Notch signaling in CD66+ cells drives the progression of human cervical cancers.

Human epithelial tumor progression and metastasis involve cellular invasion, dissemination in the vasculature, and regrowth at metastatic sites. Notch signaling has been implicated in metastatic progression but its roles have yet to be fully understood. Here we report the important role of Notch signaling in maintaining cells expressing the carcinoembryonic antigen cell adhesion molecule CEACAM (CD66), a known mediator of metastasis. CD66 and Notch1 were studied in clinical specimens and explants of human cervical cancer, including specimens grown in a pathophysiologically relevant murine model. Gene expression profiling of CD66(+) cells from primary tumors showed enhanced features of Notch signaling, metastasis, and stemness. Significant differences were also seen in invasion, colony formation, and tumor forming efficiency between CD66(+) and CD66(-) cancer cells. Notably, CD66(+) cells showed a marked sensitivity to a Notch small molecule inhibitor. In support of studies in established cell lines, we documented the emergence of a tumorigenic CD66(+) cell subset within a metastatic lesion-derived cervical-cancer cell line. Similar to primary cancers, CD66 expression in the cell line was blocked by chemical and genetic inhibitors of ligand-dependent nuclear Notch signaling. Collectively, our work on the oncogenic properties of CD66(+) cells in epithelial cancers provides insights into the nature of tumor progression and offers a mechanistic rationale to inhibit the Notch signaling pathway as a generalized therapeutic strategy to treat metastatic cancers.

Substrate specificity-conferring regions of the nonribosomal peptide synthetase adenylation domains involved in albicidin pathotoxin biosynthesis are highly conserved within the species Xanthomonas albilineans.

Albicidin is a pathotoxin produced by Xanthomonas albilineans, a xylem-invading pathogen that causes leaf scald disease of sugarcane. Albicidin is synthesized by a nonribosomal pathway via modular polyketide synthase and nonribosomal peptide synthetase (NRPS) megasynthases, and NRPS adenylation (A) domains are responsible for the recognition and activation of specific amino acid substrates. DNA fragments (0.5 kb) encoding the regions responsible for the substrate specificities of six albicidin NRPS A domains from 16 strains of X. albilineans representing the known diversity of this pathogen were amplified and sequenced. Polymorphism analysis of these DNA fragments at different levels (DNA, protein, and NRPS signature) showed that these pathogenicity loci were highly conserved. The conservation of these loci most likely reflects purifying selective pressure, as revealed by a comparison with the variability of nucleotide and amino acid sequences of two housekeeping genes (atpD and efp) of X. albilineans. Nevertheless, the 16 strains of X. albilineans were differentiated into several groups by a phylogenetic analysis of the nucleotide sequences corresponding to the NRPS A domains. One of these groups was representative of the genetic diversity previously found within the pathogen by random fragment length polymorphism and amplified fragment length polymorphism analyses. This group, which differed by three single synonymous nucleotide mutations, contained only four strains of X. albilineans that were all involved in outbreaks of sugarcane leaf scald. The amount of albicidin produced in vitro in agar and liquid media varied among the 16 strains of X. albilineans. However, no relationship among the amount of albicidin produced in vitro and the pathotypes and genetic diversity of the pathogen was found. The NRPS loci contributing to the synthesis of the primary structure of albicidin apparently are not involved in the observed pathogenicity differences among strains of X. albilineans.

Heterologous production of albicidin: a promising approach to overproducing and characterizing this potent inhibitor of DNA gyrase.

The phytotoxin and polyketide antibiotic albicidin produced by Xanthomonas albilineans is a highly potent DNA gyrase inhibitor. Low yields of albicidin production have slowed studies of its chemical structure. Heterologous expression of albicidin biosynthetic genes in X. axonopodis pv. vesicatoria resulted in a sixfold increase in albicidin production, offering promising strategies for engineering overproduction.

Xanthomonas albilineans HtpG is required for biosynthesis of the antibiotic and phytotoxin albicidin.

Xanthomonas albilineans, the causal agent of leaf scald disease of sugarcane, produces a highly potent polyketide-peptide antibiotic and phytotoxin called albicidin. Previous studies established the involvement of a large cluster of genes in the biosynthesis of this toxin. We report here the sub-cloning and sequencing of an additional gene outside of the main cluster and essential for albicidin biosynthesis. This gene encodes a 634-amino-acid protein that shows high identity with the Escherichia coli heat shock protein HtpG. Complementation studies of X. albilineans Tox- mutants confirmed the requirement of htpG for albicidin biosynthesis and revealed functional interchangeability between E. coli and X. albilineans htpG genes. HtpG was co-localised with albicidin in the cellular membrane, i.e., the cellular fraction where the toxin is most probably biosynthesised. Here we show the requirement of an HtpG protein for the biosynthesis of a polyketide-peptide antibiotic.

Albicidin pathotoxin produced by Xanthomonas albilineans is encoded by three large PKS and NRPS genes present in a gene cluster also containing several putative modifying, regulatory, and resistance genes.

Xanthomonas albilineans, which causes leaf scald disease of sugarcane, produces a highly potent pathotoxin called albicidin. We report here sequencing and homology analysis of the major gene cluster, XALB1 (55,839 bp), and a second, smaller region, XALB2 (2,986 bp), involved in albicidin biosynthesis. XALB1 contains 20 open reading frames, including i) three large genes with a modular architecture characteristic of polyketide synthases (PKSs) and nonribosomal peptide synthases (NRPSs) and ii) several putative modifying, regulatory, and resistance genes. Sequencing and complementation studies of six albicidin-defective mutants enabled us to confirm the involvement of the three PKS and NRPS genes encoded by XALB1 in albicidin production. XALB2 contains only one gene that is required for post-translational activation of PKS and NRPS enzymes, confirming the involvement of these enzymes in albicidin biosynthesis. In silico analysis of these three PKS or NRPS enzymes allowed us to propose a model for the albicidin backbone assembly and to gain insight into the structural features of this pathotoxin. This is the first description of a complete mixed PKS-NRPS gene cluster for toxin production in the genus Xanthomonas.