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. 2020 Dec 16:10:571088.
doi: 10.3389/fcimb.2020.571088. eCollection 2020.

Genomic Properties and Temporal Analysis of the Interaction of an Invasive Escherichia albertii With Epithelial Cells

Fabiano T Romão  1   2   3 , Fernando H Martins  2   3   4 , Rodrigo T Hernandes  5 , Tadasuke Ooka  6 , Fernanda F Santos  1 , Denise Yamamoto  1   7 , Alexis Bonfim-Melo  1 , Nina Jones  8 , Tetsuya Hayashi  9 , Waldir P Elias  4 , Vanessa Sperandio  2   3 , Tânia A T Gomes  1
Affiliations

Genomic Properties and Temporal Analysis of the Interaction of an Invasive Escherichia albertii With Epithelial Cells

Fabiano T Romão et al. Front Cell Infect Microbiol. .

Abstract

Diarrhea is one of the main causes of infant mortality worldwide, mainly in the developing world. Among the various etiologic agents, Escherichia albertii is emerging as an important human enteropathogen. E. albertii promote attaching and effacing (AE) lesions due to the presence of the locus of enterocyte effacement (LEE) that encodes a type three secretion system (T3SS), the afimbrial adhesin intimin and its translocated receptor, Tir, and several effector proteins. We previously showed that E. albertii strain 1551-2 invades several epithelial cell lineages by a process that is dependent on the intimin-Tir interaction. To understand the contribution of T3SS-dependent effectors present in E. albertii 1551-2 during the invasion process, we performed a genetic analysis of the LEE and non-LEE genes and evaluated the expression of the LEE operons in various stages of bacterial interaction with differentiated intestinal Caco-2 cells. The kinetics of the ability of the 1551-2 strain to colonize and form AE lesions was also investigated in epithelial HeLa cells. We showed that the LEE expression was constant during the early stages of infection but increased at least 4-fold during bacterial persistence in the intracellular compartment. An in silico analysis indicated the presence of a new tccP/espFU subtype, named tccP3. We found that the encoded protein colocalizes with Tir and polymerized F-actin during the infection process in vitro. Moreover, assays performed with Nck null cells demonstrated that the 1551-2 strain can trigger F-actin polymerization in an Nck-independent pathway, despite the fact that TccP3 is not required for this phenotype. Our study highlights the importance of the T3SS during the invasion process and for the maintenance of E. albertii 1551-2 inside the cells. In addition, this work may help to elucidate the versatility of the T3SS for AE pathogens, which are usually considered extracellular and rarely reach the intracellular environment.

Keywords: Escherichia albertii; Tir cytoskeleton-coupling protein/EspFu; attaching and effacing lesion; diarrhea; invasion; locus of enterocyte effacement; pathogenicity; type three secretion system.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Amino acid sequence comparison of Tir (A) and TccP/EspFU (B) proteins of E. albertii 1551-2 with known protein sequences. Strain names are shown in parentheses. In panel (A), the intimin-binding domain (IBD) and two predicted transmembrane domains (TMD) of the Tir protein are indicated by dashed and solid lines, respectively. Black triangles indicate the tyrosine (Y) residues that are phosphorylated by host cell kinase(s). The underlines identified with *1, *2, *3, and *4 indicate the regions containing a sequence equivalent to EPEC Tir S434, O157 EHEC Tir Y458/EPEC Tir Y454, EPEC Tir Y474, and O157 EHEC Tir519-524, respectively. In panel (B), the N-terminal region (56 amino acids) and central region of TccP/EspFU family proteins are indicated by gray lines. Proline-rich repeats (PRRs) and a partial repeat are indicated by arrows and a dashed arrow, respectively. See Table S1 for sequence identities of the whole proteins, N-terminal regions, central regions, and PRR1s between the proteins shown in this figure.
Figure 2
Figure 2
Kinetics of pedestal formation and number of pedestals produced by E. albertii strain 1551-2 during the initial interaction with HeLa cells. (A) Actin pedestals were detected after the early point (2 h) and increased in the later time-points. In green, host cell F-actin was labeled with phalloidin-FITC, and, in red, bacteria harbored the mCherry plasmid. In the Control panel, F-actin pedestals and bacteria were not detected. Arrows indicate some F-actin pedestals. (B) Pedestals were enumerated in ten distinct fields from the same slide and expressed as the mean number of pedestals per cell. The results at 3 and 6 h were compared with 2 h. **p < 0.01 and ***p < 0.001. Original magnification: 63 x.
Figure 3
Figure 3
Evaluation of TccP3 production and colocalization with F-actin accumulation underneath adherent E. albertii 1551-2 strain. (A) Detection of TccP3 production by the 1551-2 strain by ELISA. The recombinant protein TccP-His and atypical EPEC BA589 (TccP2+) were used as positive controls, while the atypical EPEC strain BA1768 (TccP-/TccP2-) was used as a negative control. ***p < 0.001. (B) HeLa cells were incubated with E. albertii 1551-2 for 6 h, fixed and stained with anti-TccP sera, phalloidin-FITC, and DAPI, then evaluated by confocal microscopy. Actin filaments (green) clearly accumulate underneath adherent bacteria and colocalize (arrowheads) with TccP3 (red). Confocal microscopy image of a single focal plane; DAPI (blue), scale bar = 5 μm.
Figure 4
Figure 4
Immunoblotting and Immunofluorescence to demonstrate TccP3-Myc production and colocalization with polymerized F-actin underneath adhered bacteria in infected HeLa cells. Immunoblotting with α-Myc antibodies (A) with the following strains: E. albertii 1551-2 (lane 1), 1551-2 (pTccP3) (lane 2), EHEC 86-24 (lane 3), and EHEC (pKC471) (lane 4). Immunofluorescence with α-Myc (B) and α-Tir (C) antibodies demonstrating the colocalization (arrowheads) of TccP3-Myc and Tir proteins with polymerized F-actin in infected epithelial cells. Scale bar = 10 μm.
Figure 5
Figure 5
Evaluation of the efficiency in F-actin polymerization in HeLa cells due to TccP3 production in the EHECΔtccP background. The efficiency of the TccP3 produced by E. albertii 1551-2 strain to promote F-actin aggregation was evaluated by quantitative assays to determine the percentage (%) of cell-associated bacteria with intense F-actin staining (A) and number of sites of F-actin staining in epithelial cells (B) infected with the strains: O157:H7 EHEC 86-24, EHEC 86-24ΔtccP, EHEC 86-24ΔtccP (pKC471), and EHEC 86-24ΔtccP (pTccP3). Representative images used in quantitative fluorescence actin staining (FAS) assays are shown in panel (C) and arrowheads indicate intense F-actin staining. These data demonstrate that TccP3 does not restore the efficiency of the EHEC 86-24 strain to promote F-actin accumulation underneath adherent bacteria. **p < 0.01 and ***p < 0.001, and ns: not significant. Scale bar = 10 μm.
Figure 6
Figure 6
Quantitative bacterial adherence and invasion indexes of E. albertii 1551-2 in differentiated Caco-2 cells at 1.5, 3, and 6 h. (A) E. albertii 1551-2 can associate with Caco-2 cells efficiently within 3 h after infection. The number of associated bacteria did not change between 3 and 6 h, suggesting that, at 3 h, bacteria have occupied most of the host sites available in vitro under this condition. **p < 0.01 and ***p < 0.001. (B) No intracellular bacteria were recovered after 1.5 h of infection. A low number of bacteria was detected at 3 h (0.2% of invasion). *p < 0.05. The results at 3 and 6 h were compared with those obtained in 1.5 h.
Figure 7
Figure 7
Relative gene expression of the LEE region operons during the interaction of E. albertii 1551-2 strain with differentiated intestinal Caco-2 cells at the time-points 1.5, 3, and 6 h. No differences were detected in the expression of all genes tested among the three time-points. p > 0.05.
Figure 8
Figure 8
Relative gene expression of the LEE operons by internalized E. albertii 1551-2 strain in Caco-2 cells in the period of 24 h, as compared with internalized bacteria at 6 h. The ler (LEE1), escJ (LEE2), escV (LEE3), escN (LEE3), eae (LEE5), espB (LEE4), and espD (LEE4) genes were overexpressed by intracellular bacteria at least 4-fold at 24 h in comparison with 6 h. No alteration of espA (LEE4) expression was detected. **p < 0.01 and ***p < 0.001.
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