A ToxR-based two-hybrid system for the detection of periplasmic and cytoplasmic protein-protein interactions in Escherichia coli: minimal requirements for specific DNA binding and transcriptional activation

The Vibrio cholerae transcriptional regulator ToxR is anchored in the cytoplasmic membrane by a single transmembrane segment, its C-terminal domain facing the periplasm. Most of its N-terminal cytoplasmic domain shares sequence similarity with the winged helix-turn-helix (wHTH) motif of OmpR-like transcriptional regulators. In the heterologous host Escherichia coli ToxR activates transcription at the V.cholerae ctx promoter in a dimerization-dependent manner, which has led to its employment as a genetic indicator for protein-protein interactions. However, although offering a broader potential application range than other prokaryotic two-hybrid systems described to date, ToxR has so far only been used to study interactions between heterologous transmembrane segments or to monitor homodimerization of C-terminal fusion partners in the periplasm and the cytoplasm of E.coli. Here we show that the ToxR-system also allows the detection of heterodimerization in both cellular compartments of E.coli. In addition, to better understand ToxR's mode of action at ctx in E.coli, we have investigated the minimal requirements for its function as a transcriptional activator. We show that the wHTH motif of ToxR's N-terminal domain constitutes the minimal structural element required to activate transcription at ctx in E.coli when fused to a dimerizing protein modul

Longitudinal claudin gene expression analyses in canine mammary tissues and thereof derived primary cultures and cell lines

Human and canine mammary tumours show partial claudin expression deregulations. Further, claudins have been used for directed therapeutic approaches. However, the development of claudin targeting approaches requires stable claudin expressing cell lines. This study reports the establishment and characterisation of canine mammary tissue derived cell lines, analysing longitudinally the claudin-1, -3, -4 and -7 expressions in original tissue samples, primary cultures and developed cell lines. Primary cultures were derived from 17 canine mammary tissues: healthy, lobular hyperplasia, simple adenoma, complex adenoma, simple tubular carcinoma, complex carcinoma, carcinoma arising in a benign mixed tumour and benign mixed tissue. Cultivation was performed, if possible, until passage 30. Claudin mRNA and protein expressions were analysed by PCR, QuantiGene Plex Assay, immunocytochemistry and immunofluorescence. Further, cytokeratin expression was analysed immunocytochemically. Cultivation resulted in 11 established cell lines, eight showing epithelial character. In five of the early passages the claudin expressions decreased compared to the original tissues. In general, claudin expressions were diminished during cultivation. Three cell lines kept longitudinally claudin, as well as epithelial marker expressions, representing valuable tools for the development of claudin targeted anti-tumour therapies

PqsBC, a condensing enzyme in the biosynthesis of the Pseudomonas aeruginosa quinolone signal: crystal structure, inhibition, and reaction mechanism

Pseudomonas aeruginosa produces a number of alkylquinolone-type secondary metabolites best known for their antimicrobial effects and involvement in cell-cell communication. In the alkylquinolone biosynthetic pathway, the β-ketoacyl-(acyl carrier protein) synthase III (FabH)-like enzyme PqsBC catalyzes the condensation of octanoyl-coenzyme A and 2-aminobenzoylacetate (2-ABA) to form the signal molecule 2-heptyl-4(1H)-quinolone. PqsBC, a potential drug target, is unique for its heterodimeric arrangement and an active site different from that of canonical FabH-like enzymes. Considering the sequence dissimilarity between the subunits, a key question was how the two subunits are organized with respect to the active site. In this study, the PqsBC structure was determined to a 2 Å resolution, revealing that PqsB and PqsC have a pseudo-2-fold symmetry that unexpectedly mimics the FabH homodimer. PqsC has an active site composed of Cys-129 and His-269, and the surrounding active site cleft is hydrophobic in character and approximately twice the volume of related FabH enzymes that may be a requirement to accommodate the aromatic substrate 2-ABA. From physiological and kinetic studies, we identified 2-aminoacetophenone as a pathway-inherent competitive inhibitor of PqsBC, whose fluorescence properties could be used for in vitro binding studies. In a time-resolved setup, we demonstrated that the catalytic histidine is not involved in acyl-enzyme formation, but contributes to an acylation-dependent increase in affinity for the second substrate 2-ABA. Introduction of Asn into the PqsC active site led to significant activity toward the desamino substrate analog benzoylacetate, suggesting that the substrate 2-ABA itself supplies the asparagine-equivalent amino function that assists in catalysis

Synthesis and biological activity of methylated derivatives of the Pseudomonas metabolites HHQ, HQNO and PQS

Selectively methylated analogues of naturally occurring 2-heptyl-4(1H)-quinolones, which are alkaloids common within the Rutaceae family and moreover are associated with quorum sensing and virulence of the human pathogen Pseudomonas aeruginosa, have been prepared. While the synthesis by direct methylation was successful for 3-unsubstituted 2-heptyl-4(1H)-quinolones, methylated derivatives of the Pseudomonas quinolone signal (PQS) were synthesized from 3-iodinated quinolones by methylation and iodine–metal exchange/oxidation. The two N- and O-methylated derivatives of the PQS showed strong quorum sensing activity comparable to that of PQS itself. Staphylococcus aureus, another pathogenic bacterium often co-occurring with P. aeruginosa especially in the lung of cystic fibrosis patients, was inhibited in planktonic growth and cellular respiration by the 4-O-methylated derivatives of HQNO and HHQ, respectively

Chemical Modification and Detoxification of the <i>Pseudomonas aeruginosa</i> Toxin 2‑Heptyl-4-hydroxyquinoline <i>N</i>‑Oxide by Environmental and Pathogenic Bacteria

2-Heptyl-4-hydroxyquinoline <i>N</i>-oxide (HQNO), a major secondary metabolite and virulence factor produced by the opportunistic pathogen <i>Pseudomonas aeruginosa</i>, acts as a potent inhibitor of respiratory electron transfer and thereby affects host cells as well as microorganisms. In this study, we demonstrate the previously unknown capability of environmental and pathogenic bacteria to transform and detoxify this compound. Strains of <i>Arthrobacter</i> and <i>Rhodococcus</i> spp. as well as <i>Staphylococcus aureus</i> introduced a hydroxyl group at C-3 of HQNO, whereas <i>Mycobacterium abscessus</i>, <i>M. fortuitum</i>, and <i>M. smegmatis</i> performed an <i>O</i>-methylation, forming 2-heptyl-1-methoxy-4-oxoquinoline as the initial metabolite. <i>Bacillus</i> spp. produced the glycosylated derivative 2-heptyl-1-(β-d-glucopyranosydyl)-4-oxoquinoline. Assaying the effects of these metabolites on cellular respiration and on quinol oxidase activity of membrane fractions revealed that their EC₅₀ values were up to 2 orders of magnitude higher than that of HQNO. Furthermore, cellular levels of reactive oxygen species were significantly lower in the presence of the metabolites than under the influence of HQNO. Therefore, the capacity to transform HQNO should lead to a competitive advantage against <i>P. aeruginosa.</i> Our findings contribute new insight into the metabolic diversity of bacteria and add another layer of complexity to the metabolic interactions which likely contribute to shaping polymicrobial communities comprising <i>P. aeruginosa.</i

A Comparison of Fresh Frozen vs. Formalin-Fixed, Paraffin-Embedded Specimens of Canine Mammary Tumors via Branched-DNA Assay

Mammary neoplasms are the tumors most affecting female dogs and women. Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable source of archived biological material. Fresh frozen (FF) tissue is considered ideal for gene expression analysis. However, strategies based on FFPE material offer several advantages. Branched-DNA assays permit a reliable and fast workflow when analyzing gene expression. The aim of this study was to assess the comparability of the branched-DNA assay when analyzing certain gene expression patterns between FF and FFPE samples in canine mammary tumors. RNA was isolated from 109 FFPE samples and from 93 FF samples of different canine mammary tissues. Sixteen (16) target genes (Tp53; Myc; HMGA1; Pik3ca; Mcl1; MAPK3; FOXO3; PTEN; GATA4; PFDN5; HMGB1; MAPK1; BRCA2; BRCA1; HMGA2; and Her2) were analyzed via branched-DNA assay (b-DNA). ACTB, GAPDH, and HPRT1 were used as data normalizers. Overall, the relative gene expression of the two different origins of samples showed an agreement of 63%. Still, care should be taken, as FFPE specimens showed lower expression of the analyzed targets when compared to FF samples. The fact that the gene expression in FFPE proved to be lower than in FF specimens is likely to have been caused by the effect of storage time. ACTB had the best performance as a data normalizer

Prevalence of the Prefoldin Subunit 5 Gene Deletion in Canine Mammary Tumors.

A somatic deletion at the proximal end of canine chromosome 27 (CFA27) was recently reported in 50% of malignant mammary tumors. This region harbours the tumor suppressor gene prefoldin subunit 5 (PFDN5) and the deletion correlated with a higher Ki-67 score. PFDN5 has been described to repress c-MYC and is, therefore, a candidate tumor-suppressor and cancer-driver gene in canine mammary cancer. Aim of this study was to confirm the recurrent deletion in a larger number of tumors.Droplet digital PCR for PFDN5 was performed in DNA from 102 malignant, 40 benign mammary tumors/dysplasias, 11 non-neoplastic mammary tissues and each corresponding genomic DNA from leukocytes. The copy number of PFDN5 was normalized to a reference amplicon on canine chromosome 32 (CFA32). Z-scores were calculated, based on Gaussian distributed normalized PFDN5 copy numbers of the leukocyte DNA. Z-scores ≤ -3.0 in tissue were considered as being indicative of the PFDN5 deletion and called as such. The Ki-67 proliferation index was assessed in a subset of 79 tissue samples by immunohistochemistry.The deletion was confirmed in 24% of all malignant tumors, detected in only 7.5% of the benign tumors and was not present in any normal mammary tissue sample. The subgroup of solid carcinomas (n = 9) showed the highest frequency of the deletion (67%) and those malignomas without microscopical high fraction of benign tissue (n = 71) had a 32% frequency (p<0.01 vs. benign samples). The Ki-67 score was found to be significantly higher (p<0.05) in the PFDN5-deleted group compared to malignant tumors without the deletion.A somatic deletion of the PFDN5 gene is recurrently present in canine mammary cancer, supporting a potential role in carcinogenesis. The association of this deletion with higher Ki-67 indicates an increased proliferation rate and thus a link to tumor aggressiveness can be hypothesized. The confirmation of earlier results warrants further studies on PFDN5 as cancer-driver gene