Targeting of Regulators as a Promising Approach in the Search for Novel Antimicrobial Agents

Since the discovery of penicillin in the first half of the last century, antibiotics have become the pillars of modern medicine for fighting bacterial infections. However, pathogens resistant to antibiotic treatment have increased in recent decades, and efforts to discover new antibiotics have decreased. As a result, it is becoming increasingly difficult to treat bacterial infections successfully, and we look forward to more significant efforts from both governments and the scientific community to research new antibacterial drugs. This perspective article highlights the high potential of bacterial transcriptional and posttranscriptional regulators as targets for developing new drugs. We highlight some recent advances in the search for new compounds that inhibit their biological activity and, as such, appear very promising for treating bacterial infections

Feeling the heat: The campylobacter jejuni HrcA transcriptional repressor is an intrinsic protein thermosensor

The heat-shock response, a universal protective mechanism consisting of a transcriptional reprogramming of the cellular transcriptome, results in the accumulation of proteins which coun-teract the deleterious effects of heat-stress on cellular polypeptides. To quickly respond to thermal stress and trigger the heat-shock response, bacteria rely on different mechanisms to detect temperature variations, which can involve nearly all classes of biological molecules. In Campylobacter jejuni the response to heat-shock is transcriptionally controlled by a regulatory circuit involving two re-pressors, HspR and HrcA. In the present work we show that the heat-shock repressor HrcA acts as an intrinsic protein thermometer. We report that a temperature upshift up to 42°C negatively affects HrcA DNA-binding activity to a target promoter, a condition required for de-repression of regu-lated genes. Furthermore, we show that this impairment of HrcA binding at 42°C is irreversible in vitro, as DNA-binding was still not restored by reversing the incubation temperature to 37°C. On the other hand, we demonstrate that the DNA-binding activity of HspR, which controls, in combi-nation with HrcA, the transcription of chaperones’ genes, is unaffected by heat-stress up to 45°C, portraying this master repressor as a rather stable protein. Additionally, we show that HrcA binding activity is enhanced by the chaperonin GroE, upon direct protein–protein interaction. In conclu-sion, the results presented in this work establish HrcA as a novel example of intrinsic heat-sensing transcriptional regulator, whose DNA-binding activity is positively modulated by the GroE chap-eronin

Changes in microphytobenthos fluorescence over a tidal cycle: implications for sampling designs

Intertidal microphytobenthos (MPB) are important primary producers and provide food for herbivores in soft sediments and on rocky shores. Methods of measuring MPB biomass that do not depend on the time of collection relative to the time of day or tidal conditions are important in any studies that need to compare temporal or spatial variation, effects of abiotic factors or activity of grazers. Pulse amplitude modulated (PAM) fluorometry is often used to estimate biomass of MPB because it is a rapid, non-destructive method, but it is not known how measures of fluorescence are altered by changing conditions during a period of low tide. We investigated this experimentally using in situ changes in minimal fluorescence (F) on a rocky shore and on an estuarine mudflat around Sydney (Australia), during low tides. On rocky shores, the time when samples are taken during low tide had little direct influence on measures of fluorescence as long as the substratum is dry. Wetness from wave-splash, seepage from rock pools, run-off, rainfall, etc., had large consequences for any comparisons. On soft sediments, fluorescence was decreased if the sediment dried out, as happens during low-spring tides on particularly hot and dry days. Surface water affected the response of PAM and therefore measurements used to estimate MPB, emphasising the need for care to ensure that representative sampling is done during low tide

A convenient and robust in vivo reporter system to monitor gene expression in the human pathogen helicobacter pylori

Thirty years of intensive research have significantly contributed to our understanding of Helicobacter pylori biology and pathogenesis. However, the lack of convenient genetic tools, in particular the limited effectiveness of available reporter systems, has notably limited the toolbox for fundamental and applied studies. Here, we report the construction of a bioluminescent H. pylori reporter system based on the Photorhabdus luminescens luxCDABE cassette. The system is constituted of a promoterless lux acceptor strain in which promoters and sequences of interest can be conveniently introduced by double homologous recombination of a suicide transformation vector. We validate the robustness of this new lux reporter system in noninvasive in vivo monitoring of dynamic transcriptional responses of inducible as well as repressible promoters and demonstrate its suitability for the implementation of genetic screens in H. pylori. © 2012, American Society for Microbiology

Protecting the environment through insect farming as a means to produce protein for use as livestock, poultry, and aquaculture feed

Securing protein for the approximate 10 billion humans expected to inhabit our planet by 2050 is a major priority for the global community. Evidence has accrued over the past 30 years that strongly supports and justifies the sustainable use of insects as a means to produce protein products as feed for pets, livestock, poultry, and aquacultured species. Researchers and entrepreneurs affiliated with universities and industries, respectively, from 18 nations distributed across North and South America, Europe, Asia, Africa and Australia contributed to the development of this article, which is an indication of the global interest on this topic. A brief overview of insects as feed for the aquaculture industry along with a review of the black soldier fly, Hermetia illucens (Diptera: Stratiomyidae), as a model for such systems is provided

A dualistic model of primary anal canal adenocarcinoma with distinct cellular origins, etiologies, inflammatory microenvironments and mutational signatures: implications for personalised medicine.

Primary adenocarcinoma of the anal canal is a rare and aggressive gastrointestinal disease with unclear pathogenesis. Because of its rarity, no clear clinical practice guideline has been defined and a targeted therapeutic armamentarium has yet to be developed. The present article aimed at addressing this information gap by in-depth characterising the anal glandular neoplasms at the histologic, immunologic, genomic and epidemiologic levels. In this multi-institutional study, we first examined the histological features displayed by each collected tumour (n = 74) and analysed their etiological relationship with human papillomavirus (HPV) infection. The intratumoural immune cell subsets (CD4, CD8, Foxp3), the expression of immune checkpoints (PD-1, PD-L1), the defect in mismatch repair proteins and the mutation analysis of multiple clinically relevant genes in the gastrointestinal cancer setting were also determined. Finally, the prognostic significance of each clinicopathological variable was assessed. Phenotypic analysis revealed two region-specific subtypes of anal canal adenocarcinoma. The significant differences in the HPV status, density of tumour-infiltrating lymphocytes, expression of immune checkpoints and mutational profile of several targetable genes further supported the separation of these latter neoplasms into two distinct entities. Importantly, anal gland/transitional-type cancers, which poorly respond to standard treatments, displayed less mutations in downstream effectors of the EGFR signalling pathway (i.e., KRAS and NRAS) and demonstrated a significantly higher expression of the immune inhibitory ligand-receptor pair PD-1/PD-L1 compared to their counterparts arising from the colorectal mucosa. Taken together, the findings reported in the present article reveal, for the first time, that glandular neoplasms of the anal canal arise by HPV-dependent or independent pathways. These etiological differences leads to both individual immune profiles and mutational landscapes that can be targeted for therapeutic benefits

Regulatory (pan-)genome of an obligate intracellular pathogen in the PVC superphylum.

Like other obligate intracellular bacteria, the Chlamydiae feature a compact regulatory genome that remains uncharted owing to poor genetic tractability. Exploiting the reduced number of transcription factors (TFs) encoded in the chlamydial (pan-)genome as a model for TF control supporting the intracellular lifestyle, we determined the conserved landscape of TF specificities by ChIP-Seq (chromatin immunoprecipitation-sequencing) in the chlamydial pathogen Waddlia chondrophila. Among 10 conserved TFs, Euo emerged as a master TF targeting >100 promoters through conserved residues in a DNA excisionase-like winged helix-turn-helix-like (wHTH) fold. Minimal target (Euo) boxes were found in conserved developmentally-regulated genes governing vertical genome transmission (cytokinesis and DNA replication) and genome plasticity (transposases). Our ChIP-Seq analysis with intracellular bacteria not only reveals that global TF regulation is maintained in the reduced regulatory genomes of Chlamydiae, but also predicts that master TFs interpret genomic information in the obligate intracellular α-proteobacteria, including the rickettsiae, from which modern day mitochondria evolved

(Re)-definition of the holo- and apo-Fur direct regulons of Helicobacter pylori

: Iron homeostasis is a critical process for living organisms because this metal is an essential co-factor for fundamental biochemical activities, like energy production and detoxification, albeit its excess quickly leads to cell intoxication. The protein Fur (ferric uptake regulator) controls iron homeostasis in bacteria by switching from its apo- to holo-form as a function of the cytoplasmic level of ferrous ions, thereby modulating gene expression. The Helicobacter pylori HpFur protein has the rare ability to operate as a transcriptional commutator; apo- and holo-HpFur function as two different repressors with distinct DNA binding recognition properties for specific sets of target genes. Although the regulation of apo- and holo-HpFur in this bacterium has been extensively investigated, we propose a genome-wide redefinition of holo-HpFur direct regulon in H. pylori by integration of RNA-seq and ChIP-seq data, and a large extension of the apo-HpFur direct regulon. We show that in response to iron availability, new coding sequences, non-coding RNAs, toxin-antitoxin systems, and transcripts within open reading frames are directly regulated by apo- or holo-HpFur. These new targets and the more thorough validation and deeper characterization of those already known provide a complete and updated picture of the direct regulons of this two-faced transcriptional regulator

Paradigm Shift in Gastric Cancer Prevention: Harnessing the Potential of Aristolochia olivieri Extract

Gastric cancer, particularly adenocarcinoma, is a significant global health concern. Environmental risk factors, such as Helicobacter pylori infection and diet, play a role in its development. This study aimed to characterize the chemical composition and evaluate the in vitro antibacterial and antitumor activities of an Aristolochia olivieri Colleg. ex Boiss. Leaves’ methanolic extract (AOME). Additionally, morphological changes in gastric cancer cell lines were analyzed. AOME was analyzed using HPLC-MS/MS, and its antibacterial activity against H. pylori was assessed using the broth microdilution method. MIC and MBC values were determined, and positive and negative controls were included in the evaluation. Anticancer effects were assessed through in vitro experiments using AGS, KATO-III, and SNU-1 cancer cell lines. The morphological changes were examined through SEM and TEM analyses. AOME contained several compounds, including caffeic acid, rutin, and hyperoside. The extract displayed significant antimicrobial effects against H. pylori, with consistent MIC and MBC values of 3.70 ± 0.09 mg/mL. AOME reduced cell viability in all gastric cancer cells in a dose- and time-dependent manner. Morphological analyses revealed significant ultrastructural changes in all tumor cell lines, suggesting the occurrence of cellular apoptosis. This study demonstrated that AOME possesses antimicrobial activity against H. pylori and potent antineoplastic properties in gastric cancer cell lines. AOME holds promise as a natural resource for innovative nutraceutical approaches in gastric cancer management. Further research and in vivo studies are warranted to validate its potential clinical applications

Synergic Activity of FGFR2 and MEK Inhibitors in the Treatment of FGFR2-Amplified Cancers of Unknown Primary

Patients with cancer of unknown primary (CUP) carry the double burden of an aggressive disease and reduced access to therapies. Experimental models are pivotal for CUP biology investigation and drug testing. We derived two CUP cell lines (CUP#55 and #96) and corresponding patient-derived xenografts (PDXs), from ascites tumor cells. CUP cell lines and PDXs underwent histological, immune-phenotypical, molecular, and genomic characterization confirming the features of the original tumor. The tissue-of-origin prediction was obtained from the tumor microRNA expression profile and confirmed by single-cell transcriptomics. Genomic testing and fluorescence in situ hybridization analysis identified FGFR2 gene amplification in both models, in the form of homogeneously staining region (HSR) in CUP#55 and double minutes in CUP#96. FGFR2 was recognized as the main oncogenic driver and therapeutic target. FGFR2-targeting drug BGJ398 (infigratinib) in combination with the MEK inhibitor trametinib proved to be synergic and exceptionally active, both in vitro and in vivo. The effects of the combined treatment by single-cell gene expression analysis revealed a remarkable plasticity of tumor cells and the greater sensitivity of cells with epithelial phenotype. This study brings personalized therapy closer to CUP patients and provides the rationale for FGFR2 and MEK targeting in metastatic tumors with FGFR2 pathway activation