Effects of lactoferrin derived peptides on simulan

Authors: Tjitske Sijbrandij Antoon J Ligtenberg Kamran Nazmi Enno C I Veerman Jan G M Bolscher Floris J Bikker

Publish Date: 2016/11/10

Volume: 33, Issue: 1, Pages: 3-

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Abstract

Lactoferrin LF is an important immune protein in neutrophils and secretory fluids of mammals Bovine LF bLF harbours two antimicrobial stretches lactoferricin and lactoferampin situated in close proximity in the N1 domain To mimic these antimicrobial domain parts a chimeric peptide LFchimera has been constructed comprising parts of both stretches LFcin17–30 and LFampin265–284 To investigate the potency of this construct to combat a set of Gram positive and Gram negative bacteria which are regarded as simulants for biological warfare agents the effect on bacterial killing membrane permeability and membrane polarity were determined in comparison to the constituent peptides and the native bLF Furthermore we aimed to increase the antimicrobial potency of the bLF derived peptides by cationic amino acid substitutions Overall the bactericidal activity of the peptides could be related to membrane disturbing effects ie membrane permeabilization and depolarization Those effects were most prominent for the LFchimera Arginine residues were found to be crucial for displaying antimicrobial activity as lysine to arginine substitutions resulted in an increased antimicrobial activity affecting mostly LFampin265–284 whereas arginine to lysine substitutions resulted in a decreased bactericidal activity predominantly in case of LFcin17–30LF is a multifunctional 80 kDa glycoprotein and secreted as innate immunity factor in secretory fluids eg tears saliva and milk and also found in the granules of the neutrophils Farnaud and Evans 2003 It is known to have bactericidal fungicidal and antiviral activity as well as antitumor antiinflammatory and immunoregulatory properties Brock 1995 Most of these activities reside in the basic N1domain of LF LactoferricinB LFcinB released by gastric pepsin cleavage of bovine LF bLF Bellamy et al 1992 is a positively charged looped peptide consisting of amino acid residues 17–41 with more potent bactericidal and fungicidal activity than the native protein Studies on a shorter variant consisting of amino acid residues 17–30 LFcin17–30 revealed a broadspectrum bactericidal activity against Grampositive and Gramnegative bacteria including Bacillus subtilis Escherichia coli and Pseudomonas aeruginosa Groenink et al 1999 Besides LFcinB the N1domain contains a second stretch designated LFampin which also shows features that are characteristic for AMPs including the presence of positively charged residues and a hydrophobic domain Previously two bovine LFampin variants LFampin268–284 and LFampin265–284 were compared with respect to their bactericidal activities It was found that LFampin265–284 killed a set of Grampositive bacteria that were resistant to LFampin268–284 It appeared that the presence of the Nterminal sequence 265AspLeuIle268 enhanced the propensity of LFampin to adopt an αhelix leading to an improved activity Bolscher et al 2006 Van der Kraan et al 2004The mutual orientation of the two antimicrobial stretches localized at the edge of the N1domain prompted us to investigate the activities in conjunction as well as in separation Therefore a chimerical structure consisting of LFcin17–30 and LFampin265–284 was generated by linking both peptides to the two amino group of a lysine residue Bolscher et al 2009 NMR analysis showed that this lysine linker forced both peptides to extended αhelixes Haney et al 2012 This LFchimera exhibited stronger bactericidal activity than its constituent peptides either individually or as a mixture not hampered by the shielding effects of high ionic strength Bolscher et al 2009The potency of this chimeric peptide provoked us to test the peptide against various human pathogens like Staphylococcus aureus FloresVillasenor et al 2010 Escherichia coli O157H7 FloresVillasenor et al 2012 Streptococcus pneumoniae LeonSicairos et al 2014 and Burkholderia pseudomallei Kanthawong et al 2009 Puknun et al 2013 2016 the latter which is classified by the Centers for Disease Control and Prevention as a category B biological warfare agent BWA Rotz et al 2002However development of new AMPs for combatting BWA entails several safety security and logistical drawbacks including culturing and testing of virulent bacteria under adequate biocontainment conditions hampering large screenings of peptides In order to overcome these difficulties most of the research activities and tests reported so far are performed using simulants BWA simulants BWAS which are phylogenetically or structurally related to BWA Therefore for the present study we have chosen a set of both Gram positive and Gram negative bacteria which are considered BWAS Adducci et al 2016 Bikker et al 2006 Dawson and Liu 2008 Kaman et al 2011a These include Bacillus cereus and Bacillus globigii considered safer simulants for Bacillus anthracis the etiologic agent of anthrax Jansen et al 2014 Furthermore we included two strains which are closely related to Yersinia pestis ie Yersinia enterocolitica and Yersinia pseudotuberculosis the latter causing severe fever and abdominal pain Atkinson and Williams 2016 Kaman et al 2011a Finally the effects on two Salmonella spp were studied including a distinct strain of Salmonella enterica serotype typhimurium known as definitive type 104 DT104 a major cause of gastroenteritis in humans and animals Hall 2010The antimicrobial activities of LFchimera and its constituent peptides as well as the native bLF were mapped by studying 1 the minimum bactericidal concentration MBC 2 membrane integrity by using the propidium iodide PI assay and 3 membrane polarity using a DiSC35 assay Furthermore we aimed to increase the antimicrobial potency of the bLF derived peptides by cationic amino acid substitutions As cationic amino acid residues attract the peptide to the anionic bacterial membrane positively charged residues including arginine Arg R lysine Lys K and histidine His H play an important role within AMPs in general Despite their identical charge Arg residues appear more prevalent in naturally occurring AMPs than Lys and His Cutrona et al 2015 Henriques et al 2006 Hristova and Wimley 2011 Joliot and Prochiantz 2004 Yeaman and Yount 2003 Besides by in vitro experiments it was found that Lys to Arg substitutions increased the antimicrobial effects of peptides implying that the guanidinium group in Arg may be preferable for activity than the amine group found in Lys Cutrona et al 2015 Strom et al 2003 In line we analyzed variants of LFcin17–30 LFampin265–284 and LFchimera in which all Arg residues were replaced for Lys residues and vice versa Arg to His and Lys to His substitutions were omitted as LF peptides do not contain His

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