Identification and recombinant production of antimicrobial peptides from the Black Soldier Fly, Hermetia illucens (L.)
The need for new antimicrobial therapies to treat drug-resistant bacterial infections is one of the leading risks to global public health. Previous discovery strategies concentrated on the isolation of secondary metabolite antibiotics that are secreted by soil-dwelling bacteria. However, the exhaustion of such discovery platforms has led to an interest in other avenues of research. In recent years, antimicrobial peptides (AMPs) have emerged as promising alternatives to conventional antibiotics that possess potent antimicrobial abilities and are less prone to resistance. AMPs are important components of innate immune systems found ubiquitously in nature. The Black Soldier Fly, Hermetia illucens (L.), has been recognised as a rich source of AMPs and several AMPs have been characterised for their antibacterial activities. This thesis aims to identify and produce AMPs from H. illucens and build on our collective repertoire of antimicrobial strategies against drug-resistant bacterial infections. To achieve this, an approach was taken that amalgamated experimental and theoretical studies and led to the identification of several novel putative AMPs from H. illucens. AMPs are typically small proteins under 10kDa. Low molecular weight proteins were identified by mass spectrometry from the haemolymph of H. illucens larvae. At the same time, AMP sequences were computationally predicted from the coding sequences of the H. illucens genome. Translated protein sequences from 3 of the coding sequences predicted to be AMPs were contained in the haemolymph mass spectrometry data set. These 3 putative AMPs alongside 6 other H. illucens AMPs from published studies encompassed several families of AMPs including attacins, cecropins, and defensins. The 9 candidate putative AMPs were carried forward to in silico and in vitro phenotypic studies. During this project, there have been major advancements in computational protein structure predictions that allowed us to theoretically characterise the 9 candidate putative AMPs. Such analyses led to several important discoveries. The 3 novel putative AMPs (named LF01, LF07, and LF10) were identified as members of paralogous gene clusters with closely related protein structures which is indicative of an evolutionarily conserved family. Also, a predicted attacin AMP (named LF06) was proposed to adopt a homo-trimeric structure similar to a Gram-negative bacterial porin. Genes encoding the 9 candidate putative AMPs were cloned into a pET expression system that was under the control of the inducible T7 promoter. Recombinant proteins were heterologously expressed and purified from Escherichia coli BL21(DE3) cells. Of the 9 proteins, 5 putative AMPs were screened for antimicrobial activities against a panel of 12 bacterial strains. Two of the candidate putative AMPs were recognised for their antimicrobial activities against the serious Gram-negative pathogen Pseudomonas aeruginosa. The 2 AMPs were the LF06 attacin which had been predicted to function as a homo-trimeric porin and LF10 which was one of the novel putative AMPs that was encoded in a paralogous gene cluster. The 2 AMPs were characterised by their activities against 2 strains of P. aeruginosa including the highly virulent Pa14 strain. Membrane damage induced by the AMPs was visualised using fluorescent and transmission electron microscopy, and electrochemical impedance spectroscopy. In summary, this work has used modern and traditional approaches to identify and engineer 2 new AMPs with antibacterial activities. These important AMPs warrant further development and could be used for the treatment of infections caused by the serious pathogen P. aeruginosa.