Identification of the cis -Acting Site Involved in Activation of Promoters Regulated by Activity of the Type III Secretion Apparatus in Shigella flexneri

Abstract

Bacteria of Shigella spp. use a virulence plasmid-encoded type III secretion (TTS) system to invade the colonic epithelium in humans. The activity of the TTS apparatus is tightly regulated in the wild-type strain and is induced upon contact of bacteria with epithelial cells, whereas it is deregulated, i.e., constitutively active, in some mutants. Under conditions of deregulated secretion, approximately 20 proteins are secreted, including VirA, OspB to OspG, and at least three members of the IpaH family, all of which are encoded by the virulence plasmid. Conditions inducing or deregulating the activity of secretion also induce the transcription of virA and four ipaH genes. The transcription of virA and ipaH9.8 requires both MxiE, a transcriptional activator of the AraC family, and IpgC, the chaperone of IpaB and IpaC, acting as a coactivator. Using reporter plasmids containing lacZ transcriptional fusions, we showed that the ipaH7.8, ipa4.5, ospC1, and ospF promoters are activated under conditions of deregulated secretion and that both MxiE and IpgC are necessary and sufficient for their activation in both Shigella flexneri and Escherichia coli. Promoter mapping and deletion analysis of the ipaH9.8, virA, and ospC1 promoters identified a 17-bp motif, the MxiE box, which overlaps the ؊35 region and is essential for the activation of these promoters. The presence of eight MxiE boxes on the virulence plasmid suggests that 11 genes encoding secreted proteins may be regulated by the activity of secretion. We also present evidence that at least one ipaH gene that is carried by the chromosome is controlled by MxiE and IpgC.

Analysis of virulence plasmid gene expression defines three classes of effectors in the type III secretion system of Shigella flexneri

“…The increased expression of ospF, ipaH4, ipaH7, ospC1, virA and ipaH9 detected in the present analysis is consistent with previous results obtained using lacZ transcriptional fusions (Demers et al, 1998;Mavris et al, 2002b) and with the presence, in the 59 region of these genes, of a putative MxiE box exhibiting at least 14 matches out of 16 positions with the MxiE box consensus sequence (Fig. 3).…”

“…Control of promoter activity by MxiE correlates with the presence of nine MxiE boxes on the VP. MxiE boxes are also present in the 59 region of chromosomal ipaH genes, consistent with the observation that these genes are also controlled by MxiE and IpgC (Mavris et al, 2002b).…”

“…There are five chromosomal ipaH genes in the S. flexneri strain M90T of serotype 5 and we showed previously that at least some of these genes are controlled by MxiE and IpgC (Mavris et al, 2002b). The recent elucidation of the genomic sequence of two S. flexneri strains of serotype 2a, CP301 and 2457T, revealed the presence of seven chromosomal ipaH genes in these strains, including four complete copies and three copies inactivated by insertion sequences or frameshifting mutations (Jin et al, 2002;Wei et al, 2003).…”

“…The cis-acting element required for increased transcription of ospC1, virA and ipaH9 promoters was shown to be a 17 bp motif (59-GTATCGTTTTTTTANAG), designated the MxiE box, which is located between positions 249 and 233 with respect to the transcription start site (Mavris et al, 2002b). The increased expression of ospF, ipaH4, ipaH7, ospC1, virA and ipaH9 detected in the present analysis is consistent with previous results obtained using lacZ transcriptional fusions (Demers et al, 1998;Mavris et al, 2002b) and with the presence, in the 59 region of these genes, of a putative MxiE box exhibiting at least 14 matches out of 16 positions with the MxiE box consensus sequence (Fig.…”

“…In each case, nucleotides identical to those present in the MxiE box consensus sequence are shown in upper-case bold characters and nucleotides identical to those in the "10 box consensus sequence are shown in upper-case. The nucleotides corresponding to the transcription start sites determined for ospC1, virA and ipaH9.8 (Mavris et al, 2002b) are underlined. The numbers of nucleotides present between the determined or putative transcription start and the translational start are indicated.…”