Down-regulation of the kps region 1 capsular assembly operon following attachment of Escherichia coli type 1 fimbriae to D-mannose receptors - PubMed (original) (raw)

Down-regulation of the kps region 1 capsular assembly operon following attachment of Escherichia coli type 1 fimbriae to D-mannose receptors

William R Schwan et al. Infect Immun. 2005 Feb.

Abstract

A differential-display PCR procedure identified the capsular assembly gene kpsD after Escherichia coli type 1 fimbrial binding to mannose-coated Sepharose beads. Limiting-dilution reverse-transcribed PCRs confirmed down-regulation of the kpsD gene, and Northern blot and lacZ fusion analyses showed down-regulation of the kpsFEDUCS region 1 operon. KpsD protein levels fell, and an agglutination test showed less K capsular antigen on the surface following the bacterial ligand-receptor interaction. These data show that binding of type 1 fimbriae (pili) to d-mannose receptors triggers a cross talk that leads to down-regulation of the capsule assembly region 1 operon in uropathogenic E. coli.

PubMed Disclaimer

Figures

FIG. 1.

FIG. 1.

Quantitative determination of kpsD and ftsZ transcript levels by LD-RT-PCR analysis. The cDNAs generated from strain NU149 cells mixed with plain Sepharose or

d

-mannose-coated Sepharose beads were examined at 0, 10, 25, 60, and 120 min. The KpsD1-KpsD2 and ECFtsZ1-ECFtsZ2 primer pairs were used to amplify serially twofold-diluted cDNAs targeting kpsD (367-bp product) and ftsZ (302-bp product) transcripts, respectively. The PCR amplifications were done a minimum of three times. All PCR products were electrophoresed on 1.5% agarose gels. The following dilutions of cDNAs were used: undiluted (lane 1), 1:2 (lane 2), 1:4 (lane 3), 1:8 (lane 4), 1:16 (lane 5), 1:32 (lane 6), 1:64 (lane 7), 1:128 (lane 8), and 1:256 (lane 9).

FIG. 2.

FIG. 2.

Regulation of the kpsFEDUCS promoter following attachment to plain Sepharose (−),

l

-mannose-coated Sepharose (+L),

d

-mannose-coated Sepharose (+D), or plain Sepharose plus 50 mM free

d

-mannose (+F). The assays were done using a kpsFEDUCS promoter fused to lacZYA as a transcriptional fusion on a single-copy-number plasmid placed in strain AAEC189. The β-galactosidase (β-Gal) activity is expressed in Miller units; means ± standard deviations are indicated from three separate runs.

FIG. 3.

FIG. 3.

Quantitative determination of kpsD transcript levels in strains NU14 (wild type) and NU14-1 (fimH mutant) by LD-RT-PCR analysis. The cDNAs generated from strain NU14 or NU14-1 cells mixed with

d

-mannose-coated Sepharose beads at 0 and 120 min were examined. The KpsD1-KpsD2 primer pair was used to amplify serially twofold-diluted cDNAs targeting kpsD (367-bp product) transcripts. The PCR amplifications were done a minimum of three times. All PCR products were electrophoresed on 1.5% agarose gels. The following dilutions of cDNAs were used: undiluted (lane 1), 1:2 (lane 2), 1:4 (lane 3), 1:8 (lane 4), 1:16 (lane 5), 1:32 (lane 6), 1:64 (lane 7), 1:128 (lane 8), and 1:256 (lane 9).

FIG. 4.

FIG. 4.

Northern hybridization analysis of transcription of the kpsFEDUCS operon from total RNAs isolated from NU149 cells mixed with plain Sepharose (−) or

d

-mannose-coated Sepharose (+) beads after 0 and 2 h of exposure. Ten micrograms of total RNA from NU149 cells mixed with plain Sepharose beads or

d

-mannose-coated Sepharose beads was probed with a kpsD DNA fragment. After 6 days, the blot was developed with a phosphorimager and the amounts of kpsFEDUCS RNA (approximate size, 7.9 kb) were compared between lanes by using ImageQuant 5.2 software.

FIG. 5.

FIG. 5.

Enzyme-linked immunosorbent assay analysis of the KpsD protein from strain NU149 cells mixed with plain or

d

-mannose-coated Sepharose beads for 0, 8, and 24 h. Optical densities at 405 nm (O.D.405) were determined, and means ± standard deviations are indicated from three runs. (−), plain Sepharose; (+),

d

-mannose-coated Sepharose.

FIG. 6.

FIG. 6.

Directagen analysis of K1 capsular expression in strain NU14. Bacterial cells were mixed with plain Sepharose (−) or

d

-mannose-coated Sepharose (+) beads and assayed after 0 and 24 h. White clumps of bacteria indicate a positive response.

Similar articles

Cited by

References

    1. Abu Kwaik, Y., and L. L. Pederson. 1996. The use of differential display-PCR to isolate and characterize a Legionella pneumophila locus induced during the intracellular infection of macrophages. Mol. Microbiol. 21:543-556. - PubMed
    1. Arrecubieta, C., T. C. Hammarton, B. Barrett, S. Chareonsudjai, N. Hodson, D. Rainey, and I. S. Roberts. 2001. The transport of group 2 capsular polysaccharides across the periplasmic space in Escherichia coli. J. Biol. Chem. 276:4245-4250. - PubMed
    1. Bahrani-Mougeot, F. K., S. Pancholi, M. Daoust, and M. S. Donnenberg. 2001. Identification of putative urovirulence genes by subtractive cloning. J. Infect. Dis. 182:S21-S23. - PubMed
    1. Bahrani-Mougeot, F. K., E. L. Buckles, C. V. Lockatell, J. R. Hebel, D. E. Johnson, C. M. Tang, and M. S. Donnenberg. 2002. Type 1 fimbriae and extracellular polysaccharides are preeminent uropathogenic Escherichia coli virulence determinants in the murine urinary tract. Mol. Microbiol. 45:1079-1093. - PubMed
    1. Blomfield, I. C., M. S. McClain, J. A Prine, P. J. Calie, and B. I. Eisenstein. 1991. Type1 fimbriation and fimE mutants of Escherichia coli K-12. J. Bacteriol. 173:5298-5307. - PMC - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources