Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS

Genetic regulation of the LEE: the Pathogenicity Island of AEEC strains

The specific objectives of this work package are: 1) investigating molecular aspects of the LEE thermoregulation, 2) determination of the pattern of expression of LEE genes during the infection of organ culture and 3) testing the role of IHF and Ler during infection of organ culture.

Major finding during the project:
Concerted regulation of the different virulence genes is essential for successful colonization of the host by pathogens. The genes encoding the TTSS and some of the effector proteins are located within a unique pathogenicity island, the locus of enterocyte effacement (LEE). Work in several groups as well as work in our laboratory indicate that the 41 LEE genes are organized in 11 transcriptional units including: LEE1, LEE2, LEE3, LEE4, LEE5, LEE6 (rorf1,rorf2), LEE7 (orf10, orf11), rorf3, map, cesF, escD, and map. Moreover, we found that some of these transcriptional units contain internal promoters as well. Our long-term goal is to decipher the complex regulatory network coordinating the expression of the genes encoding the TTSS and the effector proteins.

We discovered that the first gene in the LEE1 operon encode a regulator termed Ler. Ler positively regulates the transcription of all the LEE transcriptional units except LEE1 (Friedberg et. al., 1999 and unpublished data). Therefore, the decision of whether to activate the LEE1 promoter is critical to the initiation of a regulatory cascade leading to the expression of other LEE operons. Entry of the pathogen into the host is associated with a shift in temperature from below 30 degrees Celsius to 37 degrees Celsius. We showed that EPEC exploits this temperature shift as a signal for activating the expression of its virulence genes.

We further investigated the molecular mechanism of this thermoregulation. We found that the thermostat, which senses differences in temperature, is the LEE1 promoter (Umanski et. al., 2002). This property of the LEE1 promoter is dependent on H-NS, a nucleoid-associated protein. We also discovered that H-NS represses the expression of the LEE1 operons at 27 degrees Celsius, but not at 37 degrees Celsius, leading to thermo-regulated expression of all the Ler-regulated operons. In addition, the expression of LEE2, LEE3, LEE4 and LEE5 is repressed by H-NS at both 27 degrees C and 37 degrees Celsius. Upon shifting the culture temperature from 27 degrees Celsius to 37 degrees Celsius, Ler is synthesized, it in turn activating the expression of LEE2, LEE3, LEE4 and LEE5 by releasing the H-NS-mediated repression (Umanski et al., 2002).

While H-NS represses the LEE1 operon, Integration Host Factor (IHF), another nucleoid-associated protein, is a positive regulator of the LEE1 promoter. We found that LEE1 expression is strictly dependent on binding IHF upstream to the LEE1 promoter (Friedberg et al., 1999, Yona-Nadler et al., 2003). Interestingly, IHF is also required for repression of the flhDC operon in EPEC. FlhDC are positive regulators of the flagellum encoding genes (numbering about 40). Thus, IHF represses flagellum synthesis (Yona-Nadler et al., 2003). Moreover, we showed that a unique EPEC factor is required for IHF-mediated flagellum repression (Yona-Nadler et al., 2003).

We also investigated the mechanism that determines the Ler steady state concentration. We found that Ler acts as a specific auto-repressor of LEE1 transcription (Berdichevski et al., submitted). We further show that Ler specifically binds upstream of the LEE1 operon in vivo and in vitro. Comparison of the Ler affinities to different DNA regions suggest that the auto-regulation mechanism limits the steady state level of Ler to concentrations that are just sufficient for activation of the LEE2 and LEE3 promoters and probably other LEE promoters. This mechanism may reflect the need of EPEC to balance between maximizing colonization efficiency by increasing the expression of the virulence genes and minimizing the immune response of the host by limiting their expression. In addition, we found that the auto-regulation mechanism reduces the cell-to-cell variability in the levels of LEE1 expression (Berdichevski et al., submitted). These findings point to a new negative regulatory circuit that suppresses the noise, and optimises the expression levels of Ler and other LEE1 genes.

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POB 12272
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