Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2000 Jul;182(14):4077-86.
doi: 10.1128/JB.182.14.4077-4086.2000.

A PhoP-regulated outer membrane protease of Salmonella enterica serovar typhimurium promotes resistance to alpha-helical antimicrobial peptides

T Guina  1 , E C Yi, H Wang, M Hackett, S I Miller
Affiliations

A PhoP-regulated outer membrane protease of Salmonella enterica serovar typhimurium promotes resistance to alpha-helical antimicrobial peptides

T Guina et al. J Bacteriol. 2000 Jul.

Abstract

The outer membrane protein contents of Salmonella enterica serovar Typhimurium strains with PhoP/PhoQ regulon mutations were compared by two-dimensional gel electrophoresis. At least 26 species of outer membrane proteins (OMPs) were identified as being regulated by PhoP/PhoQ activation. One PhoP/PhoQ-activated OMP was identified by semiautomated tandem mass spectrometry coupled with electronic database searching as PgtE, a member of the Escherichia coli OmpT and Yersinia pestis Pla family of outer membrane proteases. Salmonella PgtE expression promoted resistance to alpha-helical cationic antimicrobial peptides (alpha-CAMPs). Strains expressing PgtE cleaved C18G, an 18-residue alpha-CAMP present in culture medium, indicating that protease activity is likely to be the mechanism of OmpT-mediated resistance to alpha-CAMPs. PhoP/PhoQ did not regulate the transcription or export of PgtE, indicating that another PhoP/PhoQ-dependent mechanism is required for PgtE outer membrane localization. PgtE is a posttranscriptionally regulated component of the PhoP/PhoQ regulon that contributes to Salmonella resistance to innate immunity.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
2-D PAGE map of PhoP/PhoQ-regulated OMPs. OMPs were separated by IEF on a linear pH gradient of 4 to 7 and on SDS–12% PAGE gels. Proteins were visualized by staining in Coomassie brilliant blue. 2-D map positions of the porins OmpA, OmpC/OmpF, and PhoP-regulated phase I flagellin (Fla) were determined by comparison with previous studies (38). Unlabeled arrowheads point to protein species present exclusively in the absence (PhoP null) or presence (PhoP constitutive) of an active PhoP/PhoQ regulatory system. PgtE is a protein that was sequenced and analyzed in this study.
FIG. 2
FIG. 2
Base peak capillary HPLC mass chromatogram from the tryptic digest of Salmonella PgtE. In this type of HPLC trace, the signal intensity of the most abundant ion is plotted for each scan. Each scan of the mass spectrometer takes about 1 s. Peptides identified by SEQUEST are indicated by arrows and contain the following partial sequences: (A) ELVYDTDTGR; (B) ELVYDTDGRK; (C) KLSQLDWK; (D) GWLLQGDNYK; (E) FSWTAR; (F) YIGNFPHGVR; (G) GIGYSQR; (H) YSDWVNAHDNDEHYMR; and (I) IFAEFAYSK.
FIG. 3
FIG. 3
Peptide sequencing by tandem mass spectrometry and identification of Salmonella PgtE. Fragment ions observed are indicated in boldface above (b-type ions) and below (y-type ions) the peptide sequence. (A) CID mass spectrum of GWLLQGDNYK. (B) CID mass spectrum of IFAEFAYSK. (C) CID mass spectrum of FSWTAR. The nomenclature used for b and y peptide fragment ions has been described by Biemann (4a). Low-mass ions that are indicative of amino acid composition but not sequence are described by the amino acid single-letter code.
FIG. 4
FIG. 4
pgtE expression does not require PhoP. The expression of pgtE was measured throughout the growth of bacterial cultures by quantitating the amount of luciferase activity produced by strains containing the pgtE-f-luc transcriptional fusion. Bacterial cultures were grown in rich medium (LB). The graph depicts one experiment performed in triplicate and is representative of several experiments. Error bars represent the standard deviation (SD); no bars indicate that the SD is insignificant. FLU, firefly luciferase light units. const, constitutive.
FIG. 5
FIG. 5
High levels of pgtE expression increase Salmonella survival in the presence of C18G. Mid-log-phase bacterial cultures were incubated with the indicated concentrations of C18G, and the number of CFU was determined. Expression of pgtE from a high-copy plasmid (in TG73) increased survival in the presence of C18G compared to the parental strain (CS022), while the strain with the pgtE deletion (TG61) did not display significant sensitivity to C18G. A pagP mutant (CS435) was sensitive to C18G.
FIG. 6
FIG. 6
Salmonella cells expressing PgtE cleave C18G peptide present in the culture supernatants. Culture supernatants were collected after an MIC experiment, and their contents were analyzed by reversed-phase HPLC as described in Materials and Methods. A strain expressing pgtE from a high-copy plasmid (TG73) efficiently cleaved C18G (B), while a strain carrying a mutation in ompT (TG61) did not cleave C18G (C). The control sample contained C18G in buffer without bacteria present (A).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Adams P, Fowler R, Howell G, Kinsella N, Skipp P, Coote P, O'Connor C D. Defining protease specificity with proteomics: a protease with a dibasic amino acid recognition motif is regulated by a two-component signal transduction system in Salmonella. Electrophoresis. 1999;20:2241–2247. - PubMed
    1. Bairoch A, Apweiler R. The SWISS-PROT protein sequence data bank and its supplement TrEMBL in 1999. Nucleic Acids Res. 1999;27:49–54. - PMC - PubMed
    1. Bals R, Wang X, Zasloff M, Wilson J M. The peptide antibiotic LL-37/hCAP-18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface. Proc Natl Acad Sci USA. 1998;95:9541–9546. - PMC - PubMed
    1. Belden W J, Miller S I. Further characterization of the PhoP regulon: identification of new PhoP-activated virulence loci. Infect Immun. 1994;62:5095–5101. - PMC - PubMed
    1. Biemann K. Contributions of mass spectrometry to peptide and protein structure. Biomed Environ Mass Spectrom. 1988;16:99–111. - PubMed

Publication types

MeSH terms

Associated data

-