The Secreted Effector Protein EspZ Is Essential for Virulence of Rabbit Enteropathogenic Escherichia coli

Abstract

e Attaching and effacing (A/E) pathogens adhere intimately to intestinal enterocytes and efface brush border microvilli. A key virulence strategy of A/E pathogens is the type III secretion system (T3SS)-mediated delivery of effector proteins into host cells. The secreted protein EspZ is postulated to promote enterocyte survival by regulating the T3SS and/or by modulating epithelial signaling pathways. To explore the role of EspZ in A/E pathogen virulence, we generated an isogenic espZ deletion strain (⌬espZ) and corresponding cis-complemented derivatives of rabbit enteropathogenic Escherichia coli and compared their abilities to regulate the T3SS and influence host cell survival in vitro. For virulence studies, rabbits infected with these strains were monitored for bacterial colonization, clinical signs, and intestinal tissue alterations. Consistent with data from previous reports, espZtransfected epithelial cells were refractory to infection-dependent effector translocation. Also, the ⌬espZ strain induced greater host cell death than did the parent and complemented strains. In rabbit infections, fecal ⌬espZ strain levels were 10-fold lower than those of the parent strain at 1 day postinfection, while the complemented strain was recovered at intermediate levels. In contrast to the parent and complemented mutants, ⌬espZ mutant fecal carriage progressively decreased on subsequent days. ⌬espZ mutant-infected animals gained weight steadily over the infection period, failed to show characteristic disease symptoms, and displayed minimal infection-induced histological alterations. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining of intestinal sections revealed increased epithelial cell apoptosis on day 1 after infection with the ⌬espZ strain compared to animals infected with the parent or complemented strains. Thus, EspZ-dependent host cell cytoprotection likely prevents epithelial cell death and sloughing and thereby promotes bacterial colonization.

Type III secreted effectors that target mitochondria

“…The LEE encoded E . coli ‐secreted protein Z ( EspZ ) is an essential virulence effector (Wilbur et al, 2015). It is an integral membrane protein containing two predicted transmembrane segments.…”

“…Another function is the enforcement of a robust anti‐apoptotic effect on the host (Serapio‐Palacios & Finlay, 2020). Infection with an EPEC‐∆ espZ strain results in rapid host cell death and ∆Ψm disruption, compared to EPEC‐ wt infected cells, while ectopic expression of EspZ antagonised these effects (Roxas et al, 2012; Shames et al, 2010; Shames et al, 2011; Wilbur et al, 2015). The translocated effector inhibited the increase in Cytochrome c release from mitochondria, the activation of caspase‐3 and ‐7 and the loss of ∆Ψm (Roxas et al, 2012; Shames et al, 2011), suggesting that it reaches host mitochondria where it antagonises the pro‐apoptotic activities of other effectors, such as EspF and Map (Ramachandran et al, 2020; Serapio‐Palacios & Finlay, 2020).…”

RNA-seq profiles of chicken type II pneumocyte in response to Escherichia coli infection

“…Recently, the EPEC T3SS effector protein has been shown to regulate apoptosis and promote host cell survival, such as T3SS effector proteins such as EspZ, NleH1 and NleH2[42, 43]. Some studies show that EPEC can activate at least three separate anti-apoptotic pathways, such as tryptophan kinase pathway, protein kinase C pathway and NF-κB transcription factor, which may induce IL-8 expression and activate anti-apoptotic pathway.…”

On the offense and defense: mitochondrial recovery programs amidst targeted pathogenic assault

“…EspF, on the other hand, is reported to stimulate cell death by triggering the release of cytochrome c from mitochondria [128,129]. EPEC produces another mitochondrial-associated virulence factor, EspZ, but in contrast to EspF, EspZ delays apoptosis during infection [130,131]. This effector protein delays apoptosis in part through its ability to associate with the inner mitochondrial membrane where it prevents fluctuations in mitochondrial membrane potential [130].…”