Motility increase of adherent invasive Escherichia coli (AIEC) induced by a sub-inhibitory concentration of recombinant endolysin LysPA90

Abstract

Endolysins are bacteriophage enzymes required for the eruption of phages from inside host bacteria via the degradation of the peptidoglycan cell wall. Recombinant endolysins are increasingly being seen as potential antibacterial candidates, with a number currently undergoing clinical trials. Bacteriophage PBPA90 infecting Pseudomonas aeruginosa harbors a gene encoding an endolysin, lysPA90. Herein, recombinant LysPA90 demonstrated an intrinsic antibacterial activity against Escherichia coli in vitro. It was observed that a sub-inhibitory concentration of the recombinant protein induced the upregulation of genes related to flagella biosynthesis in a commensal E. coli strain. Increases in the number of bacterial flagella, and in motility, were experimentally substantiated. The treatment caused membrane stress, leading to the upregulation of genes rpoE, rpoH, dnaK, dnaJ, and flhC, which are upstream regulators of flagella biosynthesis. When adherent invasive Escherichia coli (AIEC) strains were treated with subinhibitory concentrations of the endolysin, bacterial adhesion and invasion into intestinal epithelial Caco-2 cells was seen to visibly increase under microscopic examination. Bacterial counting further corroborated this adhesion and invasion of AIEC strains into Caco-2 cells, with a resultant slight decrease in the viability of Caco-2 cells then being observed. Additionally, genes related to flagella expression were also upregulated in the AIEC strains. Finally, the enhanced expression of the proinflammatory cytokine genes TNF-α, IL-6, IL-8, and MCP1 in Caco-2 cells was noted after the increased invasion of the AIEC strains. While novel treatments involving endolysins offer great promise, these results highlight the need for the further exploration of possible unanticipated and unintended effects.

Keywords: AIEC; bacteriophage; endolysin; flagella; membrane stress.

Figure 1

Antibacterial activity of recombinant endolysin LysPA90. (A) Domain structure of endolysin LysPA90. Numbers indicate amino acid positions. (B) Zymogram assay of purified recombinant endolysin LysPA90. Left; zymogram on E. coli ATCC8739, right; SDS-PAGE analysis of purified LysPA90. (C) Antibacterial activity of purified recombinant LysPA90 against E. coli ATCC8739. Antibacterial activity was tested at concentrations of 0 ~ 2 μM for 30 to 90 min. Experiments were performed in triplicate. Data are expressed as mean ± standard deviation.

Figure 2

Comprehensive transcriptome analysis of E. coli upon treatment with LysPA90. (A) Differentially expressed genes (DEGs) were grouped based on COG analysis. Genes upregulated (red bars) or downregulated (blue bars) more than six-fold were functionally grouped. The number of DEGs found in each category was divided by the total number of genes in the same category and is shown as a percentile. (B) Genes associated with flagella and fimbriae were sorted from the RNA-Seq data and the expression ratio (LysPA90/non-treated) is displayed in a colorimetric gradient: downregulation in blue and upregulation in red.

Figure 3

Upregulation of genes related to flagella biosynthesis and resulting phenotypical changes. (A) Realtime RT-PCR of genes related to flagella biosynthesis. Total RNA from wildtype (WT) or flhDC deletion mutant (ΔflhDC) strains were isolated with or without stimulation of the endolysin LysPA90. flhC, flagellar transcriptional regulator (Class I); fliA, flagella-specific sigma factor (Class II); flgF, flagella basal body rod protein (Class II), and flgK, flagella hook-associated protein (Class III). Experiments were performed in triplicate. Data are expressed as mean ± standard deviation. (B) Western blot analysis of FliC, flagellin protein (Class III) expression. GroEL was used as an internal control. (C) Transmission electron microscopic observation of flagella expression. Note that multi-flagella structure was seen only from the wildtype strain in the presence of LysPA90. (D) Observation of swarming motilities. Wild type (WT) or flhDC mutant strains (Δ flhDC) were spotted with or without preincubation with LysPA90 or denatured LysPA90 (top two rows). Wild type strain preincubated with LysPA90 was spotted next to a disc soaked in a solution containing 14.7 mg/ml of LysPA90 (bottom row). The latter plate was incubated longer than the former to see the halo extending beyond the disc.

Figure 4

Elucidation of a possible mechanism of flagella overexpression. (A) Comparison of NPN uptake of bacteria in the presence of various antibacterials. Control, no antibiotics; LysPA90, endolysin (14.7 μg/ml); AZM; azithromycin (16 μg/ml), VAN; vancomycin (128 μg/ml), CP; chloramphenicol (4 μg/ml), CST; colistin (0.25 μg/ml). (B) Realtime RT-PCR analysis of genes involved in membrane stress in either the presence of, or in the absence of LysPA90. (C) Realtime RT-PCR analysis of genes involved in membrane stress from either wildtype or rpoE deletion mutant (ΔrpoE). (D) Realtime RT-PCR analysis of genes involved in membrane stress from either wildtype or rpoH deletion mutant (ΔrpoH). Experiments were performed in triplicate. Data are expressed as mean ± standard deviation.

Figure 5

Motility increase in AIEC strains (A) Swarming motility of two AIEC strains, AIEC43 and AIEC52, in the presence or absence of LysPA90 (left two panels). AIEC strains preincubated with LysPA90 were spotted next to a disc soaked in a solution containing 14.7 mg/ml of LysPA90 (right panels). The latter plate was incubated longer than the former to see the halo extending beyond the disc. (B) Realtime RT-PCR analysis of selected genes involved in flagella biosynthesis from AIEC strains in the presence of LysPA90. flhC; flagellar transcriptional regulator (Class I); flhE, flagella protein (Class II); fliC, flagellin (Class III); flgK, flagella hook-associated protein (Class III). (C) Realtime RT-PCR analysis of type I fimbriae gene expression from two AIEC strains in the presence of LysPA90. ydeQ, putative fimbrial adhesin protein; yehD, putative Yeh fimbriae subunit; yfcV, major subunit of putative chaperon-usher fimbria. Experiments were performed in triplicate. Data are expressed as mean ± standard deviation.

Figure 6

Changes in epithelial Caco-2 cells co-cultured with AIEC strains in the presence of LysPA90. (A) Microscopic observation of adhesion or invasion of AIEC strains to Caco-2 cells. Co-cultured AIEC and Caco-2 cells were treated with PBS or LysPA90 to examine any change in the adherence of the bacteria. The culture was further treated with PBS or gentamycin to remove any bacteria outside Caco-2 cells to examine any change in invasion. (B) Counting of AIEC cells which adhered to (left) or invaded Caco-2 cells (right). (C) MTT assay of Caco-2 cells co-cultured with AIEC strains in the absence or presence of LysPA90. LysPA90 only (without AIEC strains) was used as a control. (D) Realtime RT-PCR analysis of gene expression related to inflammation from Caco-2 cells in the presence of AIEC strains and LysPA90. Experiments were performed in triplicate. Data are expressed as mean ± standard deviation.