Identification of Key Amino Acid Residues Modulating Intracellular and In vitro Microcin E492 Amyloid Formation

Abstract

Microcin E492 (MccE492) is a pore-forming bacteriocin produced and exported by Klebsiella pneumoniae RYC492. Besides its antibacterial activity, excreted MccE492 can form amyloid fibrils in vivo as well as in vitro. It has been proposed that bacterial amyloids can be functional playing a biological role, and in the particular case of MccE492 it would control the antibacterial activity. MccE492 amyloid fibril’s morphology and formation kinetics in vitro have been well-characterized, however, it is not known which amino acid residues determine its amyloidogenic propensity, nor if it forms intracellular amyloid inclusions as has been reported for other bacterial amyloids. In this work we found the conditions in which MccE492 forms intracellular amyloids in Escherichia coli cells, that were visualized as round-shaped inclusion bodies recognized by two amyloidophilic probes, 2-4′-methylaminophenyl benzothiazole and thioflavin-S. We used this property to perform a flow cytometry-based assay to evaluate the aggregation propensity of MccE492 mutants, that were designed using an in silico prediction of putative aggregation hotspots. We established that the predicted amino acid residues 54–63, effectively act as a pro-amyloidogenic stretch. As in the case of other amyloidogenic proteins, this region presented two gatekeeper residues (P57 and P59), which disfavor both intracellular and in vitro MccE492 amyloid formation, preventing an uncontrolled aggregation. Mutants in each of these gatekeeper residues showed faster in vitro aggregation and bactericidal inactivation kinetics, and the two mutants were accumulated as dense amyloid inclusions in more than 80% of E. coli cells expressing these variants. In contrast, the MccE492 mutant lacking residues 54–63 showed a significantly lower intracellular aggregation propensity and slower in vitro polymerization kinetics. Electron microscopy analysis of the amyloids formed in vitro by these mutants revealed that, although with different efficiency, all formed fibrils morphologically similar to wild-type MccE492. The physiological implication of MccE492 intracellular amyloid formation is probably similar to the inactivation process observed for extracellular amyloids, and could be used as a mean of sequestering potentially toxic species inside the cell when this bacteriocin is produced in large amounts.

Amyloid‐like aggregation of designer bolaamphiphilic peptides: Effect of hydrophobic section and hydrophilic heads

“…These short peptides could self-assemble into typical amyloid fibrils on their own and played determinative roles for the amyloidogenic property of their full-length versions. [12][13][14][15][16][17][18][19] On the other hand, some de novo designed short peptides have also shown the ability to form amyloid fibrils, providing useful models for investigating the molecular basis of this intriguing process. [20][21][22][23][24][25] Although these peptides were still quite different from each other considering their exact amino acid sequences, a large proportion of them showed a common feature of amphiphilic structure, suggesting the essential role of hydrophobic interaction in the formation of amyloid fibrils.…”

“…[20][21][22][23][24][25] Although these peptides were still quite different from each other considering their exact amino acid sequences, a large proportion of them showed a common feature of amphiphilic structure, suggesting the essential role of hydrophobic interaction in the formation of amyloid fibrils. [26][27][28][29][30] Among all these amyloid-like peptides, Aβ [16][17][18][19][20][21][22] with the sequence of KLVFFAE might be the most widely studied one. 31 A lot of work have been done on this peptide, but its bolaamphiphilic feature, ie, 2 hydrophilic heads connected by a hydrophobic section, has not received enough attention.…”

Exploiting Zebrafish Xenografts for Testing the in vivo Antitumorigenic Activity of Microcin E492 Against Human Colorectal Cancer Cells

“…Escherichia coli BL21 DE3 cells were transformed with the pMccE492 plasmid carrying a 13-kbp segment of the microcin E492 production cluster, originally cloned from Klebsiella pneumoniae RYC492 (Aguilera et al, 2016). An overnight culture of these cells grown in LB broth (10 g/L tryptone, 10 g/L NaCl and 5 g/L yeast extract) were typically used to inoculate (1:1000) 2 L of M9 medium supplemented with citrate and glucose (2 g/L each) and ampicillin (100 µg/mL).…”

“…The gene cluster encoding for active MccE492 is located in the genomic island GI-E492 (Marcoleta et al, 2013b(Marcoleta et al, , 2016, which was found to be highly prevalent among liver abscessassociated strains of K. pneumoniae (Marcoleta et al, 2016(Marcoleta et al, , 2018Lam et al, 2018). A striking characteristic of MccE492 is that it forms amyloid fibers both in vivo and in vitro ( Figure 1A; Bieler et al, 2005;Arranz et al, 2012;Marcoleta et al, 2013a;Aguilera et al, 2016). Regarding its cytotoxic activity, MccE492 was toxic to different malignant cell lines (HeLa, Jurkat, and RJ2.25), and did not have effect on KG-1 and a primary culture from human tonsils (Hetz et al, 2002).…”

Techniques to Analyze sRNA Protein Cofactor Self-Assembly In Vitro

“…Indeed, these functional amyloids have been widely reported in different organisms, including bacteria. In prokaryotes, diverse functions have been described for these amyloids, such as a role in biofilm development [11], formation of curli and pili [12], or creation of pores in the host membrane [13]. In this chapter we intend to provide a guideline to develop approaches that can be used to detect the fibers of sRNA protein cofactors, precisely molecular imaging, such as atomic force microscopy and transmission electron microscopy, infrared spectroscopy, synchrotron radiation circular dichroism, and small angle X-ray scattering.…”