pubmed.ncbi.nlm.nih.gov

OmpT outer membrane proteases of enterohemorrhagic and enteropathogenic Escherichia coli contribute differently to the degradation of human LL-37 - PubMed

OmpT outer membrane proteases of enterohemorrhagic and enteropathogenic Escherichia coli contribute differently to the degradation of human LL-37

Jenny-Lee Thomassin et al. Infect Immun. 2012 Feb.

Abstract

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are food-borne pathogens that cause serious diarrheal diseases. To colonize the human intestine, these pathogens must overcome innate immune defenses such as antimicrobial peptides (AMPs). Bacterial pathogens have evolved various mechanisms to resist killing by AMPs, including proteolytic degradation of AMPs. To examine the ability of the EHEC and EPEC OmpT outer membrane (OM) proteases to degrade α-helical AMPs, ompT deletion mutants were generated. Determination of MICs of various AMPs revealed that both mutant strains are more susceptible than their wild-type counterparts to α-helical AMPs, although to different extents. Time course assays monitoring the degradation of LL-37 and C18G showed that EHEC cells degraded both AMPs faster than EPEC cells in an OmpT-dependent manner. Mass spectrometry analyses of proteolytic fragments showed that EHEC OmpT cleaves LL-37 at dibasic sites. The superior protection provided by EHEC OmpT compared to EPEC OmpT against α-helical AMPs was due to higher expression of the ompT gene and, in turn, higher levels of the OmpT protein in EHEC. Fusion of the EPEC ompT promoter to the EHEC ompT open reading frame resulted in decreased OmpT expression, indicating that transcriptional regulation of ompT is different in EHEC and EPEC. We hypothesize that the different contributions of EHEC and EPEC OmpT to the degradation and inactivation of LL-37 may be due to their adaptation to their respective niches within the host, the colon and small intestine, respectively, where the environmental cues and abundance of AMPs are different.

PubMed Disclaimer

Figures

Fig 1
Fig 1

Proteolytic degradation of LL-37 and C18G. OmpT-mediated degradation of LL-37 (A) and C18G (B). LL-37 or C18G (10 μg) was incubated with the indicated strains for the indicated times. The resulting AMP cleavage products were separated by Tris-Tricine SDS-PAGE and visualized by Coomassie staining. Asterisks, migration of the dye front; pound sign, an aberrantly migrating band that is observed only after complete C18G cleavage and may correspond to cleavage product aggregates. Data shown are representative of at least three independent experiments.

Fig 2
Fig 2

Mass spectrometry analysis of LL-37 degradation products. OmpT-dependent LL-37 cleavage products were detected by liquid chromatography and analyzed by MS/MS. Shown is a schematic of the LL-37 amino acid sequence. Dibasic sequences are shown in black. Vertical filled arrows, OmpT cleavage sites; horizontal open arrows, fragments detected by MS/MS analysis.

Fig 3
Fig 3

Proteolytic cleavage of a synthetic FRET peptide. The synthetic FRET peptide containing the dibasic sequence RK was incubated with various EHEC and EPEC strains. Peptide cleavage, indicated by the increase in fluorescence, was measured over time. (A) Fluorescence of the FRET peptide incubated with EHEC wild type (black), EHEC ΔompT mutant (red), and EHEC ΔompT(pEHompT) mutant (blue); (B) fluorescence of the FRET peptide incubated with EPEC wild type (black), EPEC ΔompT mutant (red), and EPEC ΔompT(pEPompT) mutant (blue). All samples were normalized against a PBS blank; data shown are representative of three independent experiments.

Fig 4
Fig 4

Expression of the ompT gene. (A) Transcription of ompT in the EHEC and EPEC wild-type and ΔompT strains; (B) transcription of ompT in the EPEC wild-type, ΔompT, ΔompT(pEPompT), and ΔompT(pEHompT) strains. Expression of ompT was quantified by RT-qPCR. Relative mRNA expression is representative of ompT expression normalized against 16S RNA. Results are expressed as means ± SDs. Statistical significance was assessed using a one-way analysis of variance and Tukey's post hoc comparison test. Unless otherwise indicated, asterisks indicate statistical significance versus wild type (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Fig 5
Fig 5

Detection of the OmpT protein by Western blotting. (A and B) OmpT-FLAG was detected using a polyclonal anti-FLAG antibody; filled arrows, OmpT-FLAG protein species. (A) Whole-cell lysates (WCL), soluble fractions (CYT), inner membrane fractions (IM), and OM fractions (OM) from the EPEC ΔompT mutant with the empty vector (ΔompT) or pEPompT-FLAG (pEPompT); (B) whole-cell lysates of the EHEC ΔompT mutant with the empty vector (ΔompT), pEHompT-FLAG (pEHompT), or pEPompT-FLAG (pEPompT) and of the EPEC ΔompT mutant with the empty vector (ΔompT), pEPompT-FLAG (pEPompT), or pEHompT-FLAG (pEHompT); (C) OmpT was detected using a polyclonal anti-CroP antibody. Whole-cell lysates of EHEC wild type, ΔompT mutant, and ΔompT(pEHompT) mutant and of EPEC wild type, ΔompT mutant, and ΔompT(pEPompT) mutant. All samples were normalized (by OD600) to ensure that the same number of cells was used. Data shown are representative of three independent experiments. Filled arrows, OmpT protein species; asterisks, cross-reactive bands.

Fig 6
Fig 6

The EPEC ompT promoter lowers the expression of EHEC OmpT. (A and B) OmpT from various EPEC (A) and EHEC (B) strains was detected by Western blotting of whole-cell lysates using a polyclonal anti-CroP antibody. Asterisks, cross-reactive bands. Data shown are representative of three independent experiments. (C and D) Cleavage of the FRET peptide was measured over time. (C) Fluorescence of the FRET peptide incubated with EPEC wild type and ΔompT, ΔompT(pEPompT), ΔompT(pEHompT), ΔompT(pEPpromEHompT), and ΔompT(pEHpromEPompT) mutants; (D) fluorescence of the FRET peptide incubated with EHEC wild type and ΔompT, ΔompT(pEHompT), ΔompT(pEPompT), ΔompT(pEPpromEHompT), and ΔompT(pEHpromEPompT) mutants. All samples were normalized against a PBS blank; data shown are representative of two independent experiments.

Similar articles

Cited by

References

    1. Bevins CL, Salzman NH. 2011. Paneth cells, antimicrobial peptides and maintenance of intestinal homeostasis. Nat. Rev. Microbiol. 9:356–368 - PubMed
    1. Burton MF, Steel PG. 2009. The chemistry and biology of LL-37. Nat. Prod. Rep. 26:1572–1584 - PubMed
    1. Campos MA, et al. 2004. Capsule polysaccharide mediates bacterial resistance to antimicrobial peptides. Infect. Immun. 72:7107–7114 - PMC - PubMed
    1. Darveau RP, et al. 1992. Peptides related to the carboxyl terminus of human platelet factor IV with antibacterial activity. J. Clin. Invest. 90:447–455 - PMC - PubMed
    1. Dekker N, Cox RC, Kramer RA, Egmond MR. 2001. Substrate specificity of the integral membrane protease OmpT determined by spatially addressed peptide libraries. Biochemistry 40:1694–1701 - PubMed

Publication types

MeSH terms

Substances