Type III secretion-dependent cell cycle block caused in HeLa cells by enteropathogenic Escherichia coli O103.

Rabbit enteropathogenic Escherichia coli (EPEC) O103 induces in HeLa cells an irreversible cytopathic effect characterized by the recruitment of focal adhesions, formation of stress fibers, and inhibition of cell proliferation. We have characterized the modalities of the proliferation arrest and investigated its underlying mechanisms. We found that HeLa cells that were exposed to the rabbit EPEC O103 strain E22 progressively accumulated at 4C DNA content and did not enter mitosis. A significant proportion of the cells were able to reinitiate DNA synthesis without division, leading to 8C DNA content. This cell cycle inhibition by E22 was abrogated in mutants lacking EspA, -B, and -D and was restored by transcomplementation. In contrast, intimin and Tir mutants retained the antiproliferative effect. The cell cycle arrest was not a direct consequence of the formation of stress fibers, since their disruption by toxins during exposure to E22 did not reverse the cell cycle inhibition. Likewise, the cell cycle arrest was not dependent on the early tyrosine dephosphorylation events triggered by E22 in the cells. Two key partner effectors controlling entry into mitosis were also investigated: cyclin B1 and the associated cyclin-dependent kinase 1 (Cdk1). Whereas cyclin B1 was not detectably affected in E22-exposed cells, Cdk1 was maintained in a tyrosine-phosphorylated inactive state and lost its affinity for p13(suc1)-agarose beads. This shows that Cdk1 is implicated in the G2/M arrest caused by EPEC strain E22.

Cytolethal distending toxin (CDT): a bacterial weapon to control host cell proliferation?

Cytolethal distending toxins (CDT) constitute a family of genetically related bacterial protein toxins able to stop the proliferation of numerous cell lines. This effect is due to their ability to trigger in target cells a signaling pathway that normally prevents the transition between the G2 and the M phase of the cell cycle. Produced by several unrelated Gram-negative mucosa-associated bacterial species, CDTs are determined by a cluster of three adjacent genes (cdtA, cdtB, cdtC) encoding proteins whose respective role is not yet fully elucidated. The CDT-B protein presents sequence homology to several mammalian and bacterial phosphodiesterases, such as DNase I. The putative nuclease activity of CDT-B, together with the activation by CDT of a G2 cell cycle checkpoint, strongly suggests that CDT induces an as yet uncharacterized DNA alteration. However, the effective entry of CDT into cells and subsequent translocation into the nucleus have not yet been demonstrated by direct methods. The relationship between the potential DNA-damaging properties of this original family of toxins and their role as putative virulence factors is discussed.

Study of the cytolethal distending toxin (CDT)-activated cell cycle checkpoint. Involvement of the CHK2 kinase.

The bacterial cytolethal distending toxin (CDT) triggers a G2/M cell cycle arrest in eukaryotic cells by inhibiting the CDC25C phosphatase-dependent CDK1 dephosphorylation and activation. We report that upon CDT treatment CDC25C is fully sequestered in the cytoplasmic compartment, an effect that is reminiscent of DNA damage-dependent checkpoint activation. We show that the checkpoint kinase CHK2, an upstream regulator of CDC25C, is phosphorylated and activated after CDT treatment. In contrast to what is observed with other DNA damaging agents, we demonstrate that the activation of CHK2 can only take place during S-phase. Use of wortmannin and caffeine suggests that this effect is not dependent on ATM but rather on another as yet unidentified PI3 kinase family member. These results confirm that the CDT is therefore responsible for specific genomic injuries that block cell proliferation by activating a cell cycle checkpoint.

Study of the cytolethal distending toxin-induced cell cycle arrest in HeLa cells: involvement of the CDC25 phosphatase.

HeLa cells exposed to Escherichia coli cytolethal distending toxins (CDT) arrest their cell cycle at the G2/M transition. We have shown previously that in these cells the CDK1/cyclin B complex is inactive and can be reactivated in vitro using recombinant CDC25 phosphatase. Here we have investigated in vivo the effects of CDC25 on this cell cycle checkpoint. We report that overexpression of CDC25B or CDC25C overrides an established CDT-induced G2 cell cycle arrest and leads the cells to accumulate in an abnormal mitotic stage with condensed chromatin and high CDK1 activity. This effect can be counteracted by coexpression of the WEE1 kinase. In contrast, overexpression of CDC25B or C prior to CDT treatment prevents G2 arrest and allows most of the cells to progress through mitosis with only a low percentage of cells arrested in abnormal mitosis. The implications of these results on the biochemical nature of the CDT-induced cell cycle arrest are discussed.

Sequence of lethal events in HeLa cells exposed to the G2 blocking cytolethal distending toxin.

The bacterial cytolethal distending toxin (CDT) was previously shown to block the cell cycle of several cell lines at stage G2 through inactivation of the cyclin-dependent kinase Cdkl and without induction of DNA strand breaks. In the present study, we have analyzed, using various methods of analytical cytometry, the progressive transformation and delayed lethal events in the tumor-derived HeLa cell line temporarily exposed to CDT. The cell proliferation arrest induced by CDT was irreversible but, starting about two days after exposure, the G2 block released partially, concomitantly with a decline in the level of Cdkl phosphorylation. This partial release resulted in endoreduplication, leading to the emergence of a significant subpopulation of cells with a 8C DNA content, and by multipolar abortive mitosis which accounted for the mortality recorded 2 and 3 days after exposure. The other major lethal event was a micronucleation process which started to be significant about 3 days after exposure and amplified later on. Both multipolar abortive mitosis and micronucleation appeared topologically related to centrosomal amplification.

Role of tir and intimin in the virulence of rabbit enteropathogenic Escherichia coli serotype O103:H2.

Attaching and effacing (A/E) rabbit enteropathogenic Escherichia coli (REPEC) strains belonging to serogroup O103 are an important cause of diarrhea in weaned rabbits. Like human EPEC strains, they possess the locus of enterocyte effacement clustering the genes involved in the formation of the A/E lesions. In addition, pathogenic REPEC O103 strains produce an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton characterized by the formation of focal adhesion complexes and the reorganization of the actin cytoskeleton into bundles of stress fibers. To investigate the role of intimin and its translocated coreceptor (Tir) in the pathogenicity of REPEC, we have used a newly constructed isogenic tir null mutant together with a previously described eae null mutant. When human HeLa epithelial cells were infected, the tir mutant was still able to induce the formation of stress fibers as previously reported for the eae null mutant. When the rabbit epithelial cell line RK13 was used, REPEC O103 produced a classical fluorescent actin staining (FAS) effect, whereas both the eae and tir mutants were FAS negative. In a rabbit ligated ileal loop model, neither mutant was able to induce A/E lesions. In contrast to the parental strain, which intimately adhered to the enterocytes and destroyed the brush border microvilli, bacteria of both mutants were clustered in the mucus without reaching and damaging the microvilli. The role of intimin and Tir was then analyzed in vivo by oral inoculation of weaned rabbits. Although both mutants were still present in the intestinal flora of the rabbits 3 weeks after oral inoculation, neither mutant strain induced any clinical signs or significant weight loss in the inoculated rabbits whereas the parental strain caused the death of 90% of the inoculated rabbits. Nevertheless, an inflammatory infiltrate was present in the lamina propria of the rabbits infected with both mutants, with an inflammatory response greater for the eae null mutant. In conclusion, we have confirmed the role of intimin in virulence, and we have shown, for the first time, that Tir is also a key factor in vivo for pathogenicity.

Comparison of necrotoxigenic Escherichia coli isolates from farm animals and from humans.

Necrotoxigenic Escherichia coli (NTEC) isolated from animals and humans can belong to the same serogroups/types and produce or carry the genes coding for fimbrial and afimbrial adhesins of the same family, P, S, F17, and/or AFA, raising the question of a potential zoonotic source of human infection. The main purpose of this study was to compare 239 NTEC1 strains (45 from cattle, 65 from humans and 129 from piglets) and 98 NTEC2 strains from cattle, using a uniform and standardized typing scheme. The O serogroups and the biotypes recognized amongst NTEC1 and NTEC2 strains were quite varied, although some were more frequently observed (serogroups O2, O4, O6, O8, O18, O78, and O83 and biotypes 1, 2, 5, 6, and 9). Hybridization, results with gene probes for the P family (PAP probe), S family (SFA probe), AFA family (AFA probe), F17 family (F17 probe) of fimbrial and afimbrial adhesins, could differentiate most NTEC1 strains, which are PAP-, SFA- and/or AFA-positive, from NTEC2 strains, which are mainly F17- and/or AFA-positive, but were of no help in differentiating between NTEC1 strains from cattle, humans, and piglets. All but seven (98%) NTEC1 and NTEC2 strains were serum resistant, 199 (59%) produced an aerobactin, and colicin (I, V, or unidentified) was produced by 22-34% of them. On the other hand, more than 90% of the NTEC1 strains were haemolytic on sheep blood agar compared with only 40% of the NTEC2 strains. Production of a classical haemolysin, active on sheep erythrocytes, and hybridization with the PAP probe were associated in a majority of NTEC1 strains (63-81%), but very rarely in NTEC2 strains (3%). Production of enterohaemolysin and hybridization with the PAP probe were much less frequently associated in NTEC strains (1-9%). It was thus possible neither to completely differentiate NTEC1 strains from cattle, humans, and pigs, nor to define a signature for the NTEC strains. Necrotoxigenic E. coli must still be identified on the basis of the production of the Cytotoxic Necrotizing Factors 1 or 2 (or of their encoding genes) and complete differentiation of NTEC1 strains from cattle, humans, and piglets, use additionnal methods.

The bacterial cytolethal distending toxin (CDT) triggers a G2 cell cycle checkpoint in mammalian cells without preliminary induction of DNA strand breaks.

The bacterial cytolethal distending toxin (CDT) was previously shown to arrest the tumor-derived HeLa cell line in the G2-phase of the cell cycle through inactivation of CDK1, a cyclin-dependent kinase whose state of activation determines entry into mitosis. We have analysed the effects induced in HeLa cells by CDT, in comparison to those induced by etoposide, a prototype anti-tumoral agent that triggers a G2 cell cycle checkpoint by inducing DNA damage. Both CDT and etoposide inhibit cell proliferation and induces the formation of enlarged mononucleated cells blocked in G2. In both cases, CDK1 from arrested cells could be reactivated both in vitro by dephosphorylation by recombinant Cdc25B phosphatase and in vivo by caffeine. However, the cell cycle arrest triggered by CDT, unlike etoposide, did not originate from DNA strand breaks as demonstrated in the single cell gel electrophoresis assay and by the absence of slowing down of S phase in synchronized cells. Together with additional observations on synchronized HeLa cells, our results suggest that CDT triggers a G2 cell cycle checkpoint that is initiated during DNA replication and that is independent of DNA damage.

Use of CDC2 from etoposide-treated cells as substrate to assay CDC25 phosphatase activity.

Cyclin-dependent kinases (CDKs) regulate the key transition of the cell cycle in all organisms. In response to Etoposide (VP-16) induced DNA damage, cells undergo a G2-phase arrest resulting in the accumulation of inactive CDK1 (CDC2) kinase complexes. Here we report that upon Etoposide treatment CDC2 is phosphorylated on tyrosine 15 and is dephosphorylated and activated in vitro by recombinant CDC25 phosphatase. We also show that inactive CDC2 kinase from Etoposide-treated cells can be used as a substrate in a sensitive two-step assay of CDC25 phosphatase. This assay, which is very simple to set-up, is based on the monitoring of CDC2 kinase activity after CDC25-dependent dephosphorylation. It provides the possibility to use a highly physiological substrate in antimitotic drugs screening.

Rabbit EPEC: a model for the study of enteropathogenic Escherichia coli.

Colibacillosis has become, in rational rabbit breeding units of western Europe, one of the most economically and pathologically important issues since the beginning of the 1980s. Data on the virulence mechanisms and the phenotypic characters of the E. coli strains that are responsible for lethal diarrhoea epizootics have been gathered throughout the years. These strains are representative of a pathovar called enteropathogenic E. coli (EPEC) in diarrhoeagenic strains of human origin. EPEC are mainly characterized by their ability to induce a typical lesion called attachment/effacement, whose determinism lies in a pathogenicity island: the locus of enterocyte effacement. The understanding of the pathogenesis mechanisms of this type of bacteria should lead to new tools helping to control the disease in rabbit farming.

Necrotoxic Escherichia coli (NTEC): two emerging categories of human and animal pathogens.

Necrotoxic Escherichia coli (NTEC) were originally defined as strains of E. coli producing a toxin called cytotoxic necrotising factor (CNF). Two types of CNF have been identified, each of them being genetically linked to several other specific virulence markers, a situation that allows the definition of two distinct homogeneous categories of NTEC called NTEC-1 and NTEC-2. CNF1 and CNF2 are highly homologous holoproteins containing 1,014 amino acids that exert both lethal and necrotic activities in vivo and induce multinucleation and actin stress fibres in cell cultures. The activity of CNFs on mammal cells is due to their ability to constitutively activate by deamidation the Rho proteins, a family of small GTPases that regulate the physiology of the cell cytoskeleton. In NTEC-1, the gene encoding CNF1 belongs to a pathogenicity island which also comprises the genes encoding for alpha-haemolysin and P-fimbriae. In NTEC-2 strains, CNF2 is encoded by a plasmid that also encodes, in 100% of the isolates, a new member of the cytolethal distending toxin family (CDT-III) and in about 50% of the isolates, the F17b-fimbrial adhesin that confers the ability to adhere to calf intestinal villi. The presence of CDT is also suspected in a large majority of NTEC-1 strains. NTEC-1 strains can be found in humans and in all species of domestic mammals, whereas NTEC-2 strains have only been reported in ruminants. The implication of NTEC strains has been clearly established in extra-intestinal infections of humans and animals, for instance in urinary tract infections for NTEC-1 strains. Their role in severe dysenteric syndromes, both in humans and animals, is substantiated by several clinical reports, but there is little published information on this pathogenicity in animal models of infection. The combined production of several powerful toxins (haemolysin, CNF, CDT) by NTEC strains makes them, however, potentially aggressive pathogens which deserve to be searched for on a larger scale. Moreover, NTEC-1 from man and animals appear to be highly related according to available molecular markers, which indicates that domestic animals could constitute reservoirs of NTEC strains which are pathogenic for humans.

The long-term cytoskeletal rearrangement induced by rabbit enteropathogenic Escherichia coli is Esp dependent but intimin independent.

Attaching and effacing rabbit enteropathogenic Escherichia coli (REPEC) of the O103 serogroup adhere diffusely on HeLa cells and trigger a slow progressive cytopathic effect (CPE) characterized by the recruitment of vinculin and the assembly of actin stress fibres. In contrast to REPEC O103, the reference human EPEC strain E2348/69 is unable to trigger the CPE. In this study, we have shown first that the fimbrial adhesin AF/R2, which mediates the diffuse adhesion of REPEC O103, was not sufficient to induce the CPE capability upon E2348/69. Non-polar mutants of REPEC O103 for espA, espB, espD and eae were then constructed. The four mutants were unable to induce attaching and effacing lesions in the rabbit ileal loop model. The esp mutants were no longer able to induce the CPE, whereas the eae mutant still induced the CPE. Each espA, -B, -D mutant could be fully complemented in trans by the corresponding cloned esp genes from both the parental strain and the CPE-negative E2348/69 strain, indicating that no single esp encodes the information needed to confer the CPE phenotype. In conclusion, the CPE is the first example of an Esp-dependent but Eae (intimin)-independent alteration of the host cell cytoskeleton by certain EPEC strains.

Escherichia coli cytolethal distending toxin blocks the HeLa cell cycle at the G2/M transition by preventing cdc2 protein kinase dephosphorylation and activation.

Cytolethal distending toxins (CDT) constitute an emerging heterogeneous family of bacterial toxins whose common biological property is to inhibit the proliferation of cells in culture by blocking their cycle at G2/M phase. In this study, we investigated the molecular mechanisms underlying the block caused by CDT from Escherichia coli on synchronized HeLa cell cultures. To this end, we studied specifically the behavior of the two subunits of the complex that determines entry into mitosis, i.e., cyclin B1, the regulatory unit, and cdc2 protein kinase, the catalytic unit. We thus demonstrate that CDT causes cell accumulation in G2 and not in M, that it does not slow the progression of cells through S phase, and that it does not affect the normal increase of cyclin B1 from late S to G2. On the other hand, we show that CDT inhibits the kinase activity of cdc2 by preventing its dephosphorylation, an event which, in normal cells, triggers mitosis. This inhibitory activity was demonstrated for the three partially related CDTs so far described for E. coli. Moreover, we provide evidence that cells exposed to CDT during G2 and M phases are blocked only at the subsequent G2 phase. This observation means that the toxin triggers a mechanism of cell arrest that is initiated in S phase and therefore possibly related to the DNA damage checkpoint system.

Enteropathogenic Escherichia coli O103 from rabbit elicits actin stress fibers and focal adhesions in HeLa epithelial cells, cytopathic effects that are linked to an analog of the locus of enterocyte effacement.

Escherichia coli O103, a major agent of weaned-rabbit diarrhea in Western Europe, was previously shown to produce diarrhea and attaching-and-effacing intestinal lesions in experimentally infected rabbits and to possess a homolog of the eaeA gene of enteropathogenic E. coli (EPEC). In the present study, we have shown that although negative in the fluorescent-actin staining test on HeLa cells, prototype rabbit E. coli O103 strain B10 was able to induce an original cytopathic effect (CPE) in the same interaction model. This CPE was characterized by a generalized reorganization of the actin cytoskeleton and the formation of focal adhesions on the entire surface of the target cells. These effects amplified with time, leading to cell death about 5 days after the interaction. They were produced by all rabbit E. coli O103 strains tested, by rabbit-infecting E. coli RDEC-1, and also by two human EPEC isolates. We localized genes associated with CPE by using TnphoA insertion mutagenesis in strain B10. In all five independent CPE-negative mutants that we were able to generate, the insertion was located in a region of the genome homologous to the 35-kb locus of enterocyte effacement (LEE locus) of EPEC E2348/69. The mutants concurrently lost the ability to secrete four major supernatant proteins of 25, 37, 39, and 40 kDa, which were shown by immunoprecipitation to share antigenic determinants with secreted proteins of human EPEC E2348/69. The virulence of one of these mutants (strain B10/CA1) was compared with that of the parental strain in the weaned-rabbit diarrhea model. The mutant was totally deprived of virulence, although it colonized the intestine as efficiently as the parental strain did. This study points to a new pathogenic trait of EPEC strains, which is associated with the LEE locus and, possibly, with in vivo virulence.

A new cytolethal distending toxin (CDT) from Escherichia coli producing CNF2 blocks HeLa cell division in G2/M phase.

Escherichia coli strain 1404, isolated from a septicaemic calf, carries a transferable plasmid called pVir which codes for the cytotoxic necrotizing factor type 2 (CNF2). A 4h interaction between strain 1404 and HeLa cells induced the formation of giant mononucleated cells blocked in G2/M phase. Mating experiments between strain 1404 and a non-pathogenic recipient strain demonstrated that the factor(s) encoded by pVir mediated the cell-cycle arrest. A 3.3 kb DNA fragment isolated from a DNA bank of pVir was shown to code for the factor(s) causing the cell-cycle arrest. Nucleotide sequence analysis revealed the presence of three genes encoding proteins sharing significant amino acid homology with the cytolethal distending toxins (CDTs) previously isolated from E. coli, Campylobacter jejuni and Shigella dysenteriae. Southern hybridization experiments demonstrated that the pVir of other CNF2-producing E. coli strains contained sequences related to cdt. Although the amino acid sequences amongst CDT diverged significantly, the two other CDTs previously isolated from E. coli were also able to block the HeLa cell cycle. In conclusion, this study demonstrates the mode of action of CDT and will help us to elucidate the role of this emerging toxin family in microbial pathogenesis.

Interaction of Escherichia coli producing cytotoxic necrotizing factor with HeLa epithelial cells.

Cytotoxic necrotizing factors (CNF) constitute a group a cell-associated proteic toxins of 110-115 kDa produced by some clinical isolates of Escherichia coli from man and animals. Purified CNFs are known to exacerbate actin polymerization in exposed cells, a property that has been ascribed to their ability to modify rho a small GTP-protein involved in the regulation of the cytoskeleton. We speculated that, in spite of their lack of excretion in broth culture supernatants, CNF might be expressed upon direct interaction of organisms with infected cells. To test this hypothesis, we set up a model of interaction using epithelial cell line HeLa and the CNF1-producing strain BM2-1, which is adherent to Hela cells. An interaction of four hour duration triggers the progressive development of a dose-dependent cytopathic effect (CPE) with following characteristics: (1) intense cell enlargement with formation of a dense network of stress fibers, (2) inhibition of cell mitosis due to an irreversible block in G2/M transition phase, (3) nucleus swelling and fragmentation, and (4) cell death starting five days after infection. The three last features clearly differentiate CPE from the effect produced by CNF1 alone. In addition CPE, was not produced by cell-free culture supernatants nor abolished by an antiserum neutralizing CNF1. Tn5::PhoA insertion in the 3' end of cnf1 structural gene abolished CPE, which was not restored by trans complementation with cloned cnf1. These results demonstrate that CNF1-producing E. coli exert a specific pathogenic effect in HeLa cells, which is determined by cnf1 and at least one additional gene, located downstream cnf1.

Mitotic block and delayed lethality in HeLa epithelial cells exposed to Escherichia coli BM2-1 producing cytotoxic necrotizing factor type 1.

The cytopathic effect (CPE) of Escherichia coli producing cytotoxic necrotizing factor type 1 (CNF1) was investigated by using a human epithelial cell (HeLa) model of infection with CNF1-producing E. coli BM2-1. This strain was shown to bind loosely, but massively, to HeLa cells. A 4-h interaction between bacteria and eukaryotic cells triggered the delayed appearance of a progressive dose-dependent CPE characterized by (i) intense swelling of cells accompanied by the formation of a dense network of actin stress fibers, (ii) inhibition of cell division due to a complete block in the G2 phase of the cell cycle, and (iii) nucleus swelling and chromatin fragmentation. These alterations resulted in cell death starting about 5 days after interaction. The absence of multinucleation clearly distinguished the CPE from the effect produced by cell-free culture supernatants of infected cells nor prevented by a CNF1-neutralizing antiserum. Pathogenicity was completely abolished after Tn5::phoA insertion mutagenesis in the cnf-1 structural gene but not restored by trans complementation with a recombinant plasmid containing intact cnf-1 and its promoter. These results suggest that a gene downstream of cnf-1, essential to the induction of the CPE, was affected by the mutation. On the other hand, transformation of the wild-type strain BM2-1 with the same recombinant plasmid leads to a significant increase in both CNF1 activity and CPE, demonstrating the direct contribution of CNF1 to the CPE. In conclusion, the pathogenicity of E. coli BM2-1 for HeLa cells results from a complex interaction involving cnf-1 and associated genes and possibly requiring a preliminary step of binding of bacterial organisms to target cells.

Role of Adhesive Factor/Rabbit 2 in experimental enteropathogenic Escherichia coli O103 diarrhea of weaned rabbit.

The Adhesive Factor/Rabbit 2 (AF/R2) is found in Escherichia coli strains of serovar O103:K-:H2 and rhamnose-negative biovars isolated from weaned rabbits with diarrhea. This adhesin allows the colonization of the distal parts of the digestive tract, a first step leading to severe inflammatory diarrhea and death of the animals. In vitro, AF/R2 expression mediates diffuse adhesion of E. coli on HeLa cells, adhesion to ileal villi of newborn and weaned rabbits and the presence of a major 32 kDa subunit in bacterial surface extracts. In this work, we constructed TnphoA mutants of the prototype strain B10 and selected an isogenic clone that did not express the AF/R2 32 kDa subunit when grown in permissive conditions in vitro. The pathogenicity of the wild type strain and of the isogenic mutant was compared by oral inoculation to 35-day-old weaned rabbits. The mutant showed impaired colonization and a highly significant loss of pathogenicity. However, the occurrence of residual weight losses, and of diarrheas and mortalities in some inoculated rabbits suggest that pathogenicity of rabbit O103 enteropathogeniclike E. coli (EPEC-like) strains is due to multiple virulence factors and that other virulence traits remain to be found.