Biochemical Society Transactions (2000) Volume 28, Part 5
75,
Serotype Conversion of Shigellaflexnerr in A Patient with Shigellosis
I.-H.’
C.-S.
,Chiou*, W.-B. Hsu’
I: Institute of Molecular Biology, Natzonal Chung Hsing Universrty,
Taichung, Taiwan Republic of China; 2: The Third Branch Ofice, Center for
Disease Control, Department of Health, Taichung, Taiwan, Republic of China
Five isolates of ShigellaJlexnen were recovered successively from a patient of shigellosis. Slide agglutination test with commercial polyclonal antisera identified the first
3 isolates as serotype Za and the last two isolates serotype Y. Genomic DNAs of the
5 isolates were digested with Not1 and analyzed by pulsed-field gel electrophoresis.
The result showed identical patterns with the 5 isolates, suggesting that they were
likely derived from the same isolate and serotype conversion had occurred in the
patient.
Mavris et al (Mol. Microbiology 26: 939, 1997) had demonstrated that a 2.6-kb
DNA fragment from a lysogenic phage SfII of S. Jlexneri was capable of converting
isolates of serotype Y to that of serotype 2a. Since the lipopolysaccharides (the 0antigens) of serotypes 2a and Y differed in a glucosyl residue in their repeating units,
Mavris et al. named the genes in the 2.6-kb fragment bgt (bactoprenol glucosyl
transferase) and gtrll (glucosyl transferase 11). They could further identify the
protein product of bgt, but not that of gtrll. We were able to PCR-amplify the 2.6kb fragments from the 5 ShrgeUn isolates. The 2.6-kb fragments from the first
isolate (serotype 2a) and the fifth isolate (serotype Y) were cloned separately into
plasmid pOK12 and transformed into each of the two isolates to see any serotype
changes of the two isolates (complementation test). It was found that the 2.6-kb
fragment from the isolate of serotype 2a could convert the isolate of serotype Y into
that of serotype Za, but not vice versa. Further deletion analysis with the 2.6-kb
fragments from the two isolates indicated that gtrll instead of bgt was responsible
for the serotype conversion.
DNA sequencing of the grrll regions of the two isolates was performed and
the result indicated only one amino acid residue difference in the two predicted
GtrII protein sequences. The first isolate (serotype 2a) has cysteine and the fifth
isolate (serotype Y)has tyrosine at residue 437. It is concluded that it was the Cys
to Tyr mutation at residue 437 of GtrII that lead to loss of GtrII activity in the
isolate of serotype Y. The girl1 gene from the first isolate was cloned into PET
plasmid and overexpressed by the T7 promoter/ /T7 polymerase system using [35S]methionine labelling. We observed a faint expression signal corresponding to a
protein of 40 kDa, while the predicted size for GtrII is 55 kDa. It is assumed that a
bacterial membrane fraction was co-migrated with the GtrII protein, which
somehow changed the mobility of the protein and decreased the intensity of the
signal. At present, we are attempting to prove the assumption.
INHIBITION OF PSEUDOMONAS AERUGINOSA
753 LECTINS BY GLYCOPROTEINS OF AVIAN EGG
WHITES
B. Lerrer, N. Gilboa-Garber
Faculty of Life Sciences, Bar-Ilan University, Ramat Gan, Israel
Pigeoddove (columbidae) egg white, which was reported to
block Escherichia coli P-fimbrial agglutinin, was shown in the
present study to similarly inhibit PA-LL and PA-ILL lectins of
P. aeruginosu. PA-IL, which is a galactophilic lectin specific for
P~PIIB+Iblood group antigens, was selectively inhibited by the
columbidal albumen but not by those of chicken or quail.
PA-IIL, which binds fucosdmannose, also displayed high
sensitivity to the quail albumen while low sensitivity to that of
the chicken. Among other lectins examined for comparison, the
galactophilic galectin-1, peanut and Eryfhrinu coruIIodendron
lectins and the hcose-specific UIex and UIvu lectins were
generally resistant to those three egg whites. The
mannosdglucose-specific Con A and the GlcNAdSA-specific
wheat germ agglutinin (WGA) activities were strongly abrogated
by all of the albumens examined. Con A was found to be most
sensitive to the quail’s followed by the chicken’s, whereas WGA
was most sensitive to the chicken’s albumen. These results show
the selectivity of the inhibition of the two P. uemginosu lectins
by the columbidal albumen - due to the presence of
glycoproteins with terminal galactose of the PA-IL-specificity
and terminal mannose residues which bind to PA-IIL. They
endorse the attribution of protection of the avian embryo from
microbial infections to the egg whites.
This study is part of B. Lerrer’s Ph.D. research and is supported
by the Bar-Ilan Research Fund.
A23 I
INDUCTION OF E-SELECTIN BY VASOACTIVE
PEPTIDES ON KERATINOCYTES I N VITRO
Hagi-Pavli E,Farthing PM & Kapas S
Molecular Signalling Group, Clinical Sciences Research Centre & *Oral
Microbiology Dept, St Bartholomew’s & the Royal London School of
Medicine & Dentistry, 2 Newark Street, London E l 2AD, L’K
754
E-selectin is an adhesion molecule primarily expressed by endothelial
cells in response to cytokines, and has an important role in the recruitment of leukocytes into inflamed tissue. In the oral cavity, E-selectin
expression has been established in different inflammatory disorders such
as gingivitis and periodontitis. Recently, it has been reported that Eselectin expression is not confined to the endothelium but may also
occur on keratinocytes from the gingival epithelium. It is not known
whether E-selectin is inducible on oral keratinocytes or what factors
control this, however, epithelial cells, such as keratinocytes are exposed
to a multitude of stimuli and paracrine factors during the inflammatory
process. Therefore, the aim of this study was to determine whether Eselectin could be induced on oral keratinocytes (H357) by paracrine
factors such as the vasoactive peptides adrenomedullin (AM) and
adrenocorticotrophin hormone (ACTH). AM and ACTH were
incubated with H357 cells for 18 hours and E-selectin expression was
measured by ELISA. Both peptides significantly induced E-selectin
expression on keratinocytes. To determine induction at the gene level
total RNA was extracted from untreated and treated cells and RT- PCR
was performed. PCR products were detected in stimulated cells,
sequenced and found to be identical to endothelial. We also determined
that both AM and ACTH induced cAMP levels in a dose dependent
manner and E-selectin induction was mimicked by the cAMP analogue,
dibutyryl CAMP. Exposure of H357 cells to AM caused activation and
translocation of the cytosolic transcription factor NF-KBto the nucleus.
These results demonstrate that vasoactive peptides can induce E-selectin
expression on oral keratinocytes and that induction is possibly NF-KB
mediated.
INCREASED EXPRESSION OF ADRENOMEDULLIN I N
PATHOGEN-CHALLENGED ORAL EPITHELIAL
CELLS
Kapas S, Bansal A, Bhargava V, Maher R, Mali D, Hagi-Pavli E &
“Allaker RP
Molecular Signalling Group, Clinical Sciences Research Centre &
‘*OralMicrobiology Dept, St Bartholomew’s & the Royal London
School of Medicine & Dentistry, 2 Newark Street, London E l 2AD,
UK
755
Epithelium provides the first line of defense against invading microorganisms in animals and plants. Several antimicrobial peptides have
been isolated and shown to be expressed by mammalian and plant
surface epithelial cells, such as defensins, magainins and cecropins.
These discoveries have led to an emerging concept that these peptides
contribute to the protective barrier of the epithelium by destroying
microbes. Adrenomedullin, a multifunctional peptide, is expressed by
many epithelial cells. Previous studies, by ourselves and others, have
shown that adrenomedullin has antimicrobial activity against members
of human skin, oral, respiratory tract and gut microflora. The oral
cavity contains an epithelium that is constantly colonised by
microflora which includes bacteria, fungi and viruses. The oral
mucosa is often subject to abrasions, yet infections in a host are rare.
We exposed oral keratinocytes to culture supernatants of four
commonly found microorganisms of the oral cavity, Porpbyromonas
gingivalis, Streptococcus mutans, Candida albicans and Eikenella
corrodens for various lengths of time. We observed that the
adrenomedullin gene was upregulated in the presence of the bacteria,
but not with the yeast, Candida albicans, however, all microorganisms
induced expression of human p-defensin 2. We propose there is a
potential role for these proteins in enhancing mucosal defense
mechanisms adrenomedullin, participating in the prevention of local
infection, thus contributing to host defense mechanisms.
0 2000 Biochemical Society