The cytolethal distending toxins (CDTs) are secreted virulence proteins produced by a number of bacterial pathogens, including Escherichia coli
, Haemophilus ducreyi
spp., Salmonella enterica
serovar Typhi, Actinobacillus actinomycetemcomitans
, Shigella dysenteriae
, and Helicobacter
). CDTs consist of the three subunits CdtA, CdtB, and CdtC and form a ternary complex (40
). CdtB shares conserved residues with the active sites of DNase I-like nucleases, and purified CdtB primarily shows single-strand nicking activity on coiled plasmid DNA and subsequently produces linear DNA in vitro (15
). Enzymatically active CdtB induces cell cycle arrest at the G2
/M phase, inhibits cell proliferation, and causes cellular enlargement in mammalian cells (28
). Biochemical analysis has also demonstrated that DNA damage checkpoint machineries and Rho-type GTPase function are involved in CdtB-induced cell cycle arrest and cellular enlargement (13
). However, the entire complement of genes required for the repair of CdtB-induced DNA lesions and also those leading to cell cycle arrest, cellular enlargement, and cell death have not been fully identified. Because Hassane et al. showed that CdtB is active in yeast (Saccharomyces cerevisiae
) cells similar to mammalian cells (19
), we used the yeast genome to further elucidate and characterize the components of the CdtB response pathway and to illustrate the consequences of CdtB activity in host cells.
Genome-wide deletion strains of yeast have now been used in many studies (33
), and genome-wide analyses have a number of advantages over their classical genetic counterparts, not only in terms of the greater ease in obtaining global results but also because of their far greater comprehensiveness. Hence, if all of the yeast deletion strains in a particular set are screened for desirable phenomena, the known genes identified in this screening will reveal novel features of the required functions. Moreover, the absence of specific genes can also disclose features of unrelated functions. The value of identifying absent genes in such screening approaches has not been highly emphasized in traditional genetics, even in genome-wide analysis, because these analyses need to be sufficiently systematic to verify that such an absence is not an artifact or due to leakage from the screening filters. In our present study, we have adopted a systematic transformation method that allows us to analyze each deletion strain one by one for the CdtB-induced growth phenotype. This method also allows us to generate a comprehensive series of results for genes required for the CdtB response, which illustrates a genome-wide view of host-pathogen interactions.
In comparison with the numerous previous studies of double-strand breaks (DSBs) (26
), little is currently known about the repair mechanisms for single-strand breaks (SSBs). Even if there is no direct evidence for SSB creation induced by CdtB expression in vivo, yeast genome-wide analysis will reveal the feature of CdtB-induced DNA lesions by comparison with the responses to the direct DSB that can be created by the ectopic expression of HO endonuclease in yeast (47
) and the other DNA damage. In the present study, we report the results of our genome-wide screen of genes required for CdtB response in yeast. We show here that evolutionally conserved mechanisms involving components of homologous recombination (HR), DNA replication, chromosome maintenance, and mRNA decay are required for the response to CdtB. The genes that we identified in this analysis also indicate that there are specific features of CdtB response that do not fully overlap with the components required for direct DSB and other DNA damage.