Characterization of a highly potent antimicrobial peptide microcin N from uropathogenicEscherichia coli

Abstract

Microcin N is a low-molecular weight, highly active antimicrobial peptide produced by uropathogenic Escherichia coli In this study, the native peptide was expressed and purified from pGOB18 plasmid carrying E. coli in low yield. The pure peptide was characterized using mass spectrometry, N-terminal sequencing by Edman degradation as well as trypsin digestion. We found that the peptide is 74-residue long, cationic (+2 total charge), highly hydrophobic and consists of glycine as the first N-terminal residue. The minimum inhibitory concentration of the peptide against Salmonella enteritidis was found to be 150 nM. Evaluation of the solution conformation of the peptide using circular dichroism spectroscopy showed that the peptide is well folded in 40% trifluoroethanol with helical structure whereas the folded structure is lost in aqueous solution. To increase the yield of this potent peptide, we overexpressed GST-tagged microcin N using E. coli BL21. Recombinant GST-tagged microcin N was successfully expressed in E. coli BL21; however, the cleaved mature microcin N did not show activity against the indicator strain (S. enterica) most likely due to the extreme hydrophobic nature of the peptide. Efforts to produce active microcin N in large scale are discussed as this peptide has huge potential to be the next generation antimicrobial agent.

pMPES: A Modular Peptide Expression System for the Delivery of Antimicrobial Peptides to the Site of Gastrointestinal Infections Using Probiotics

“…With the exception of EntP and HirJM79, all of the peptides tested herein are naturally encoded with glycine-glycine leader peptides [17,19,33,35]. EntP and HirJM79 are believed to be secreted via the general Sec-type secretion pathways of their native producers [3,36].…”

Bacteriocins to Thwart Bacterial Resistance in Gram Negative Bacteria

“…MccN/24 is active against E. coli and S. enterica Typhimurium, but not against L. monocytogenes or Campylobacter jejuni (Wooley et al, 1999). It was also reported by Kaur et al (2016) that MccN/24 exhibits a potent activity against Salmonella strains. Furthermore, MccV is active against some pathogenic E. coli with MIC values ranging from 7.7 × 10 −3 to 13.25 µg/mL (0.89-1517.94 nM) (Boubezari et al, 2018).…”

“…Class II microcins form a more homogeneous group than their class I cousins ( Table 1 and Supplementary Figures S1B,C ), although they are subdivided into class IIa, encompassing MccL ( Pons et al, 2004 ), MccN/24 ( Kaur et al, 2016 ), MccPDI ( Eberhart et al, 2012 ), MccS ( Zschüttig et al, 2012 ) and MccV ( Gratia, 1925 ), and class IIb (MccE492, MccH47, MccM, Vassiliadis et al, 2010 ). MccN was formely termed Mcc24 ( O’Brien and Mahanty, 1994 ) and is termed MccN/24 in this review.…”

“…Class IIa microcins have been characterized from E. coli strains from various origins. The MccN/24 producer is an uropathogenic E. coli ( Kaur et al, 2016 ) and the MccL producer comes from poultry intestine ( Sablé et al, 2003 ), while MccS is produced by a probiotic strain, E. coli G3/10 (Symbioflor2 ® ; DSM17252) ( Zschüttig et al, 2012 ). The producing strains are in some cases multi-microcin producers, such as E. coli LR05 that secretes MccB17, MccJ25 and the uncharacterized MccD93 in addition to MccL ( Sablé et al, 2003 ).…”

Microcins reveal natural mechanisms of bacterial manipulation to inform therapeutic development

“…For these reasons, microcins are routinely obtained from bacteria. Purification is performed using the native producer or a heterologous strain carrying the genes necessary for microcin synthesis, immunity, export and modification as needed [22,125,152,[162][163][164][165]. Typically, the supernatant of the secreting strain is pre-purified by column chromatography, followed by HPLC [22,125,152,165].…”