PerspectiveMicrobiology

Arresting Features of Bacterial Toxins

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Science  13 Oct 2000:
Vol. 290, Issue 5490, pp. 287-288
DOI: 10.1126/science.290.5490.287

Many bacterial pathogens make protein toxins that work in fascinating ways to disrupt the normal processes of host cells. These bacterial toxins are key factors in determining the outcome of infection and are among the most potent poisons known to humankind. Two recent reports, one by Lara-Tejero and Galán (1) on page 354 of this issue and the other by Elwell and Dreyfus (2) in a recent issue of Molecular Microbiology, provide another example of how these powerful weapons disrupt the host cell. The two studies demonstrate that a family of bacterial toxins called the cytolethal distending toxins (CDTs) are enzymes that attack DNA in the host cell nucleus. In contrast, almost all bacterial toxins that act inside host cells either destroy or modify host cell proteins.

The CDTs are secreted by a diverse group of bacterial pathogens, including several that are important causes of gastrointestinal illness (3). As the name implies, these toxins cause marked swelling and eventual death of many types of cultured mammalian cells. The two new studies now suggest how the CDTs cause arrest of host cells in G2 phase of the cell cycle—that is, after DNA replication in S phase but before the cell divides into two daughters during mitosis (4)—leading to cell destruction (see the figure).

Enzyme activities of toxin terrorists.

The CdtB subunit of CDT, a DNase, is imported into the nucleus where it attacks the DNA being replicated during S phase, activating the DNA damage response pathway. This pathway maintains Cdc2 (a key regulatory protein) in its inactive state, resulting in arrest of the host cell in G2 phase of the cell cycle. Continued biosynthesis by the arrested host cell leads to distension of the cytoplasm. DNA damage caused by CdtB results in chromatin fragmentation and eventual cell death. [Photographs of control and CDT-treated cells courtesy of Lara-Tejero and Galán]

The CDTs are multisubunit toxins composed of three proteins—CdtA, CdtB, and CdtC. To determine which of the three subunits is able to halt the cell cycle, Lara-Tejero and Galán (1) expressed each of the three CDT genes from the intestinal pathogen Campylobacter jejuni in mammalian cells. Whereas cells expressing CdtA and CdtC appeared normal, those expressing CdtB displayed drastic alterations in their nuclei. Microinjection of small amounts of purified CdtB subunit alone recapitulated all of the morphological changes typically associated with CDTs. The investigators used an updated algorithm to search protein databases to determine whether CdtB contained any motifs that have been found in other proteins. They spotted homology between the amino acid sequences of C. jejuni CDT and mammalian deoxyribonuclease I (DNase I). This finding was noted independently by Elwell and Dreyfus (2), who pointed out the similarity between the amino acid sequences of mammalian DNase I and Escherichia coli CdtB.

DNase I proteins—enzymes that cut DNA into smaller pieces—share conserved amino acid residues that are important for enzyme activity. Both groups (1, 2) tested mutant CdtB proteins containing altered conserved residues to see whether the toxins were still potent. Each mutation resulted in a substantial decrease in CdtB toxicity. Elwell and Dreyfus (2) correlated the decrease in toxicity with a concomitant decrease in DNase I activity in vitro. Meanwhile, Lara-Tejero and Galán (1) showed that CdtB became localized in the nucleus of toxin-treated cells, consistent with its proposed role as a DNase (1).

Identification of CDT as a DNase immediately suggests a model for how the toxin arrests the host cell in G2. Damage to the DNA induces cell cycle arrest by triggering signaling cascades that keep Cdc2, a key regulatory protein, in an inactive (phosphorylated) form (5) (see the figure). Damage to the DNA inflicted by CDT results in activation of a damage response pathway, and accumulation of inactive Cdc2 resulting in arrest of cells in G2 (4). Continued biosynthesis by these arrested cells may result in the characteristic distension of the cytoplasm associated with CDTs. DNA damage inflicted by CdtB results in chromatin fragmentation and eventual cell death.

Whereas DNA damage normally results in arrest of cells at either G1 (just before DNA replication) or G2 (just before cell division), CDT-treated cells invariably halt in G2 only. Lara-Tejero and Galán propose that CdtB damages DNA only when the DNA is in a vulnerable physical state, that is, during replication in S phase. Indeed, exposure of cells to CDT during DNA replication is required for arrest at the subsequent G2; exposure to CDT after DNA replication is complete allows cells to progress through mitosis and not to halt until the next G2 (see the figure) (4).

An unusual aspect of the CDT family is that its members are made by diverse sorts of bacteria. The only common feature of all known CDT-producing bacteria is that they infect epithelial cell layers, such as those comprising the gastrointestinal or genitourinary tract. Epithelial cells would be especially sensitive to the cell cycle-arresting activity of CDTs, because they continuously proliferate and differentiate as they migrate from deeper layers toward the epithelial surface, from which they are eventually shed. Disruption of normal epithelial cell turnover could lead to breakdown of the epithelial barrier, permitting easier access of bacteria and their secreted toxins to underlying tissues. Cells of the immune system are another potential target for CDTs because they proliferate in response to antigen. Supporting this hypothesis is the finding that certain CDTs inhibit proliferation of monocytes and lymphocytes (3, 6), potentially affecting both innate and acquired immunity.

Now that CDTs have been identified as DNases, many exciting avenues for investigation should open up. For example, a next step will be to identify the host cell or bacterial factors that regulate the DNase activity of CDTs or that deliver CdtB to the nucleus (perhaps with the help of the other two CDT subunits). Further amino acid mutation studies will allow rigorous analysis of the importance of CdtB's DNase activity in animal models of infection. With the finding that CDT family members are DNases, pathogenic bacteria have provided us with yet more tools to study basic biological processes in the eukaryotic cell—in this case, control of the cell cycle.

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