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. 2024 Jul 7;16(1):36.
doi: 10.1186/s13099-024-00626-8.

The C-terminal proline-rich repeats of Enteropathogenic E. coli effector EspF are sufficient for the depletion of tight junction membrane proteins and interactions with early and recycling endosomes

Imran Ansari #  1 , Anupam Mandal #  1 , Kritika Kansal  1 , Pangertoshi Walling  1 , Sumbul Khan  1 , Saima Aijaz  2
Affiliations

The C-terminal proline-rich repeats of Enteropathogenic E. coli effector EspF are sufficient for the depletion of tight junction membrane proteins and interactions with early and recycling endosomes

Imran Ansari et al. Gut Pathog. .

Abstract

Background: Enteropathogenic E. coli (EPEC) causes acute infantile diarrhea accounting for significant morbidity and mortality in developing countries. EPEC uses a type three secretion system to translocate more than twenty effectors into the host intestinal cells. At least four of these effectors, namely EspF, Map, EspG1/G2 and NleA, are reported to disrupt the intestinal tight junction barrier. We have reported earlier that the expression of EspF and Map in MDCK cells causes the depletion of the TJ membrane proteins and compromises the integrity of the intestinal barrier. In the present study, we have examined the role of the proline-rich repeats (PRRs) within the C-terminus of EspF in the depletion of the tight junction membrane proteins and identified key endocytosis markers that interact with EspF via these repeats.

Results: We generated mutant EspF proteins which lacked one or more proline-rich repeats (PRRs) from the N-terminus of EspF and examined the effect of their expression on the cellular localization of tight junction membrane proteins. In lysates derived from cells expressing the mutant EspF proteins, we found that the C-terminal PRRs of EspF are sufficient to cause the depletion of TJ membrane proteins. Pull-down assays revealed that the PRRs mediate interactions with the TJ adaptor proteins ZO-1 and ZO-2 as well as with the proteins involved in endocytosis such as caveolin-1, Rab5A and Rab11.

Conclusions: Our study demonstrates the direct role of the proline-rich repeats of EspF in the depletion of the TJ membrane proteins and a possible involvement of the PRRs in the endocytosis of host proteins. New therapeutic strategies can target these PRR domains to prevent intestinal barrier dysfunction in EPEC infections.

Keywords: Endocytosis; Enteropathogenic E. Coli; EspF; Intestinal barrier; Proline rich repeats; Tight Junctions.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Expression of PRR domains of EspF depletes the total levels of TJ proteins. (A) Schematic representation of mutant EspF constructs. (B) Cell lysates obtained from wild type MDCK cells or stable cells lines expressing GFP vector (AcGFP), GFP-EspF (EspF), GFP-PRR-1-2-3 (containing all three PRRs), GFP-PRR-2-3 (containing PRR-2-3) and GFP-PRR-3 (containing only PRR-3) were analyzed by Western blotting. (C) Band intensities were measured by ImageJ software and fold change in the expression of each protein with respect to MDCK cells (normalized to 1) was plotted relative to GAPDH. Three cell lines were analyzed for each EspF construct and experiments were performed at least three times. A representative blot from one experiment is shown. Bars represent means ± s.e.m from three independent experiments; **p value < 0.005 and ***p value < 0.0005. No change was seen in the levels of the TJ adaptor ZO-1 or actin (not shown in panel C)
Fig. 2
Fig. 2
Expression of the EspF PRR domains affects the cellular localization of claudin-1, claudin-4 and occludin. Stable cell lines expressing GFP vector alone, GFP-EspF or GFP-tagged constructs containing one or more EspF-PRR domains were grown on coverslips and the cells were labeled with antibodies against claudin-1 (A), claudin-4 (B), occludin (C) and ZO-1 (D). The localization pattern of TJ proteins were examined by microscopy. Scale bars: 10 μm. TJ proteins are in red; nucleus is shown in blue
Fig. 3
Fig. 3
The EspF PRR domains mediate the interaction of EspF with the TJ adaptor proteins ZO-1 and ZO-2. (A) Schematic representation of mutant GST-tagged EspF constructs. (B) Pull-down assays using GST, GST-EspF or different GST-tagged constructs carrying one or more PRR domains were performed to show that EspF interacts with the TJ adaptor proteins ZO-1 and ZO-2 through the PRR domains. (C) The efficiency of the pull down products was quantified by calculating the band intensities relative to the level of expression of the corresponding GST-tagged EspF mutant proteins. Comparisons were made with the pull down efficiency seen in full length GST-EspF protein (normalized to 1) and graphs were plotted
Fig. 4
Fig. 4
The EspF PRR domains mediate the interaction of EspF with Caveolin-1, Rab5A and Rab11. (A) Schematic representation of GST-tagged EspF, GST-PRR-1-2-3, GST-PRR-2-3, GST-PRR-3 and GST-PRR1-2 constructs. (B) Pull-down assays using GST-EspF or different GST-tagged constructs carrying one or more PRR domains were performed to show that EspF interacts with Caveolin-1, Rab5a and Rab11 through the PRR domains. (C) Pull down efficiency of the different PRR proteins was compared with that of full length EspF (normalized to 1) and graphs were plotted
Fig. 5
Fig. 5
Schematic diagram showing the effect of EspF-PRR domains on tight junction membrane proteins. EspF may be recruited to the plasma membrane through its interaction with SNX9. Once at the plasma membrane, the PRR domains of EspF likely interact and activate markers of early and recycling endosomes to mediate the displacement of claudin-1, claudin-4 and occludin from the tight junctions into the cytoplasm and their depletion
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