Regulation of the Edwardsiella ictaluri Type III Secretion System by pH and Phosphate Concentration through EsrA, EsrB, and EsrC

Abstract

A recently described Edwardsiella ictaluri type III secretion system (T3SS) with functional similarity to the Salmonella pathogenicity island 2 T3SS is required for replication in channel catfish head-kidney-derived macrophages (HKDM) and virulence in channel catfish. Quantitative PCR and Western blotting identified low pH and phosphate limitation as conducive to expression of the E. ictaluri T3SS, growth conditions that mimic the phagosomal environment. Mutagenesis studies demonstrated that expression is under the control of the EsrAB two-component regulatory system. EsrB also induces upregulation of the AraC-type regulatory protein EsrC, which enhances expression of the EscB/EseG chaperone/effector operon in concert with EsrB and induces expression of the pEI1-encoded effector, EseH. EsrC also induces expression of a putative type VI secretion system translocon protein, EvpC, which is secreted under the same low-pH conditions as the T3SS translocon proteins. The pEI2-encoded effector, EseI, was upregulated under low-pH and low-phosphate conditions but not in an EsrB-or EsrC-dependent manner. Mutations of EsrA and EsrB both resulted in loss of the ability to replicate in HKDM and full attenuation in the channel catfish host. Mutation of EsrC did not affect intracellular replication but did result in attenuation in catfish. Although EsrB is the primary transcriptional regulator for E. ictaluri genes within the T3SS pathogenicity island, EsrC regulates expression of the plasmidcarried effector eseH and appears to mediate coordinated expression of the T6SS with the T3SS.Edwardsiella ictaluri, the etiological agent of enteric septicemia of catfish (ESC), was isolated, named, and characterized by Hawke (22) and Hawke et al. (23). The disease is found throughout channel catfish (Ictalurus punctatus) growing regions and is the leading cause of disease-related mortality in channel catfish production facilities (2, 3). Recent reports have expanded the host and geographic ranges to the culture of striped catfish (Pangasianodon hypophthalmus) in Vietnam (14, 51) and yellow catfish (Pelteobagrus fulvidraco) in China (29).Using signature-tagged mutagenesis, Thune et al. (52) identified mutations in 50 E. ictaluri genes that led to an attenuated phenotype in channel catfish, including a type III secretion system (T3SS) apparatus gene. Further sequencing and genome analysis led to the identification of an E. ictaluri pathogenicity island (PAI) encoding a complete T3SS, including Edwardsiella secretion apparatus (esa), chaperone (esc), effector (ese), and regulatory (esr) genes. Mutation of the apparatus gene resulted in an inability to replicate in catfish cells and loss of virulence, indicating that the T3SS is essential to the pathogenesis of E. ictaluri. These systems are widespread in Gram-negative bacteria and function to translocate effector molecules directly from the bacterial cytosol to the host cell cytoplasm.The Edwardsiella tarda T3SS, which is involved in replication within fish macrophages (38,49,50,57), is hom...

Transcriptomic dissection of the horizontally acquired response regulator EsrB reveals its global regulatory roles in the physiological adaptation and activation of T3SS and the cognate effector repertoire inEdwardsiella piscicidaduring infection toward turbot

“…8), as documented in E. ictaluri and E. tarda. 35,36,37 Our data support the idea that the regulatory circuits of SsrB/EsrB family proteins have been well conserved during evolution (Fig. 4G).…”

“…31 Like their counterparts SsrA, SsrB and SPI-2 in Salmonella, 32,33,34 EsrA and EsrB and the associated T3SS locus may be horizontally acquired and may have evolved to coordinate the expression of virulence and adaptation to host microenvironments by the reprogramming of ancestral genes unlinked to the T3SS locus. 6 Although the exact stimulus recognized by HK EsrA remains elusive, 35 EsrA-EsrB plays a key role in regulating the expression and secretion of T3SS and T6SS by directly binding to the promoters of T3SS genes and to that of EsrC, which further regulates the expression of other T3SS and T6SS genes in the bacterium. 22,36,37 It is still unknown whether other genes, especially those associated with the invasion process, and their regulatory mechanisms are controlled by EsrB during in vivo and in vitro infection of hosts.…”

Roles of plasmid-encoded proteins, EseH, EseI and EscD in invasion, replication and virulence of Edwardsiella ictaluri

“…EseI was also secreted, as detected in cytosol fraction, but its secretion was not dependent upon the T3SS, and this protein was not affected by the regulator proteins EsrBC in the E. ictaluri T3SS, as reported by Rogge and Thune (2011), which is of interest for further research. The secretion of EseH and EseI may then be deployed by E. ictaluri to interfere with host cells.…”

“…The secretion of EseH and EseI may then be deployed by E. ictaluri to interfere with host cells. Using quantitative and Western blotting analyses, Rogge and Thune (2011) found that low pH and phosphate limitation was conducive to an active T3SS in E. ictaluri, whereas they failed to detect Flag-tagged EseH. The 70-99% similarity in 33 ORFs of T3SS between E. tarda and E. ictaluri may enable the functional identification of T3SS molecules in the E. tarda surrogate.…”

“…The mutants with deletion of eseH, eseI and escD, were then built up respectively, and their roles in invasion and replication were examined in host fish cells, as well as in yellow catfish. On the other hand, as T3SS in E. ictaluri is similar to that in E. tarda in gene organization (Thune et al, 2007;Rogge and Thune, 2011;Williams et al, 2012), the T3SS in E. tarda was used to analyze the secretion and translocation of EseH, EseI and EscD in fish cells. The cellular localization of these proteins was further examined in host fish cells.…”