Invasiveness as a putative additional virulence mechanism of some atypical Enteropathogenic Escherichia colistrains with different uncommon intimin types
© Yamamoto et al; licensee BioMed Central Ltd. 2009
Received: 18 December 2008
Accepted: 21 July 2009
Published: 21 July 2009
The Erratum to this article has been published in BMC Microbiology 2009 9:235
Enteropathogenic Escherichia coli (EPEC) produce attaching/effacing (A/E) lesions on eukaryotic cells mediated by the outer membrane adhesin intimin. EPEC are sub-grouped into typical (tEPEC) and atypical (aEPEC). We have recently demonstrated that aEPEC strain 1551-2 (serotype O non-typable, non-motile) invades HeLa cells by a process dependent on the expression of intimin sub-type omicron. In this study, we evaluated whether aEPEC strains expressing other intimin sub-types are also invasive using the quantitative gentamicin protection assay. We also evaluated whether aEPEC invade differentiated intestinal T84 cells.
Five of six strains invaded HeLa and T84 cells in a range of 13.3%–20.9% and 5.8%–17.8%, respectively, of the total cell-associated bacteria. The strains studied were significantly more invasive than prototype tEPEC strain E2348/69 (1.4% and 0.5% in HeLa and T84 cells, respectively). Invasiveness was confirmed by transmission electron microscopy. We also showed that invasion of HeLa cells by aEPEC 1551-2 depended on actin filaments, but not on microtubules. In addition, disruption of tight junctions enhanced its invasion efficiency in T84 cells, suggesting preferential invasion via a non-differentiated surface.
Some aEPEC strains may invade intestinal cells in vitro with varying efficiencies and independently of the intimin sub-type.
Enteropathogenic Escherichia coli (EPEC) are important human intestinal pathogens. This pathotype is sub-grouped into typical (tEPEC) and atypical (aEPEC) EPEC [1–3]. These sub-groups differ according to the presence of the EAF plasmid, which is found only in the former group [1, 3]. Recent epidemiological studies have shown an increasing prevalence of aEPEC in both developed and developing countries [4–9].
The main characteristic of EPEC's pathogenicity is the development of a histopathologic phenotype in infected eukaryotic cells known as attaching/effacing (A/E) lesion. This lesion is also formed by enterohemorrhagic E. coli (EHEC), another diarrheagenic E. coli pathotype whose main pathogenic mechanism is the production of Shiga toxin . The A/E lesion comprises microvillus destruction and intimate bacterial adherence to enterocyte membranes, supported by a pedestal rich in actin and other cytoskeleton components . The ability to produce pedestals can be identified in vitro by the fluorescence actin staining (FAS) assay that detects actin accumulation underneath adherent bacteria indicative of pedestal generation . The genes involved in the establishment of A/E lesions are located in a chromosomal pathogenicity island named the locus of enterocyte effacement (LEE) . These genes encode a group of proteins involved in the formation of a type III secretion system (T3SS), an outer membrane adhesin called intimin , its translocated receptor (translocated intimin receptor, Tir), chaperones and several other effector proteins that are injected into the targeted eukaryotic cell by the T3SS [15, 16].
Differentiation of intimin alleles represents an important tool for EPEC and EHEC typing in routine diagnosis as well as in pathogenesis, epidemiological, clonal and immunological studies. The intimin C-terminal end is responsible for receptor binding, and it has been suggested that different intimins may be responsible for different host tissue cell tropism (reviewed in ). The 5' regions of eae genes are conserved, whereas the 3' regions are heterogeneous. Thus far 27 eae variants encoding 27 different intimin types and sub-types have been established: α1, α2, β1, β2 (ξR/β2B), β3, γ1, γ2, δ (δ/β2O), ε1, ε2 (νR/ε2), ε3, ε4, ε5 (ξB), ζ, η1, η2, θ, ι1, ι2 (μR/ι2), κ, λ, μB, νB, ο, π, ρ and σ [[18–26] and unpublished data].
In HeLa and HEp-2 cells, tEPEC expresses localized adherence (LA) (with compact bacterial microcolony formation) that is mediated by the Bundle Forming Pilus (BFP), which is encoded on the EAF plasmid. In contrast, most aEPEC express the LA-like pattern, which is often detected in prolonged incubation periods (with loose microcolonies) [, reviewed in ]. However, during the characterization of an aEPEC collection, Vieira et al.  detected 9 strains that formed characteristic LA on HeLa cells despite the absence of BFP. Further studies showed that these strains also lacked the adhesin-encoding genes of other diarrheagenic E. coli pathotypes . Therefore, an exemplary strain (aEPEC 1551-2) was studied in further detail. Subsequently, it was shown that in this strain the LA pattern actually corresponded to an invasion process mediated by the interaction of the intimin sub-type omicron . The clinical significance of these findings in the pathogenicity of aEPEC in vivo is currently unknown.
Despite the fact that EPEC is generally considered an extracellular pathogen, some studies have shown limited invasion of intestinal epithelium of humans and animals by tEPEC in vivo [30, 31]. Moreover, it has been demonstrated that some tEPEC and aEPEC strains are able to invade distinct cellular lineages in vitro [32–36]. Due to variations in the protocols used to determine the invasion indexes, it is difficult to compare the extent of the reported invasion ability among strains of tEPEC and aEPEC pathotypes. Furthermore, in the literature there are only a few studies on the ability of aEPEC strains to invade intestinal cells [34, 35]. Most tEPEC and aEPEC invasion studies have been performed on HEp-2 [32, 36, 37], and polarized intestinal Caco-2 cells [33, 35]. Invasion studies with aEPEC and intestinal T84 cells, which are phenotypically similar to human colon epithelial cells are still lacking. Since aEPEC is a heterogeneous pathotype [3, 5, 28], additional analysis of the invasive ability of aEPEC strains in vitro are necessary. These data could contribute to evaluate whether the invasion capacity might be considered as an additional virulence mechanism in other aEPEC strains. Therefore, in this study, we evaluated aEPEC strains expressing intimin sub-types omicron and non-omicron regarding their ability to invade HeLa and differentiated intestinal T84 cells. The eukaryotic cell structures involved in the initial steps of entry of aEPEC 1551-2 were also examined.
Results and Discussion
Recent studies have shown that aEPEC consist of a heterogeneous group of strains, some of which could represent tEPEC strains that lost the EAF plasmid (or part of it), EHEC/STEC strains that lost stx phage sequences, or even E. coli from the normal flora that had gained the LEE region [2, 27, 38–40]. It remains to be elucidated whether these strains bear additional and/or specific virulence properties that are not present in tEPEC.
Recently, it has been shown that aEPEC strain 1551-2 invades HeLa cells in a process dependent on intimin omicron . The aEPEC 1551-2 invasive index was about 3 folds that of tEPEC prototype strain E2348/69 tested in the same conditions. However, it is not known whether other aEPEC strains expressing intimin omicron or other intimin sub-types are also invasive. In the present study this issue was investigated.
Characteristics of the aEPEC strains studied.
It has been reported that the interaction between Afa/Dr adhesins, expressed by strains of the diarrheagenic E. coli pathotype diffusely adherent E. coli (DAEC), and α5β1 integrins also results in bacterial internalization . Adaptation to the intracellular environment help bacteria to avoid physical stresses (such as low pH or flow of mucosal secretions or blood) and many other host defense mechanisms including cellular exfoliation, complement deposition, antibody opsonization and subsequent recognition by macrophages or cytotoxic T cells . Thus, the development of mechanisms for host cell invasion, host immune response escape, intracellular replication and/or dissemination to the neighboring cells is an important strategy for intracellular bacteria .
In conclusion, we showed that aEPEC strains expressing distinct intimin sub-types are able to invade both HeLa and differentiated T84 cells. At least for the invasive aEPEC 1551-2 strain, HeLa cell invasion requires actin filaments but does not involve microtubules. In differentiated T84 cells, disruption of tight junctions increases the invasion capacity of aEPEC 1551-2. This observation could be significant in infantile diarrhea since in newborns and children the gastrointestinal epithelial barrier might not be fully developed . As observed in uropathogenic E. coli , besides representing a mechanism of escape from the host immune response, invasion could also be a strategy for the establishment of persistent disease. It is possible, that the previously reported association of aEPEC with prolonged diarrhea  is the result of limited invasion processes. However, the in vivo relevance of our in vitro observations remains to be established. Moreover, further analyses of the fate of the intracellular bacteria such as persistence, multiplication and spreading to neighboring cells are necessary.
In this study we verified that aEPEC strains, carrying distinct intimin sub-types, including three new ones, may invade eukaryotic cells in vitro. HeLa cells seem to be more susceptible to aEPEC invasion than differentiated and polarized T84 cells, probably due to the absence of tight junctions in the former cell type. We also showed that actin microfilaments are required for efficient invasion of aEPEC strain 1551-2 thus suggesting that A/E lesion formation is an initial step for the invasion process of HeLa cells, while microtubules are not involved in such phenomenon. Our results also showed that tight junctions' disruption increased significantly the invasion of T84 cells by aEPEC strain 1551-2. Altogether, our findings suggest that aEPEC strains may invade intestinal cells in vitro with varying efficiencies and that the invasion process proceeds apparently independently of the intimin sub-type.
Bacterial strains and cell culture conditions
Six aEPEC strains (two carrying intimin subtype omicron and four carrying unknown intimin sub-types randomically chosen from our collection) isolated from children with diarrhea and potentially enteropathogenic due to a positive FAS assay (Table 1), and the prototype tEPEC strain E2348/69 were studied. Strains were cultured statically in Luria Bertani broth for 18 h at 37°C. Under this condition cultures reached an OD600 of 0.5–0.6. Salmonella enterica serovar Typhimurium (a gift from J.R.C. Andrade, Universidade do Estado do Rio de Janeiro) and Shigella flexneri M90T  were used as controls in some experiments in infection assays of 4 and 6 h, respectively. All strains were shown to be susceptible to 100 μg/mL of gentamicin prior to the invasion experiments. HeLa cells (105 cells) were cultured in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% bovine fetal serum (Gibco Invitrogen) and 1% antibiotics (Gibco Invitrogen), and kept for 48 h at 37°C and 5% CO2. T84 cells (105 cells) were cultured in DMEM-F12 medium (Gibco Invitrogen) supplemented with 10% bovine fetal serum (Gibco Invitrogen), 1% non-essential amino acids (Gibco Invitrogen) and 1% antibiotics (Gibco Invitrogen), and kept for 14 days at 37°C and 5% CO2 for differentiation. For some transmission electron microscopy analysis, T84 cells (105 cells) were cultivated on the lower surface of Corning Transwell polycarbonate membrane inserts pore size 3.0 μm, membrane diameter 12 mm. In addition to apical adhesion this procedure allowed bacterial inoculation directly at the basolateral surface of the cells avoiding the use of chemical treatment to expose such surface.
The determination of O and H antigens was carried out by the method described by Guinée et al.  employing all available O (O1-O185) and H (H1-H56) antisera. All antisera were obtained and absorbed with the corresponding cross-reacting antigens to remove the nonspecific agglutinins. The O antisera were produced in the Laboratorio de Referencia de E. coli (LREC) (Lugo, Spain) and the H antisera were obtained from the Statens Serum Institut (Copenhagen, Denmark).
Typing of intimin (eae) genes
Intimin typing was performed by sequencing a fragment of the 1,125 bp from 3' variable region of the eae genes from four aEPEC strains included in this study. The complete nucleotide sequences of the new θ2 (FM872418), τ (FM872416) and ν (FM872417) variant genes were determined. The nucleotide sequence of the amplification products purified with a QIAquick DNA purification kit (Qiagen) was determined by the dideoxynucleotide triphosphate chain-termination method of Sanger, with the BigDye Terminator v3.1 Cycle Sequencing Kit and an ABI 3100 Genetic Analyzer (Applied Bio-Systems). The new eae sequences of strains analyzed were deposited in the European Bioinformatics Institute (EMBL Nucleotide Sequence Database).
Quantitative invasion assay
Quantitative assessment of bacterial invasion was performed as described previously  with modifications. Briefly, washed HeLa and polarized and differentiated T84 cells were infected with 107 colony-forming units (c.f.u.) of each aEPEC strain for 6 h or 3 h for tEPEC E2348/69. The different incubation-periods used were due to the more efficient colonization of tEPEC in comparison with the aEPEC strains; moreover, tEPEC E2348/69 induced cell-detachment in 6 h. Thereafter, cell monolayers were washed five times with PBS, and lysed in 1% Triton X-100 for 30 min at 37°C. Following cell lysis, bacteria were re-suspended in PBS and quantified by plating serial dilutions onto MacConkey agar plates to obtain the total number of cell-associated bacteria (TB). To obtain the number of intracellular bacteria (IB), a second set of infected wells was washed five times and further incubated in fresh media with 100 μg/mL of gentamicin for one hour. Following this incubation period, cells were washed five times, lysed with 1% Triton X-100 and re-suspended in PBS for quantification by plating serial dilutions. The invasion indexes were calculated as the percentage of the total number of cell-associated bacteria (TB) that was located in the intracellular compartment (IB) after 6 h (or 3 h for tEPEC E2348/69) (IBx100/TB) of infection. Assays were carried out in duplicate, and the results from at least three independent experiments were expressed as the percentage of invasion (mean ± standard error).
Cytoskeleton polymerization inhibitor
In order to evaluate the participation of cytoskeleton components in the invasion of aEPEC 1551-2, HeLa cell monolayers were incubated with 1 and 5 μg/mL of Cytochalasin-D or Colchicine (Sigma-Aldrich, St. Louis, MO) 60 min prior to bacterial inoculation . After that, cells were washed three times with PBS and the invasion assay was performed as described above. S. enterica sv Typhimurium and S. flexneri were used as controls.
EGTA treatment for tight junction disruption
In order to evaluate the interaction of aEPEC 1551-2 with the basolateral surfaces of T84 cells, differentiated cell monolayers (14 days) were incubated with 1 or 5 mM of EGTA (Sigma-Aldrich, St. Louis, MO) 60 min prior to bacterial inoculation . After that, cells were washed three times with PBS and the invasion assay was performed as describe above. S. enterica sv Typhimurium and S. flexneri were used as controls.
Detection of actin aggregation
To detect actin aggregation the Fluorescence Actin Staining (FAS) assay was performed as described previously . Briefly, cell monolayers were infected for 3 h, washed three times with PBS and incubated for further 3 h with fresh medium. Subsequently, monolayers were washed five times with PBS, fixed with 3.5% paraformaldehyde, and lysed in 1% Triton X-100 for 5 min at room temperature. Monolayers were then washed three times, incubated in a dark chamber with 5 μg/mL phalloidin (20 min), and washed. Coverslips were mounted in glycerol with 0.1% para-phenylenediamine to reduce bleaching.
Transmission Electron Microscopy
T84 cells were cultured in Transwell membranes (Costar) for 14 days and infected as described above. Then they were washed 3 times (10 min each) with D-PBS (Sigma) and fixed with 2% glutaraldehyde (Serva) for 24 h at 4°C. After fixation, cells were washed 3 times with D-PBS (10 min) and post-fixed with 1% osmium tetroxide (Plano). Cells were dehydrated through a graded ethanol series (30%, 50% and 70%), then filters were cut out from the cell culture system holder and preparations were treated with ethanol (90%, 96% and 99.8%), followed by propylenoxid (100%), Epon:Propylenoxid (1:1, Serva), and Epon 100%. Afterward, filters were embedded in flat plates and kept for 2 days for polymerization. Ultrathin sections were prepared, stained with 4% uranyl acetate (Merck) and Reynold's lead citrate (Merck), and were examined with a Tecnai G2 Spirit Twin, Fei Company at 80 kV.
Alternatively, T84 cells were cultured on 35 mm diameter plates for 14 days. Infection, fixation and dehydration were performed as described above. Subsequently, the cells were examined with a LEO 906E transmission electron microscope (Zeiss, Germany) at 80 kV.
Differences in the percentages of invasion were assessed for significance by using an unpaired, two-tailed t test (GraphPad Prism 4.0).
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant 08/53812-4), and Programa de Apoio a Núcleos de Excelência – PRONEX MCT/CNPq/FAPERJ supported this work. DY received a fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, fellowship 141708/04); DY and RTH received sandwich fellowships from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior and Programa Brasil Alemanha (CAPES – Probral 281/07). Additional funding of this work was obtained from DAAD PPP-Brasilien (D/06/33942) and the European Network ERA-NET PathoGenoMics (Project 0313937C) and from Spanish Ministry of Health and Consumer Affairs (Fondo de Investigación Sanitaria, Spanish Network for the Research in Infectious Diseases, REIPI, RD06/0008-1018), Spanish Ministry of Education and Science (AGL-2008-02129) and the Autonomous Government of Galicia (Xunta de Galicia, PGIDIT065TAL26101P, 07MRU036261PR). A. Mora acknowledges the Ramón y Cajal programme from The Spanish Ministry of Education and Science. We also thank Dr. Cecilia Mari Abe for her help in some of the TEM procedures and J.R.C. Andrade for donating the Salmonella enterica serovar Typhimurium control strain.
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