Thermoregulated expression of virulence genes in enteropathogenic Escherichia coli

Abstract

Enteropathogenic Escherichia coli (EPEC) causes severe diarrhoea in young children. The locus of enterocyte effacement (LEE) pathogenicity island comprises a cluster of operons encoding a type III secretion system and related proteins that are associated with EPEC virulence. The LEE1 operon encodes Ler that positively regulates the LEE2, LEE3, LEE4, LEE5 and espG transcriptional units. The LEE operons are repressed at 27 SC and expressed at 37 SC. This paper describes a regulatory cascade of the thermoregulation of LEE operons. LEE1 including ler is repressed by H-NS at 27 SC but not at 37 SC. In contrast, the expression of the LEE2, LEE3, LEE4, LEE5 and espG transcriptional units is repressed by H-NS at both 27 SC and 37 SC. Upon shifting the culture temperature from 27 SC to 37 SC, Ler is synthesized and in turn activates the expression of LEE2, LEE3, LEE4 and espG by releasing the H-NS mediated repression. In the case of LEE5, Ler acts both by alleviating the H-NS mediated repression and by an additional mechanism, as yet to be defined.

Ler of pathogenic Escherichia coli forms toroidal protein–DNA complexes

“…The Lerbound complexes were clearly visible upon addition of 1 mM Ler, the concentration of pre-bound H-NS (Fig. 5), consistent with the higher affinity of Ler, or lower K D for DNA than those of H-NS (Umanski et al, 2002). The LEE5 and proU DNA species pre-bound to 1 mM H-NS with 1.0 and 1.5 mM added Ler co-migrated with those complexes formed in the presence of Ler alone (Fig.…”

“…To date, LEE5 is the only operon for which Ler can increase transcriptional activity in the absence of H-NS (Haack et al, 2003;Sánchez-SanMartín et al, 2001;Umanski et al, 2002). We suggest this is in part due to the location of Ler binding, between positions 2221 and 260, and thus it is probable that this regulatory protein interacts with RNA polymerase, which binds to s 70 -dependent promoter regions extending from positions~255 to +20 in relation to the start of transcription (Craig et al, 1995).…”

“…Apparent dissociation constants (K D ) for Ler and H-NS were calculated by a modification of a published method (Carey, 1988;Umanski et al, 2002). The half-maximal binding point of each protein, which should correspond to its K D , was found by plotting the decrease in free DNA against increasing protein concentration.…”

“…Genetic and biochemical data indicate that H-NS binds both upstream and downstream of the LEE5 promoter (Haack et al, 2003;our unpublished results). Ler relieves silencing by binding with higher affinity than H-NS (Umanski et al, 2002) to the same region upstream of LEE5, disrupting H-NS-containing nucleoprotein complexes. The LEE2 operon, encoding membrane components of the T3SS, is controlled by a similar mechanism Sperandio et al, 2000).…”

In Vitro and In Vivo Model Systems for Studying Enteropathogenic Escherichia coli Infections

“…In addition, EPEC is shown to elicit a protective response in C. elegans through induction of genes regulating nematode innate immunity in response to pathogen exposure (Anyanful et al 2009). C. elegans provides a straightforward in vivo system in which to study EPEC attachment and colonization factors (Mellies et al 2006), however, practical limitations to this model do exist as C. elegans cannot survive at the optimal temperature required for expression of the virulence machinery of A/E pathogens (Umanski et al 2002). Furthermore, administration of precise bacterial inocula can be technically difficult (Mylonakis and Aballay 2005).…”