Carol L. Pickett

The cytolethal distending toxin family

Cytolethal distending toxins are produced by a small but diverse group of bacterial pathogens. This newly discovered toxin family can cause a variety of mammalian cells to become irreversibly blocked in the G2 phase of the cell cycle. How this novel effect is accomplished is unknown but the study of these fascinating toxins promises to reveal new methods of host-pathogen interaction.

Interactions of Campylobacter jejuni Cytolethal Distending Toxin Subunits CdtA and CdtC with HeLa Cells

ABSTRACT Campylobacter jejuni produces a toxin, called cytolethal distending toxin (CDT), which causes direct DNA damage leading to invocation of DNA damage checkpoint pathways. The affected cells arrest in G1 or G2 and eventually die. CDT consists of three protein subunits, CdtA, CdtB, and CdtC, with CdtB recently identified as a nuclease. However, little is known about the functions of CdtA or CdtC. In this work, enzyme-linked immunosorbent assay-based experiments were used to show, for the first time, that both CdtA and CdtC bound with specificity to the surface of HeLa cells, whereas CdtB did not. Varying the order of the addition of subunits for reconstitution of the holotoxin had no effect on activity. In addition, mutants containing deletions of conserved regions of CdtA and CdtC were able to bind to the surface of HeLa cells but were not able to participate in holotoxin assembly. Finally, both Cdt mutant subunits were able to effectively compete with CDT holotoxin in the HeLa cell binding assay.

Campylobacter jejuni Cytolethal Distending Toxin Promotes DNA Repair Responses in Normal Human Cells

ABSTRACT Cytolethal distending toxin (CDT) is a multisubunit protein found in various gram-negative bacterial pathogens of humans which is thought to cause cell death by direct DNA damage of host cells. We sought to determine if a cellular response to DNA damage could be detected by exogenous addition of the holotoxin. Exogenous addition of the Campylobacter jejuni 81-176 CDT to primary human fibroblasts resulted in formation of Rad50 foci, which are formed around double-stranded-DNA breaks. Moreover, such foci are formed in both proliferating and nonproliferating cells that are treated with C. jejuni CDT. Fibroblasts that were intoxicated and later stimulated to proliferate failed to divide and remained arrested in the G1 phase of the cell cycle.

Cytolethal Distending Toxin Demonstrates Genotoxic Activity in a Yeast Model

ABSTRACT Cytolethal distending toxins (CDTs) are multisubunit proteins produced by a variety of bacterial pathogens that cause enlargement, cell cycle arrest, and apoptosis in mammalian cells. While their function remains uncertain, recent studies suggest that they can act as intracellular DNases in mammalian cells. Here we establish a novel yeast model for understanding CDT-associated disease. Expression of the CdtB subunit in yeast causes a G2/M arrest, as seen in mammalian cells. CdtB toxicity is not circumvented in yeast genetically altered to lack DNA damage checkpoint control or that constitutively promote cell cycle progression via mutant Cdk1, because CdtB causes a permanent type of damage that results in loss of viability. Finally, we establish that CDTs are likely to be potent genotoxins, as indicated by in vivo degradation of chromosomal DNA associated with expression of CdtB—suggesting that the varied distribution of CDT in bacteria implicates many human pathogens as possessors of genotoxic activity.

Genetic organization and enzymatic activity of a superoxide dismutase from the microaerophilic human pathogen, Helicobacter pylori

Helicobacter pylori (Hp) is a microaerobic human pathogen that has been implicated as a factor in the development of chronic type-B gastritis, gastric ulcers and gastric carcinoma. The enzyme superoxide dismutase (SOD), a major defense mechanism against oxidative damage, catalyzes the breakdown of superoxide radicals to hydrogen peroxide and dioxygen. A search for sod genes in Hp, employing PCR, revealed that this bacterium contained at least one sod gene. We cloned and sequenced a sod from this organism and determined that the deduced protein encoded by this gene was most similar to an iron SOD (FeSOD). Northern blot and primer extension analysis of Hp RNA showed that the cloned gene is monocistronic and is probably transcribed from a sigma 70-like promoter. Assays for SOD activities, accompanied by inhibition studies, demonstrated that Hp produces an FeSOD. No other SOD activities were seen.

Patterns of Variations in Escherichia coli Strains That Produce Cytolethal Distending Toxin

ABSTRACT A collection of 20 Escherichia coli strains that produce cytolethal distending toxin (CDT) were analyzed for their virulence-associated genes. All of these strains were serotyped, and multiplex PCR analysis was used to ascertain the presence of genes encoding other virulence factors, including Shiga toxin, intimin, enterohemolysin, cytotoxic necrotizing factor type 1 (CNF1) and CNF2, heat-stable toxin, and heat-labile toxin. These CDT-producing strains possessed various combinations of known virulence genes, some of which have not been noted before. Partial cdtB sequences were obtained from 10 of these strains, and their predicted CdtB sequences were compared to known E. coli CdtB sequences; some of the sequences were identical to known CdtB sequences, but two were not. PCR primers based on sequence differences between the known cdt sequences were tested for their ability to detect CDT producers and to determine CDT type. Correlations between the type of CDT produced, the presence of other virulence properties, and overall strain relatedness revealed that the CDT producers studied here can be divided into three general groups, with distinct differences in CDT type and in their complement of virulence-associated genes.

Reduced synthesis of the Ybt siderophore or production of aberrant Ybt-like molecules activates transcription of yersiniabactin genes in Yersinia pestis

Synthesis of the siderophore yersiniabactin (Ybt) proceeds by a mixed nonribosomal peptide synthetase/polyketide synthase mechanism. Transcription of ybt genes encoding biosynthetic and transport functions is repressed under excess iron conditions by Fur, but is also activated by Ybt via the transcriptional regulator YbtA. While mutations in most biosynthetic genes and ybtA negate transcription activation from the regulated promoters, three biosynthetic mutations do not reduce this transcriptional activation. Here we show that two of these mutants, one lacking the putative type II thioesterase (TE) YbtT and the other with a mutation in the TE domain of HMWP1, produce reduced levels of authentic Ybt that are capable of signalling activity. Alanine substitutions in two residues of YbtT that are essential for catalytic activity in other type II TEs reduced the ability of Yersinia pestis to grow under iron-chelated conditions. The third mutant, which lacks the salicylate synthase YbtS, did not make authentic Ybt but did produce a signalling molecule. Finally, a Δpgm strain of Y. pestis, which lacks essential Ybt biosynthetic genes, also produced a signalling molecule that can activate transcription of ybt genes. The non-Ybt signal molecules from these two mutants are likely separate compounds. While these compounds are not biologically relevant to normal Ybt regulation, a comparison of the structures of Ybt and other signalling molecules will help in determining the chemical structures recognized as a Ybt signal.

Genetic organization of the region upstream from the Campylobacter jejuni flagellar gene flhA

Campylobacter jejuni (Cj) is a Gram-bacterium that causes a diarrheal disease in humans. A Cj homolog of the LcrD/FlhA family of proteins was recently described [Miller et al., Infect. Immun. 61 (1993) 2930-2936]. This family includes proteins that are involved in flagellar biogenesis, such as the Cj FlhA protein, but also includes proteins found in invasive pathogens, such as the Yersinia pestis LcrD protein, that play a role in the regulation and/or secretion of virulence-related proteins. Hybridization studies indicated that both the flhA gene and upstream DNA are present in several bacterial species closely related to Cj, including C. fetus, C. lari, C. upsaliensis and C. hyointestinalis. The presence of a second flhA/lcrD homolog was not detected in Cj, indicating that a a separate homolog involved in secretion of virulence proteins may not be present. The 4-kb region immediately upstream from Cj flhA was analyzed. Three open reading frames (ORFs) were found: a 408-nucleotide (nt) gene encoding a homolog of proteins present in Escherichia coli and Desulfovibrio vulgaris, but of unknown function, a 266-nt rpsO gene and a 2823-nt gene encoding a homolog of the Bacillus subtilis SpoIIIE protein. The Cj SpoIIIE homolog had 53% similar or identical amino acids when compared to the B. subtilis protein, and like the B. subtilis protein contained a nt-binding domain and potential transmembrane (TM) regions. All three ORFs were expressed in E. coli minicells, apparently from their own promoters.(ABSTRACT TRUNCATED AT 250 WORDS)

The Trophic Life Cycle Stage of the Opportunistic Fungal Pathogen Pneumocystis murina Hinders the Ability of Dendritic Cells To Stimulate CD4+ T Cell Responses

ABSTRACT The life cycle of the opportunistic fungal pathogen Pneumocystis murina consists of a trophic stage and an ascus-like cystic stage. Infection with the cyst stage induces proinflammatory immune responses, while trophic forms suppress the cytokine response to multiple pathogen-associated molecular patterns (PAMPs), including β-glucan. A targeted gene expression assay was used to evaluate the dendritic cell response following stimulation with trophic forms alone, with a normal mixture of trophic forms and cysts, or with β-glucan. We demonstrate that stimulation with trophic forms downregulated the expression of multiple genes normally associated with the response to infection, including genes encoding transcription factors. Trophic forms also suppressed the expression of genes related to antigen processing and presentation, including the gene encoding the major histocompatibility complex (MHC) class II transactivator, CIITA. Stimulation of dendritic cells with trophic forms, but not a mixture of trophic forms and cysts, reduced the expression of MHC class II and the costimulatory molecule CD40 on the surface of the cells. These defects in the expression of MHC class II and costimulatory molecules corresponded with a reduced capacity for trophic form-loaded dendritic cells to stimulate CD4+ T cell proliferation and polarization. These data are consistent with the delayed innate and adaptive responses previously observed in immunocompetent mice inoculated with trophic forms compared to responses in mice inoculated with a mixture of trophic forms and cysts. We propose that trophic forms broadly inhibit the ability of dendritic cells to fulfill their role as antigen-presenting cells.