcium-dependent, pattern-recognition opsonin that binds 53 a range of carbohydrate molecules associated with the surfaces or cell walls of 54 many different types of pathogens [5]. Collectively these microbial surface 55 carbohydrate molecules, including for example, lipoteichoic acid (LTA) and 56 lipopolysaccharide endotoxin (LPS), are referred to as pathogen-associated 57 molecular patterns (PAMPs) [6,7]. MBL has the intrinsic ability to distinguish 58 foreign PAMPs from self, subsequently activating the complement system and 59 providing protection via antibody-dependent and independent mechanisms [8,9]. 60 Due to the evolutionary conserved recognition carbohydrate moieties of 61 PAMPs, MBL is a broad spectrum opsonin that can bind over 90 different species 62 of pathogens, including Gram-negative and Gram-positive bacteria, fungi, 63 viruses, and parasites [10-14]. MBL binding to these various pathogens has been 64 demonstrated by means of flow cytometry [14,15], radio-immunoassay [13,16], 65 enzyme-linked immunosorbent assay (ELISA) [13,17], immunofluorescence and 66 scanning electron microscopy (SEM) [18], and Saccharomyces cerevisiae-67 induced MBL activation and ...