Translocation of the Helicobacter pylori (Hp) cytotoxin-associated gene A (CagA) effector protein via the cag-Type IV Secretion System (cag-T4SS) into host cells is a hallmark of infection with Hp and a major risk factor for severe gastric diseases, including gastric cancer. To mediate the injection of CagA, Hp uses a membrane-embedded syringe-like molecular apparatus extended by an external pilus-like rod structure that binds host cell surface integrin heterodimers. It is still largely unclear how the interaction of the cag-T4SS finally mediates translocation of the CagA protein into the cell cytoplasm. Recently certain carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), acting as receptor for the Hp outer membrane adhesin HopQ, have been identified to be involved in the process of CagA host cell injection. Here, we applied the CRISPR/Cas9-knockout technology to generate defined human gastric AGS and KatoIII integrin knockout cell lines. Although confocal laser scanning microscopy revealed a co-localization of Hp and β1 integrin heterodimers on gastric epithelial cells, Hp infection studies using the quantitative and highly sensitive Hp β-lactamase reporter system clearly show that neither β1 integrin heterodimers (α1β1, α2β1 or α5β1), nor any other αβ integrin heterodimers on the cell surface are essential for CagA translocation. In contrast, deletion of the HopQ adhesin in Hp, or the simultaneous knockout of the receptors CEACAM1, CEACAM5 and CEACAM6 in KatoIII cells abolished CagA injection nearly completely, although bacterial binding was only reduced to 50%. These data provide genetic evidence that the cag-T4SS-mediated interaction of Hp with cell surface integrins on human gastric epithelial cells is not essential for CagA translocation, but interaction of Hp with CEACAM receptors is facilitating CagA translocation by the cag-T4SS of this important microbe.
Secretion systems of Gram-negative bacteria have evolved to mediate the passage of macromolecules across two or more cellular membranes, either into the extracellular space, or directly into selected host target cells [1]. A highly versatile group represents the bacterial Type IV secretion systems (T4SS), which can transport diverse components in a contact-dependent manner, ranging from single proteins to protein-protein and protein-DNA complexes [2,3]. One of these bacteria is Helicobacter pylori (Hp), which is recognized as one of the most prevalent bacterial pathogens worldwide and very efficiently utilizes the cytotoxin-associated gene (cag) type IV secretion system (cag-T4SS) as a major virulence determinant [4,5]. The effector protein CagA, together with a set of 27 proteins acting as structural and/or regulatory elements of the T4SS, are encoded on the cag pathogenicity island (cagPAI), approximately 40 kb in size. Upon host cell contact the cag-T4SS forms needle-like surface appendages, the T4SS pili [6–8], which are involved in the translocation of CagA from cell-adherent Hp across the bacterial and epithelial membranes into the host cell cytoplasm [8]. Our view on these fascinating nanomachines was extended recently by ultrastructural insights into the cag-T4SS-dependent membranous pilus-like appendages by in vivo electron cryotomography [9]. Injected CagA is tyrosine-phosphorylated on multiple Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs in the C-terminal region, allowing its interaction with a set of cellular target proteins [10,11]. This results in dysregulation of the homeostatic signal transduction events in gastric epithelial cells, in loss of cell polarity, chronic inflammation and malignancy, qualifying CagA as a bacterial oncoprotein [12]. The cag-T4SS targets host cells via β1 integrin receptors [13,14], and induces in these cells the production and secretion of proinflammatory cytokines and chemokines, such as interleukin-8 (IL-8) [15]. The pilus-associated protein CagL has originally been reported to interact via an arginine-glycine-aspartate (RGD) motif with the α5β1 integrin heterodimer and thereby to activate Src and focal adhesion kinase, however, the requirement of the RGD motif for T4SS functionality was assessed differently [13,14]. Other cagPAI proteins, including CagY, CagI and CagA, have also been identified as interacting with the α5β1 integrin and an integrin binding domain for CagA was identified [16,17]. In addition to receptor binding by the cagT4SS itself, the outer membrane protein HopQ was identified to support CagA translocation by acting as a non-cagPAI-encoded cofactor of T4SS function [18]. Later on, HopQ was found to selectively bind a set of receptors from the carcinoembryonic antigen-related cell adhesion molecule family (CEACAMs). CEACAM1, CEACAM3, CEACAM5 and CEACAM6 were identified as functional receptors for Hp via the outer membrane protein HopQ [19,20]. Hp-CEACAM binding not only plays a role for Hp adherence, but this interaction deeply contributes to the process of CagA translocation. Thus, the human embryonic kidney cell line (HEK293), which is devoid of CEACAM receptors on its surface, was resistant for CagA injection by Hp, but became readily susceptible upon functional expression of CEACAM1 or CEACAM5 on its surface [19,20]. The purpose of this study was to further dissect the role of integrin receptors versus the function of CEACAM receptors for the cagT4SS in the process of CagA translocation. Using the CRISPR/Cas9 system, we systematically generated single to multiple integrin knockout epithelial human cell lines (AGS and KatoIII) ending up with KatoIII cells without any integrin heterodimers on their surface. Unexpectedly, CagA translocation into these completely integrin-deficient cells was not significantly changed, suggesting that other integrin-independent Hp–host cell interactions must be important. In contrast, CRISPR/Cas9-mediated knockout of CEACAM receptors (CEACAM1, CEACAM5 and CEACAM6 simultaneously) generated in KatoIII cells resulted in a strong reduction of CagA translocation capacity by Hp, suggesting that β-integrin receptors play a minor role in the T4SS-mediated CagA translocation, but the Hp-CEACAM interaction is of major importance.
The integrin receptor family is composed of 24 distinct integrin heterodimers, generated by different α and β subunits. Generally, integrin receptors follow a distinct tissue- and cell type-specific expression pattern in epithelial cells, leukocytes or platelets [21]. Thus, six β1 integrin heterodimers (α1β1, α2β1, α3β1, α5β1, α6β1 and α9β1), two αv integrins (αvβ5 and αvβ6) and the integrin α6β4 are known to be epithelial-specific (Fig 1A) [21]. AGS and KatoIII cell lines are generally used as model systems for the evaluation of CagA translocation, since both cell lines were derived from human gastric epithelial cells. To get an overview of integrin expression on the surface of these cells, we stained them with different integrin-specific antibodies and determined the integrin expression profile by flow cytometry. AGS and KatoIII cells indeed produced β1 integrins (including α1β1, α2β1, α3β1, α5β1, α6β1 and α9β1), αv integrins (αvβ5 and αvβ6) (αvβ8 only by KatoIII) and the β4 integrin (α6β4) on their surface, however with varying expression levels (Fig 1B and 1C). We planned to generate a β1 gene knockout in AGS cells that should lack surface expression of all potential β1 containing integrins, since the targeting of either subunit of a given integrin heterodimer should ultimately result in the depletion of the targeted integrin heterodimer [22,23]. In order to obtain integrin knockout cell lines without undesired off-target mutagenesis, the double nicking strategy was applied [24]. For design of paired short guide RNAs (sgRNAs) targeting the integrin β1 (ITGB1) gene, the online CRISPR design tool (http: //tools. genome-engineering. org) was used for optimal sgRNA analysis and identification (See further details of the method in Experimental Procedures) (Fig 1D and S1 Table). For generation of a β1 integrin deficient AGS cell line, verified CRISPR constructs targeting exon 5 of the β1 integrin gene (ITGB1) were transfected into AGS cells. Transfected cells went through a selection procedure to obtain knockout cell lines. Since CRISPR constructs contain the puromycin resistance gene, the transfected population was treated with puromycin to kill non-transfected cells. The surviving cells were stained with integrin β1 antibody for negative selection by FACS sorting. Finally, serial dilutions of the sorted negative populations resulted in stable cell lines, which could be verified as completely integrin β1-deficient by flow cytometry analysis (Fig 2A). Furthermore, the complete absence of the gene product was verified by (i) demonstrating the disruption of the targeted gene sequence by PCR amplification and sequencing of the integrin β1 alleles (S1A Fig) and (ii) by immunoblotting of cell lysates using a β1 integrin-specific antibody (S2A Fig). Next, the verified β1 integrin-deficient AGS cells were tested for CagA translocation capacity by Hp. Traditionally, CagA translocation is assessed by detecting tyrosine-phosphorylated EPIYA motifs as a phosphorylated CagA band via western blot. This can be used for quantification, but is not very sensitive and accurate. We have recently established a sensitive β-lactamase reporter system (TEM-1 reporter assay) to accurately determine Hp CagA translocation into host cells independently of its tyrosine phosphorylation and host cell kinase activity [25]. When applying the Hp strain P12[TEM-CagA] in the TEM-1 reporter assay, we surprisingly did not observe a significant difference in CagA translocation into AGS wild type versus β1 integrin-deficient cells (Fig 3A). This observation suggested that the β1-integrin interaction was apparently not essential for the bacteria or the cag-T4SS to inject CagA. One possible explanation for this unexpected result might be that other integrin heterodimers (αvβ5, αvβ6, or α6β4), which are known to be expressed on AGS cells (see Fig 1A), are able to functionally substitute β1 integrin heterodimers regarding CagA translocation. We therefore extended the CRISPR/Cas9-mediated knockout strategy to inactivate integrins αv and β4 separately, using the same procedure as for integrin β1 (see S3 Fig and S4 Fig for design of integrin gene inactivation strategy). Furthermore, by targeting different combinations of two of the aforementioned genes in the same cell, different combinations of double mutants were obtained (ΔITGAvB4, ΔITGB1B4), which did not produce the corresponding integrins on the cell surface, as determined by flow cytometry (Fig 2B–2E). A concomitant knockout of all three integrin genes, which ultimately should result in cell lines devoid of all integrins on the AGS cell surface, could not be obtained in the AGS cell background, probably because they do not survive. The correct genetic inactivation of the integrin genes was verified by PCR amplification and sequencing of the corresponding αv- or β4-specific integrin alleles (S1B and S1C Fig). The complete absence of the gene products was confirmed by immunoblotting with integrin αv- or β4-specific antibodies (S2A Fig). We next asked whether obvious differences in the morphology, physiology or function of the integrin-knockout AGS derivatives are apparent compared to wild type AGS cells. A thorough study of the general cell morphology did not show any peculiarities. AGS AvB4 cells did only grow in tissue culture when collagen was added, which indicated that the integrin receptor-mediated binding to certain integrin ligands was absent. All wild type and knockout mutant cells showed the hummingbird phenotype (S5 Fig). IL-8 induction in AGS wild type cells was slightly reduced in the P12ΔhopQ infecting strain, as compared to the P12 wild type (wt) and the complemented mutant strain (S6 Fig). Interestingly the level of IL-8 induction was generally higher when integrin knockout cells were used as compared to AGS wild type cells, but the general pattern of reaction of the knockout versus the wild type cells was well conserved (S6 Fig). This indicates that the main phenotypic characteristics of the knockout cells are still conserved in comparison to wild-type cells, arguing against unexpected compensatory mutations or significant alterations in signal transduction networks in the knockout cells. Interestingly, infection experiments based on the TEM-1 reporter assay showed no statistically significant difference in CagA translocation efficiency into AGS wild type versus single or multiple integrin αv- or β deficient cells (Fig 3A). Similar results were obtained by the conventional tyrosine phosphorylation experiments upon infection of the mutant AGS epithelial cell lines (Fig 3C). In conclusion, we demonstrate here that Hp is able to translocate its CagA protein into gastric epithelial AGS cells devoid of most integrin receptors on their surface, although a complete integrin-free state could not be obtained in the AGS cell background. In order to compare our data obtained from AGS gastric epithelial cells with another independent human gastric cell line we chose the KatoIII cells for integrin gene knockout experiments. The same strategy and knockout plasmids were applied. We finally obtained a total of seven stable integrin-deficient KatoIII cell lines, all of which could be verified to be completely devoid of their corresponding cell surface integrins, as determined by flow cytometry (Fig 2F–2M). These included the single knockout cell lines (ΔITGB1, ΔITGAv, ΔITGB4), the double knockout cells (ΔITGB1B4, ΔITGAvB4, ΔITGAvB1) as well as a triple knockout cell line (ΔITGB1AvB4). The latter cell line indeed lacks all integrins we tested for by specific antibodies, as demonstrated by the absence of the αv and all individual β integrin subunits (β1 –β8) on the cell surface (see scheme Fig 1A and S7B Fig). Next, KatoIII wild type and the corresponding knockout cell lines were analyzed by immunoblotting with the corresponding anti-integrin antibodies to confirm the complete absence of the gene product (S2B Fig). On the genetic level knockout mutations could be verified by sequencing of each gene (S1 Fig). We then performed infection experiments to quantify CagA translocation for all seven different KatoIII integrin-knockout cell lines. Again, single or multiple integrin knockout cell lines did not show a significantly different CagA translocation efficiency as compared to wild type KatoIII cells (Fig 3B). Using a plate reader assay for adherent AGS cells, or a flow cytometry approach for KatoIII suspension cells, we next quantified and compared CagA translocation of different Hp P12[TEM-CagA] strains. They comprised a hopQI gene deficient strain (P12ΔhopQ[TEM-CagA]), a genetically complemented hopQI knockout strain (P12ΔhopQ: hopQ[TEM-CagA]) and a strain that served as a negative control for CagA translocation (P12ΔcagT[TEM-CagA]). The outer membrane protein HopQ has been recently identified as a major Hp adhesin binding to host cell CEACAMs and was found to be a major contributing factor for CagA translocation [19,20], whereas in AGS wild type and integrin knockout cells CagA translocation by a HopQ-deficient strain was generally reduced (Fig 3A). In contrast to AGS cells, CagA translocation by the P12ΔhopQ[TEM-CagA] strain into wild type KatoIII, as well as into integrin knockout KatoIII cell lines was almost completely abolished (Fig 3B). The genetically complemented strain (P12ΔhopQ: hopQ[TEM-CagA]) was restored in its ability for CagA translocation (Fig 3A and 3B). In summary, these data support our results obtained with AGS cells. Furthermore they suggest that in AGS cells (an) other receptor (s) distinct from CEACAMs seem (s) to support the process of CagA translocation, as shown by infection assays with a P12ΔhopQ[TEM-CagA] strain (Fig 3A). Such (a) receptor (s) is/are apparently absent in the KatoIII cell background, where CagA translocation seems to be mostly dependent on the HopQ-CEACAM interaction (Fig 3B). We next performed an integrin profiling in each integrin depletion cell line to investigate whether the depletion of individual integrins can influence the expression levels of the remaining integrins. Especially increased expression levels of remaining integrins could be a reasonable explanation for the sustained CagA translocation efficiency in the different integrin deficient cell lines. To cover all kinds of integrin combinations, also aberrant expression of non-epithelial integrin heterodimers, we analyzed integrin knockout cell lines (AGS and KatoIII background) for expression of integrin αv, β1, β2, β3, β4, β5, β6, β7 and β8 by flow cytometry using specific antibodies (S7 Fig). Among them, non-epithelial integrins β2, β3, β7 and β8 were not differently expressed by any of the mutant versus the wild type cell lines. Integrins β5 and β6 were absent only in αv knockout cells (see black arrows), probably due to the loss of their exclusive alpha integrin binding partner (S7 Fig and Fig 1A for scheme). In addition, the KatoIII β1 KO cell line showed a significant reduction in integrin αv and β5 surface localization as compared to wild type KatoIII cells (S7B Fig, black arrows). The remaining integrins expressed on the surface of each integrin-deficient cell line exhibited similar expression levels as found on wild type AGS or KatoIII cells. This reduced CEACAM expression in KatoIII knockout cells could be responsible for a slightly but not significantly reduced CagA translocation capacity of the integrin double and triple knockout cells (Fig 3B). Elevated expression levels of remaining integrins, or aberrant expression of non-epithelial integrin heterodimers could be a reasonable explanation for the sustained CagA translocation efficiency in the integrin or CEACAM deficient cell lines. However, we can safely exclude this possibility after intensive integrin profiling experiments. Most importantly, no unexpected additional integrin subunit (s) appeared on the cell surface, even in the triple integrin knockout KatoIII cells. This indicates that these cells do not bear any integrin on their surface to be exploited for CagA translocation by Hp. Thus, our data unequivocally demonstrate for the first time that the apparent complete absence of any integrin on the cell surface does not have a significant effect on the capacity of Hp to translocate CagA into these cells. So far our data clearly show that integrin receptors on the surface of AGS or KatoIII cells do not have a major impact for CagA translocation capacity of Hp, but the loss of the adhesin HopQ strongly reduced CagA translocation, especially in KatoIII cells. The question arose whether it is sufficient for CagA translocation to just mediate a physical tethering of the bacteria to the cell surface, or whether a special interplay between HopQ and CEACAMs, which may result in a very tight or close binding to the cell surface, is needed to facilitate CagA translocation? To address these questions, we next generated in KatoIII cells a triple CEACAM knockout (CEACAM1/5/6 knockout) using the CRISPR/Cas system (S8 Fig) and verified the knockout status by flow cytometry, immunoblotting and sequencing (Fig 4A and 4B and S9 Fig). Attempts to combine the integrin triple knockout and the CEACAM triple knockout mutations in KatoIII were not successful, since corresponding mutant cell lines did not survive. Notably, the triple integrin knockout KatoIII cells showed a 50% reduced expression of CEACAM5 as compared to the wild type cells (Fig 4B and 4C), but a 4. 5 fold increase in CEACAM1 expression, which might be explained by the fact that both surface receptors are usually found in the same lipid background and even might interact with each other [26]. This disturbance of CEACAM expression might have some influence on the slightly lower CagA translocation activity of Hp into the integrin double and triple knockout cells that we can always observe, although this difference is not statistically significant (Fig 3B). For CEACAM1/5/6 KO KatoIII cells no significant changes in the intrinsic integrin αv or β expression pattern could be observed (S10 Fig). We then infected the CEACAM triple knockout KatoIII cells with the P12[TEM-CagA] strain to quantify CagA translocation. As expected from the results with the P12ΔhopQ[TEM-CagA] strain, the CEACAM triple knockout showed a nearly complete loss of CagA translocation, comparable to and in support of the P12ΔhopQ[TEM-CagA] strain results (Figs 3B and 5B). Similar results were obtained in the CagA tyrosine phosphorylation assay for strain P12 and other Hp lab strains (Fig 5C and 5D). Thus, in KatoIII cells the HopQ-CEACAM interaction seems to be the major driver/mediator for CagA translocation. Next, an important question was how much of the total adhesion of the bacteria can be attributed to the HopQ-CEACAM interaction and is binding per se, independent of the type of host cell receptor, sufficient to allow CagA translocation. Interestingly, the binding capacity of a P12-GFP strain to KatoIII wild type versus the CEACAM triple knockout KatoIII cells was reduced to a level of about 75% (Fig 5A), whereas the CagA translocation was nearly completely abolished under these circumstances (Fig 5B). These data suggest that binding per se is not sufficient for Hp to induce CagA injection. It seems that the HopQ-CEACAM interaction mediates a (n) additional signal (s) to initiate CagA injection. To further study potential changes in the interaction of Hp with cells lacking all surface integrin receptors, or the relevant CEACAM receptors, we performed confocal microscopy studies using KatoIII wild type cells, KatoIIIΔαvβ1β4 and KatoIIIΔCEACAM1/5/6 cell lines infected with P12 wild type or P12ΔhopQ strains (Fig 6A–6C). We typically find a reduced number of Hp binding to KatoIIIΔαvβ1β4 and KatoIIIΔCEACAM1/5/6 cell lines as compared to KatoIII wild type cells, and the binding pattern of Hp to integrin-deficient cells appears to be different. Interestingly, KatoIIIΔCEACAM1/5/6 cell lines produce large amounts of β1 integrin (Fig 6C), and Hp is found closely attached to β1 integrin, although under these conditions very little CagA translocation was found (Fig 5B). Thus, we see for each cell line an intimate interaction of the bacteria with the host cells, independent of the capacity for CagA translocation of the strain (Fig 6A–6C, white arrowheads). Triple integrin knockout cells show a high number of adherent bacteria (Fig 5A) but a lower expression of CEACAM5 (Fig 4B and 4C). This is also visible by a lack of CEACAM5 recruitment to the bacterial surface in the triple integrin knockout cells, which is in stark contrast to the CEACAM5 receptor recruitment seen in Hp-infected KatoIII wild type cells (Fig 6A, versus B and C; yellow arrows). Notably, CagA translocation into these cells is not significantly reduced as compared to the KatoIII wild type cells (Fig 3B and 3C).
It is well established that the cagPAI-encoded T4SS is a major Hp virulence determinant, the function of which has been implicated in severity of disease and increased risk of gastric cancer [27]. A major role of the cag-T4SS is the translocation of the CagA protein into various types of host cells, where CagA interferes in a phosphorylation-dependent and phosphorylation-independent manner with signaling events to manipulate fundamental processes in the gastric epithelium [28]. Major outcomes include the suppression of innate defense mechanisms [29], changes in cell polarity and migration [30,31], and putatively oncogenic events [32,33]. The involvement of a host cell integrin heterodimer (α5β1 or any other β integrin heterodimer) acting as receptor for the Hp T4SS, especially for the pilus-associated RGD containing CagL protein, was considered as a major requirement for CagA translocation [13,14] [34,35]. Several labs have provided data showing the interaction of integrin α5β1 or other αβ integrin heterodimers with different components of the cag-T4SS, especially CagL [13,34–41], but also CagA [16], CagI [42] and CagY [14,42]. Several previous studies suggested that integrins are required for CagA translocation. The major evidence for a functional role of β1 integrins as receptor for the cag-T4SS and translocation of CagA was coming from studies using β1 integrin-deficient murine fibroblast (GD25) or epithelial (GE11) cell lines, which did not support CagA translocation, whereas the corresponding β1 integrin complemented versions resulted in CagA translocation and phosphorylation [13,14]. We report now that integrin heterodimers are not required for Hp to translocate its CagA into gastric epithelial cell lines in vitro. We were interested in a better understanding of the role and the contribution of the recently identified CEACAM receptors versus the integrin receptors for CagA translocation. Therefore we generated a set of knockout cell lines and measured their ability for CagA translocation. The β-lactamase reporter system determines Hp CagA translocation into host cells in a very sensitive, reproducible and quantitative way [25]. Most important, it measures the translocation of CagA directly, rather than its tyrosine phosphorylation. The tyrosine phosphorylation depends on the activity of host cell kinases c-Src or c-Abl, which might be affected in their activity by manipulations of the cells, such as different growth conditions, buffer treatments or procedures like the gene knockout technology. Taking advantage of the CRISPR/Cas technology we started to knock out important integrins in an additive fashion to generate single (β1), double (β1β4, αvβ4, β1αv,) AGS and KatoIII cells and finally a triple integrin gene knockout cell (αvβ1β4) in the KatoIII background. All CRISPR/Cas constructs and targeted cell lines for the generation of integrin-depletion AGS and KatoIII cell lines are summarized in S2 Table. AGS cells are adherent cells, which did not survive as triple knockout without any integrin on the surface. In AGS cells growing as attached cells on solid surfaces this phenomenon might be due to the induction of anoikis, a tissue architecture surveillance mechanism, which can be induced by the absence of integrin-ECM ligation to assure that dissociated and displaced cells are effectively eliminated, in order to prevent dysplastic growth [43,44]. KatoIII cells, which grow in a semi-adherent manner, were resistant to anoikis and allowed the generation of a triple integrin knockout. The triple integrin knockout KatoIII cell line, which is devoid of any integrin receptor on the surface, was still competent for CagA translocation. From these results we have to conclude that neither the direct interaction of components of the cag-T4SS with integrins, nor any integrin-mediated signaling event is necessary for CagA translocation. The data presented above seem to be in opposition to earlier publications [13,14], which demonstrated that CagA translocation was possible in murine β1 integrin expressing GE11 or GD25 cells, but not in the corresponding integrin knockout cells. However, neither the murine GE11 and GD25, nor the chinese hamster ovary (CHO) cell line, also used for such experiments, contain human CEACAMs for binding of the Hp adhesin HopQ. This might at least explain why in our earlier experiments only very low (background) levels of CagA translocation could be observed in the β1 integrin-complemented versions of these cells [14]. From our new perspective, the earlier integrin complementation data can be interpreted that the interaction of the cag-T4SS to integrins in cells without human CEACAM receptors has only a small supportive effect for CagA translocation. However, the integrin knockout data presented in this study clearly show that integrins are not necessary for CagA translocation in AGS or KatoIII gastric cell lines. Using hamster (CHO) cell lines devoid of human beta integrins, but genetically complemented with integrin genes encoding fully functional or partial integrins (CHO K1, CHOβ1TR) we demonstrated that the extracellular part of β1 integrin was supportive for CagA translocation, but the cytoplasmic tail of β1 integrin was not necessary [14]. We also reported that neither the RGD motif in CagL for binding the β1 integrin heterodimer, nor the function of the integrin linked kinase (ILK) were essential for CagA translocation. From that we concluded that no integrin-mediated signaling is involved in this process [14]. Interestingly, also knockdown experiments of integrin α5β1 and ILK showed that both were dispensable for NF-κB activation during Hp infection, but the bacterial adhesin HopQ promoted canonical NF-κB activation in AGS and NCI-N87 cells [45]. Combined these data suggest that integrin-mediated signaling is neither needed for CagA translocation nor NFκB activation. Earlier data also showed that a recombinant protein of CagA covering the binding site of β1 integrin can interfere with CagA translocation and phosphorylation [16]. A similar effect on CagA phosphorylation was seen with the β1 integrin-specific antibody 9EG7 [14]. These data were interpreted as direct effects of β1 integrin on CagA translocation, suggesting that β1 integrin is essential. In the context of our results in this study we would explain these data as more indirect effects, e. g. by steric hindrance exerted by the binding of the recombinant protein, or the 9EG7 antibody, on the function of other receptors, such as CEACAMs, which usually reside in the same lipid domains as integrins [26]. With these novel results the question arises why components of the cag-T4SS bind specifically and in some cases with high affinity (CagA, KD values in low nanomolar range) to α5β1 integrin heterodimers [14,42] although this binding apparently has only a very minor, functional relevance for CagA translocation? The binding of the cag-T4SS components to the extracellular domains of β1 integrin heterodimers may allow, by tethering of the T4SS to the host cell, a low level CagA translocation, but for a full CagA translocation, the HopQ adhesin–CEACAM binding is necessary. Furthermore, we cannot exclude that in an in vivo situation, when Hp interacts with primary gastric cells in tissue, integrin signalling via CagL might play a role. CagA translocation might happen independent from integrin interaction, as our in vitro data suggest, but the activation of Src kinase might be necessary in primary, untransformed cells, but dispensable in transformed cell lines, in which these kinases often are constitutively active. Our group as well as other labs have shown that certain CEACAMs act as receptors for Hp and support CagA translocation when they are reconstituted in a cell line deficient of CEACAM expression (e. g. HEK293, CHO) [18–20]. The genetic complementation of CEACAM-negative cells, such as CHO or HEK293 cells, showed a drastic effect on CagA translocation [18–20]. However, in contrast to the integrin knockouts, a complete genetic knockout of CEACAM1/5/6 in an epithelial cell line (KatoIII) more or less completely abrogated CagA translocation. This clearly suggests that CEACAM receptors are essential for CagA translocation in certain cell types. Besides CEACAMs, other surface receptors seem to exist, which can support CagA translocation. Thus, in AGS cells we see a reduction in CagA translocation to approximately 50% when a HopQ-deficient versus a wild type Hp strain is used for infection (Fig 3A) [19], which is in contrast to KatoIII cells suggesting that in AGS gastric epithelial cells an additional, so far unknown receptor might be expressed, which is probably targeted by another Hp adhesin to support CagA translocation. This receptor might be absent in other cell lines, such as KatoIII cells. Earlier work described a small BabA-Leb mediated but cag-T4SS-dependent effect on the production of proinflammatory cytokine mRNA expression (IL-8, CCL5) and a very minor effect on CagA translocation of Hp. This Leb dependent augmentation of cagPAI T4SS functions, which was seen in Leb-negative non-human and non-gastric CHO or Madin-Darby canine kidney cells (MDCK) transfected with several glycosyltransferase genes [46], was independent of CEACAMs, since these cells do not produce CEACAMs recognized by HopQ. Besides the interaction of the bacteria with protein- or oligosaccharide cell surface receptors, the translocated effector protein CagA can bind phospholipids via a K-Xn-R-X-R motif, an amino acid sequence motif conserved among various pleckstrin homology (PH) domains directly involved in the interaction with acidic phospholipids, such as phosphatidylinositol (PI) and/or phosphatidylserine (PS) [47]. Murata-Kamiya and coworkers [48] reported that physical interaction of Hp CagA with host membrane PS, which is aberrantly externalized at the site of bacterial attachment by Hp, plays a key role in the delivery and intracellular localization of CagA. How the exploitation of CEACAM receptors by the adhesin HopQ, the functional buildup of the cag-T4SS secretion apparatus and the CagA binding to PS are coordinated and function to accomplish the internalization of CagA is still not well understood and the aim of intensive future research.
For TEM-1 reporter assays Hp wild type strain P12 [49] and defined P12 knockout mutants were used. To verify CagA translocation results into integrin-deficient AGS or integrin- or CEACAM-deficient KatoIII cells by tyrosine phosphorylation also other Hp strains were applied, such as 1-20A, TN2GF4 or G27 [50]. These strains harbor a functional cag pathogenicity island (cagPAI) in their genome, encoding the Cag T4SS [4]. Hp strains and mutants used in this study are listed in S3 Table. Hp strains were grown on GC agar plates (Oxoid) supplemented with vitamin mix (1%) and horse serum (8%) (serum plates) and cultured for 16–60 h in a microaerobic atmosphere (85% N2,10% CO2,5% O2) at 37°C. Escherichia coli strains Top10 and DH5alpha were grown on Luria–Bertani (LB) agar plates or in LB liquid medium [51] supplemented with antibiotics, as appropriate. Cell lines were cultivated under standard conditions [15] in 75 cm2 tissue culture flasks (BD Falcon) and subcultivated every 2–3 days in 6-well, 48- well (tissue culture treated, Costar, Corning Inc.) or 96-well microtiter plates (black, transparent bottom, tissue culture treated, 4titude). Plasmids were introduced into Hp strains by transformation as described previously [52]. Hp transformants were selected on serum plates containing 6 mg l-1 chloramphenicol, 8 mg l-1 kanamycin, 10mg l-1 erythromycin or 250 mg l-1 streptomycin, as appropriate. AGS cells (CRL-1739) and Kato III cells (HTB-103) were obtained from ATCC. Cells were generally cultured in RPMI 1640 or DMEM supplemented with 10% (vol/vol) fetal calves serum (FCS) at 37°C and 5% CO2. For passaging of the cells, the medium was removed and the cells were gently washed once with Dulbecco´s PBS (DPBS, Life Technologies). To detach cells, 2 ml Trypsin-EDTA was added to a 75 cm2 flask for 3–5min incubation at 37°C. When detachment was observed under microscope, 8 ml of the pre-warmed RPMI1640 medium was added to neutralize the trypsin. After being gently pipetted up and down, cells were dissociated and were then reseeded into new flasks. Passages taken place in every 2–3 days with a split ratio of 1: 5 or 1: 8. Cells were discarded when the passage number reached 80. Generally, one day before transfection, 0. 5 x 105 to 2 x 105 cells were plated in 500 μl of growth medium without antibiotics in a 24-well plate so that they would be 90–95% confluent at the time of transfection. For each transfection sample, 500 μg DNA and 2 μl lipofectamine 2000 was prepared according to the manufacturer´s instructions (Lipofectamine 2000, Invitrogen). After transfection the cells were incubated at 37°C in a CO2 incubator for 24–48 hours until they were ready to test for transgene expression. Cells were counted and added to a round-bottom 96-well plate with 2x105 cells per well. After centrifugation (300 g at 4°C) primary antibodies were added to each well following the recommended concentration from the manufacturer. Dilutions of antibody, if necessary, were made in FACS buffer. Cells and antibodies were incubated at 4°C for 1 hour in the dark. Primary antibody stained cells were washed 3 times and resuspended in 200 μl to 1 ml of ice-cold FACS buffer. Subsequently, fluorochrome-labeled secondary antibodies were diluted in FACS buffer at the optimal concentration (according to the manufacturer’s instructions) and were added to each well, followed by 1 h incubation at 4°C in the dark and 3 times washing as described above. Cells were analyzed by flow cytometer right after washing or kept in the dark on ice until the scheduled time for analysis. The following antibodies were used: ITGA2 (P1E6, chemicon), ITGA3 (P1B5, chemicon), ITGAv (P2W7, Santa Cruz), ITGB1 (AIIB2, EMD Millipore), ITGB2 (MEM-48, antibodies-online GmbH) ITGB3 (VI-PL2, antibodies-online GmbH), ITGB4 (439-9B, antibodies-online GmbH), ITGB5 (AST-3T, antibodies-online GmbH), ITGB6 (437211, antibodies-online GmbH), ITGB7 (FIB504, antibodies-online GmbH), ITGB8 (416922, antibodies-online GmbH), CEACAM1 (8G5, Genovac), (CEACAM5 (26/3/13, Genovac), CEACAM6 (9A6, Genovac). This procedure is used for adherent cells, such as AGS cells. One day before infection, adherent cells were detached and 2. 5 × 104 cells were seeded in each well in a 96-well plate with black wall and transparent bottom with low fluorescence background (4ortitude). The confluence of the cells was 80% to 90% on the day of infection. Before infection, Hp strains with fusion protein of beta-lactamase TEM-1 and CagA were collected as described before. Ideally, bacteria were resuspended and pre-incubated in sterile PBS containing 10% FCS at 37°C, 10% CO2 for 1. 5 h. Subsequently, cells were infected by bacteria with an MOI of 60 for 2. 5 h at 37°C, 5% CO2 as described above. Infections were stopped by placing the plates on ice and all the supernatants were removed. Prepared substrates mix is loaded immediately on the cell surface, followed by incubation at room temperature for 120 min in the dark. Plate reader filters are set to allow excitation of wavelength around 410nm, and detection of blue emission around 450nm and green emission around 520nm. Afterwards acquired data was normalized and analyzed following manufacturer’s instruction to obtain the blue to green fluorescence ratio. For suspension and semi-adherent cell lines, CagA translocation was detected by flow cytometry. The method of CagA translocation assay with flow-cytometry detection is very similar to the plate-reader detection except following procedures. Firstly, semi-adherent cells were detached after infection with room-temperature trypsin-EDTA before incubation with CCF4-AM fluorescence substrate mix. Secondly, incubation of cells with CCF4-AM mix were implemented at 27°C with constant shaking condition to allow even loading of cells with substrate and avoid cell sedimentation. Finally, cells were washed at least 2 times with PBS by centrifugation at 200–300 × g for 5 mins after incubation with CCF4-AM substrate. Cells were then analyzed by flow cytometry for Pacific Blue fluorescence and AmCyan green fluorescence. Rabbit polyclonal antisera AK268 and AK257, directed against the CagA N-terminal and the CagA C-terminal region, respectively, have been described previously [5,53]. The mouse monoclonal antibody against TEM-1 β-lactamase was obtained from Abcam (ab12251). For immunoblotting the following antibodies were used: ITGAv, (EPR16800, abcam), ITGB1 (LM534, EMD Millipore), ITGB4, (439-9B, abcam), CEACAM1/3/4/5/6 (D14HD11, Genovac). Standard infections of AGS and KatoIII cells with Hp strains and subsequent preparations for phosphotyrosine immunoblotting were performed as described previously [23]. Briefly, cells were plated one day before infection in 6-well plates. On the day of infection, cells were infected using an MOI of 60 for 2. 5 h at 37°C and 5% CO2. After washing with PBS, cells were collected by cell scrapers in the presence of 1 ml PBS* (PBS containing 1 mM Na3V04,1 mM PMSF, 10 μg/ml leupeptin and 10 μg/ml pepstatin). Cells with adherent bacteria were collected by centrifugation and resuspended in SDS-PAGE sample solution. SDS PAGE and western blotting was performed as described previously [13]. For the development of immunoblots, polyvinylidene difluoride (PVDF) filters were blocked with 5% non-fat milk powder in TBS (50 mM Tris–HCl, pH 7. 5,150 mM NaCl), 0. 1% (v/v) Tween 20 (TBS-T), and incubated with the respective antisera at a dilution of 1: 1. 000–1: 15. 000 in TBS-T with 1% non-fat milk powder. Horseradish peroxidase-conjugated anti-rabbit IgG antiserum was used to visualize bound antibody. Standard infections of AGS and KatoIII cells with Hp strains and subsequent preparations for phosphotyrosine immunoblotting were performed as described previously [5]. Tyrosine-phosphorylated proteins were analyzed by immunoblotting with the phosphotyrosine antibody PY99 (Santa Cruz Biotechnologies). Single gel systems [54] were adapted for Stain-Free detection as described in protocol depository Protocols. io under dx. doi. org/10. 17504/protocols. io. gipbudn. Western Blot data were quantified by densitometry using ImageJ. Band intensities of strain-free gel were normalized to the band intensity of KatoIII lane. CEACAM1,5 and 6 expression was measured as area percent of the respective lane and normalized to the CEACAM1,5 and 6 expression of KatoIII cells. Comparability between cell lines was achieved by standardizing each normalized CEACAM expression to the normalized loading controls. One day prior to experiments cells were seeded at 5 x 104 cells in a 24-well plate equipped with uncoated cover slides and grown overnight at 37°C and 5% CO2. Cells were infected with Hp wild type or isogenic mutant strains with an MOI of 10 for 3h at 37°C and 5% CO2. For immunostaining cells were fixed with 4% PFA for 10 min at room temperature. Cells were washed twice with Dulbecco´s PBS (DPBS, Life Technologies) and blocked overnight with 2% FCS in PBS at 4°C. Fixed cells were incubated with mouse anti-CEACAM5 (26/3/13, Genovac, 1: 300), rabbit anti-Hp (AK175,1: 400) and rat anti-integrin beta1 (AIIB2, Millipore, 1: 200) for 1h at room temperature. After washing secondary antibodies were applied (goat anti-rat Alexa488, goat anti-mouse Alexa555 and goat anti-rabbit Alexa647 all from Invitrogen, 1: 1000) and incubated for 1h at room temperature in the dark. Cell nuclei were stained with DAPI (5μg/ml) for 10 min. Samples were mounted on the cover slip with Fluorescent Mounting Medium (DAKO). A cytospin3 (Shandon) was used to centrifuge suspension cells onto glass slides. Micrographs were taken with a confocal laser scanning microscope (LSM880, Zeiss) with Airyscan Module using a 63x oil immersion objective. Statistical analysis was performed with GraphPad Prism 7. 2. Data were analyzed with One-way or Two-way analysis of variance (ANOVA), as further specified in the legends of the corresponding figures. The significance level was set to 0. 05. If overall ANOVA tests were significant, a post hoc test (Tukey’s HSD test or Bonferroni test) was performed. Details for each experiment are described in the figure legends.
The Cag Type IV secretion system of Helicobacter pylori (Hp) interacts with host cell integrins and injects the bacterial oncoprotein CagA into host cells thereby contributing to inflammation and carcinogenesis during chronic infection. Binding of β1 integrin receptors by the CagA protein and the type IV secretion system is well described by many research groups, but its function for CagA translocation is not well understood. We report here that this interaction is not essential for the function of the secretion system and for CagA injection into the gastric epithelial cells lines AGS and KatoIII. However, the bacterial binding to a set of specific receptors called carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) by the Hp outer membrane protein HopQ is a prerequisite for CagA translocation. Interestingly, other bacterial adhesins and the mediated binding events do not have a similar effect on CagA translocation, suggesting a specific feature associated with HopQ mediated binding.
cell binding
flow cytometry
cell physiology
bacteriology
phosphorylation
medicine and health sciences
pathology and laboratory medicine
viral transmission and infection
molecular probe techniques
pathogens
microbiology
immunoblotting
epithelial cells
integrins
secretion systems
molecular biology techniques
cellular structures and organelles
research and analysis methods
microbial physiology
animal cells
cell adhesion
proteins
extracellular matrix
biological tissue
molecular biology
spectrophotometry
cytophotometry
bacterial physiology
biochemistry
host cells
cell biology
anatomy
virulence factors
post-translational modification
virology
epithelium
biology and life sciences
cellular types
spectrum analysis techniques
Integrin but not CEACAM receptors are dispensable for Helicobacter pylori CagA translocation