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Identification of AEEC genes activated upon contact with host cell

The aim of this WP is to identify new virulence genes induced during the contact of AEEC strains with the epithelium. A technique known as IVET (in vivo expression technology) will be used to positively select for bacterial genes expressed upon infection of epithelial cells. This system is aimed at identifying bacterial genes that are preferentially expressed in the host during infection and are poorly transcribed under laboratory conditions. IVET will allow us the identification of promoters (and subsequently the genes) that are activated in vivo through the use of a promoter-less reporter gene located downstream of randomly inserted bacterial genomic sequences.

Major finding during the project:
The aim of this project was to use a whole-genome micro-array (instead of IVET) to screen for alteration in EHEC gene expression at a time when bacteria are attached to plasma membranes. The array was composed of 5318 50-mer oligonucleotides. Only genes with a p-value equal to or smaller than 0.05 were analysed further. Genes showing a iY2-fold difference in the expression ratio, between the two conditions, were classified as regulated genes.

Expression profiles. Expression of 401 genes was different between RBC-associated and DMEM-grown EHEC; with 296 lower and 105 higher in the RBC-associated bacteria.

Stress responses. The mRNA levels of rpoE and rseB, were more abundant in RBC-associated EHEC. RpoE controls cell envelope stress response and is transcribed when misfolded or unfolded proteins accumulate in the periplasm or outer membrane or when cells enter stationary growth phase. ResB is a regulator of RpoE and the higher level of expression in attached bacteria is therefore consistent with the elevated mRNA level of rpoE. Elevated levels of mRNA of groES and groEL (expressed in response to a variety of stress conditions in the cytoplasm) were found in RBC-associated EHEC. Moreover, the mRNA levels of both heat shock chaperone proteins DnaK and DnaJ were also elevated in the RBC-bound bacteria, although the p value was not as significant (p = 0.15). The mechanism controlling expression of these chaperones under the experimental conditions tested is not yet defined.

mRNA of genes involved in translation. With the exception of rpsV encoding the 30S ribosomal subunit protein S22, the majority of mRNA encoding ribosomal proteins was lower in the attached bacteria. These results suggest a reduction of de novo protein synthesis in attached bacteria. This is supported by the fact that the level of tig mRNA, encoding the ribosome-bound trigger factor chaperone that maintains newly translated proteins in an open conformation, is also reduced in attached bacteria.

mRNA expression profiles of the LEE genes. Of major interest was the expression profile of mRNA of the LEE genes. In general, expression levels of most of the LEE mRNA were significantly lower in RBC-associated bacteria. In particular, the levels of mRNA encoding the translocator proteins EspA, EspB and EspD were over 20 fold lower and the mRNA encoding intimin was 69 fold lower. In contrast to the almost uniform lower abundancy of LEE mRNA in RBC-associated EHEC, mRNA levels of four genes; espG, rorf3, orf15 and escN encoding the effector EspG, two proteins of unknown function and the TTSS ATPase respectively, were unchanged, although only orf15 was also statistically significant.

Regulators. yhiE and yhiF were recently reported to be negative regulators of LEE4 gene expression and null function mutations in these genes resulted in enhanced binding of EHEC to CaCo2 cells . Moreover, over expression of yhiX (gadX) reduced the production of virulence proteins in EPEC. Consistent with this, the mRNA levels of yhiE, yhiF and yhiX were higher in RBC-associated EHEC than in DMEM-grown EHEC. These transcriptional regulators influence expression of genes implicated in the maintenance of pH homeostasis. Therefore, yhiE (gadE) in EHEC, like gadX in EPEC, may regulate expression of genes required for acid resistance and virulence. SdiA was also reported to have a negative effect on LEE gene expression. Consistent with this, the mRNA level of sdiA was higher in RBC-associated EHEC.

Identification of new virulence determinants EspJ and TccP:
Similarly to down regulation of the LEE-encoded proteins, expression of two other phage encoded genes (termed espJ and tccP) that encode type III effectors were also down in attached bacteria. By adding a carboxy terminal His tag to EspJ and TccP we have shown that both proteins are secreted and translocated by the EHEC TTSS. Mutations in espJ and tccP have shown that while the former is not requited for A/E lesion formation, the latter is essential for actin reorganisation by EHEC. TccP is only the second (after Tir) EHEC type III effector that was implicated in A/E lesion formation.

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Reported by

Department of Biochemistry
SW7 2AY London
United Kingdom
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