Bacterial multidrug efflux pumps are antibiotic resistance determinants present in all microorganisms. With few exceptions, they are chromosomally encoded and present a conserved organization both at the genetic and at the protein levels. In addition, most, if not all, strains of a given bacterial species present the same chromosomally-encoded efflux pumps. Altogether this indicates that multidrug efflux pumps are ancient elements encoded in bacterial genomes long before the recent use of antibiotics for human and animal therapy. In this regard, it is worth mentioning that efflux pumps can extrude a wide range of substrates that include, besides antibiotics, heavy metals, organic pollutants, plant-produced compounds, quorum sensing signals or bacterial metabolites, among others. In the current review, we present information on the different functions that multidrug efflux pumps may have for the bacterial behaviour in different habitats as well as on their regulation by specific signals. Since, in addition to their function in non-clinical ecosystems, multidrug efflux pumps contribute to intrinsic, acquired, and phenotypic resistance of bacterial pathogens, the review also presents information on the search for inhibitors of multidrug efflux pumps, which are currently under development, in the aim of increasing the susceptibility of bacterial pathogens to antibiotics.

Bacterial physiology is regulated at different levels, from mRNA synthesis to translational regulation and protein modification. Herein, we propose a parameter, dubbed post-transcriptional variation (PTV), that allows extracting information on post-transcriptional regulation from the combined analysis of transcriptomic and proteomic data. We have applied this parameter for getting a deeper insight in the regulon of the Pseudomonas aeruginosa post-transcriptional regulator Crc. P. aeruginosa is a free-living microorganism, and part of its ecological success relies on its capability of using a large number of carbon sources. The hierarchical assimilation of these sources when present in combination is regulated by Crc that, together with Hfq (the RNA-binding chaperon in the complex), impedes their translation when catabolite repression is triggered. Most studies on Crc regulation are based either in transcriptomics or in proteomics data, which cannot provide information on post-transcriptional regulation when analysed independently. Using the PTV parameter, we present a comprehensive map of the Crc post-transcriptional regulon. In addition of controlling the use of primary and secondary carbon sources, Crc regulates as well cell respiration, c-di-GMP mediated signalling, and iron utilization. Thus, besides controlling the hyerarchical assimilation of carbon sources, Crc is an important element for keeping bacterial homeostasis and, consequently, metabolic robustness.

Despite diagnostic and therapeutic advances, liver cancer kills more than 18 million people every year worldwide, urging new strategies to model the disease and to improve the current therapeutic options. In vitro tumor models of human cancer continue to evolve, and they represent an important screening tool. However, there is a tremendous need to improve the physiological relevance and reliability of these in vitro models to fulfill today's research requirements for better understanding of cancer progression and treatment options at different stages of the disease. This review describes the hepatocellular carcinoma microenvironmental characteristics and illustrates the current immunotherapy strategy to fight the disease. Moreover, we present a recent collection of 2D and 3D in vitro liver cancer models and address the next generation of in vitro systems recapitulating the tumor microenvironment complexity in more detail.

Pseudomonas aeruginosa is an important opportunistic pathogen with high prevalence in nosocomial infections. This microorganism is a good model for understanding biological processes such as the quorum-sensing response, the metabolic integration of virulence, the mechanisms of global regulation of bacterial physiology, and the evolution of antibiotic resistance. Till now, P. aeruginosa proteomic data, although available in several on-line repositories, were dispersed and difficult to access. In the present work, proteomes of the PAO1 strain grown under very different conditions and from diverse cellular compartments have been analyzed and joined to build the Pseudomonas PeptideAtlas. This resource is a comprehensive mass spectrometry-derived peptide and inferred protein database with 71.3% coverage of the total predicted proteome of P. aeruginosa PAO1. This is the highest published coverage among the eight bacterial PeptideAtlas datasets currently available. The proteins in the Pseudomonas PeptideAtlas cover 84% of metabolic proteins, 71% of proteins involved in genetic information processing, 72% of proteins responsible for environmental information processing, more than 80% of proteins related to quorum sensing and biofilm formation, and 81% of proteins responsible for antimicrobial resistance. It exemplifies a necessary tool for targeted proteomics studies, system-wide observations, and cross-species observational studies. Here we describe how this resource was built and some of the physiologically important proteins of this pathogen. Significance: Pseudomonas aeruginosa is among the most versatile bacterial pathogens. Studies of its proteome are very important as they can reveal virulence factors and mechanisms of antibiotic resistance. The construction of a proteomic resource such as the PeptideAtlas enables targeted proteomics studies, system-wide observations, and cross-species observational studies.

Adoptive cell transfer (ACT) has been highly efficient in targeting certain refractory cancers and remains potentially effective for other cancers as new targets of cancer-immune interactions are revealed. ACT involves isolating immunocompetent cells from cancer patients, expanding them ex vivo, and infusing them back into the patient. Cells used for ACT are often effector cells, typically T cells, isolated from the patient’s peripheral blood and then engineered to target cancer cells by incorporating a T cell receptor (TCR) or chimeric antigen receptor (CAR), with additional modifications to improve immune cell proliferation and persistence. While cell therapies for various lymphomas have led to dramatic tumor regressions, response in solid tumors remains varied. Solid tumors present unique challenges, such as tumor heterogeneity and the lack of tumor-specific targets, but also an immunosuppressive tumor microenvironment (TME) characterized by poor T cell infiltration and terminal T cell differentiation and exhaustion at the tumor. To overcome the hostile TME, lymphodepleting regimens combined with an infusion of large numbers of T cells is often used. However, introducing large numbers of T cells significantly increases the risk of on-target/off-target toxicity, neurotoxicity, and cytokine release syndrome. The use of engineered T cells that transiently express TCRs or CARs by mRNA gene transfer, reduces these risks. Additionally, they avoid the use of viral vectors, hence there is no risk of insertional mutagenesis and can be manufactured more easily at low cost and on a larger scale. Such transient-engineered T cells have shown significant antitumor activity in phase I clinical trials and in preclinical animal models. However, multiple infusions are often required, increasing patient discomfort and treatment cost. To mitigate this, we sought to improve the antitumor activity of transient engineered T cells by screening a panel of small molecules targeting epigenetic regulators for their effect on T cell cytotoxicity. Using a model for engineered T cells targeting hepatocellular carcinoma, we found that short-term inhibition of G9a/GLP increased T cell antitumor activity in in vitro models and an orthotopic mouse model. G9a/GLP inhibition increased granzyme expression without terminal T cell differentiation or exhaustion and resulted in specific changes in the expression of genes and proteins involved in pro-inflammatory pathways, T cell activation, and cytotoxicity. Citation Format: Maxine S. Lam, Jose A. Reales-Calderon, Jin Rong Ow, Joey Aw, Damien Tan, Ragavi Vijayakumar, Erica Ceccarello, Tommaso Tabaglio, Yan Ting Lim, Wang Loo Chien, Fritz Lai, Anthony Tan Tanoto, Qingfeng Chen, Radoslaw M. Sobota, Giulia Adriani, Antonio Bertoletti, Ernesto Guccione, Andrea Pavesi. G9a/GLP inhibition during ex vivo lymphocyte expansion increases in vivo cytotoxicity of engineered T cells against hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1781.