An Auto-Regulating Type II Toxin-Antitoxin System Modulates Drug Resistance and Virulence in Streptococcus suis

Toxin-antitoxin (TA) systems are ubiquitous genetic elements that play an essential role in multidrug tolerance and virulence of bacteria. So far, little is known about the TA systems in Streptococcus suis. In this study, the Xress-MNTss TA system, composed of the MNTss toxin in the periplasmic space and its interacting Xress antitoxin, was identified in S. suis. β-galactosidase activity and electrophoretic mobility shift assay (EMSA) revealed that Xress and the Xress-MNTss complex could bind directly to the Xress-MNTss promoter as well as downregulate streptomycin adenylyltransferase ZY05719_RS04610. Interestingly, the Xress deletion mutant was less pathogenic in vivo following a challenge in mice. Transmission electron microscopy and adhesion assays pointed to a significantly thinner capsule but greater biofilm-formation capacity in ΔXress than in the wild-type strain. These results indicate that Xress-MNTss, a new type II TA system, plays an important role in antibiotic resistance and pathogenicity in S. suis.

Phylogeny Reveals Novel HipA-Homologous Kinase Families and Toxin-Antitoxin Gene Organizations

Bacterial multidrug tolerance and persistence are problems of increasing scientific and medical significance. The first gene discovered to confer persistence was hipA , encoding the kinase toxin of the hipBA toxin-antitoxin (TA) module of E. coli .

Toxin–Antitoxin Systems and their Role in Maintaining the Pathogenic Potential of Causative Agents of Sapronoses

In interepidemic periods, causative agents of sapronoses typically employ a variety of mechanisms for maintaining the viability in terrestrial parasitic systems, associated with different adaptive strategies and utilized by their populations to survive. Unlike spore-forming bacteria, causative agents of sapronoses form resistant cell forms: viable but nonculturable (VBNC) cells and persistence (dormant) cells. The implementation of these strategies is mediated by the influence of various stressors of the environment and is characterized by a decrease in metabolism, a change in the morphology and physiology of the bacterial cell, and also the cessation of its replication. While most of the bacterial population is killed under antibiotic exposure, this fraction of pathogens transiently exhibits a phenotypic multidrug-tolerance, causing relapses and chronic courses of many sapronoses. It is important to note that when these resistant cell forms retain virulence and when favorable conditions occur, they are again transformed into active vegetative forms. For this reason, understanding mechanisms, allowing a fraction of the bacterial population to acquire transiently multidrug-tolerance represents an essential step to eradicate these dormant populations. The discovery of the genetic modules of bacterial toxin-antitoxin systems (TAS) in recent years, was proposed to be an ideal and promising candidate to control these complex regulatory molecular mechanisms. Overexpression of the toxins often increases persister frequency in a defined population. In this review, we summarize the scientific data regarding the TAS modules involved in bacterial persistence to be used as antibiotics for the conservation of the pathogenic potential of resistant forms of pathogens of natural focal sapronosis in interepidemic periods.

TFIIH kinase CDK7 antagonizes phenotype switching and emergence of drug tolerance in melanoma

Melanoma cells switch back-and-forth between phenotypes of proliferation and invasion in response to changing microenvironment, driving metastatic progression. We show that inhibition of the TFIIH kinase CDK7 (CDK7i) results in a melanocytic to mesenchymal phenotype switching and acquisition of targeted therapy tolerance. We identify a gene expression program controlled by the transcription factor GATA6, which participates in drug tolerance in mesenchymal-like cells and which is antagonized by CDK7 in melanocytic-like cells. This program emerges concomitantly with loss of melanocyte lineage-specific MITF protein following CDK7i. By dissecting the underlying mechanism, we observe that CDK7 accumulates at the super-enhancer regulating MITF to drive its expression. MITF itself binds to a intronic region of GATA6 to transcriptionally repress it. This molecular cascade antagonizes expression of the GATA6 regulon that only emerges in MITF-low cells of metastatic melanoma. Our work reveals a role for CDK7 in counteracting phenotype switching and activation of a gene expression program mediating multidrug tolerance in melanoma cells.

Therapy-induced lipid uptake and remodeling underpin ferroptosis hypersensitivity in prostate cancer

Background Metabolic reprograming, non-mutational epigenetic changes, increased cell plasticity, and multidrug tolerance are early hallmarks of therapy resistance in cancer. In this temporary, therapy-tolerant state, cancer cells are highly sensitive to ferroptosis, a form of regulated cell death that is caused by oxidative stress through excess levels of iron-dependent peroxidation of polyunsaturated fatty acids (PUFA). However, mechanisms underpinning therapy-induced ferroptosis hypersensitivity remain to be elucidated. Methods We used quantitative single-cell imaging of fluorescent metabolic probes, transcriptomics, proteomics, and lipidomics to perform a longitudinal analysis of the adaptive response to androgen receptor-targeted therapies (androgen deprivation and enzalutamide) in prostate cancer (PCa). Results We discovered that cessation of cell proliferation and a robust reduction in bioenergetic processes were associated with multidrug tolerance and a strong accumulation of lipids. The gain in lipid biomass was fueled by enhanced lipid uptake through cargo non-selective (macropinocytosis, tunneling nanotubes) and cargo-selective mechanisms (lipid transporters), whereas de novo lipid synthesis was strongly reduced. Enzalutamide induced extensive lipid remodeling of all major phospholipid classes at the expense of storage lipids, leading to increased desaturation and acyl chain length of membrane lipids. The rise in membrane PUFA levels enhanced membrane fluidity and lipid peroxidation, causing hypersensitivity to glutathione peroxidase (GPX4) inhibition and ferroptosis. Combination treatments against AR and fatty acid desaturation, lipase activities, or growth medium supplementation with antioxidants or PUFAs altered GPX4 dependence. Conclusions Our work provides mechanistic insight into processes of lipid metabolism that underpin the acquisition of therapy-induced GPX4 dependence and ferroptosis hypersensitivity to standard of care therapies in PCa. It demonstrates novel strategies to suppress the therapy-tolerant state that may have potential to delay and combat resistance to androgen receptor-targeted therapies, a currently unmet clinical challenge of advanced PCa. Since enhanced GPX4 dependence is an adaptive phenotype shared by several types of cancer in response to different therapies, our work might have universal implications for our understanding of metabolic events that underpin resistance to cancer therapies.

Therapy-induced lipid uptake and remodeling underpin ferroptosis hypersensitivity in prostate cancer

BackgroundMetabolic reprograming, non-mutational epigenetic changes, increased cell plasticity and multidrug tolerance are early hallmarks of therapy resistance in cancer. In this temporary, therapy-tolerant state, cancer cells are highly sensitive to ferroptosis, a form of regulated cell death that is caused by oxidative stress through excess levels of iron-dependent peroxidation of polyunsaturated fatty acids (PUFA). However, mechanisms underpinning therapy-induced ferroptosis hypersensitivity remain to be elucidated.MethodsWe used quantitative single cell imaging of fluorescent metabolic probes, transcriptomics, proteomics and lipidomics to perform a longitudinal analysis of the adaptive response to androgen receptor-targeted therapies (androgen deprivation and enzalutamide) in prostate cancer (PCa).ResultsWe discovered that cessation of cell proliferation and a robust reduction in bioenergetic processes were associated with multidrug tolerance and a strong accumulation of lipids. The gain in lipid biomass was fueled by enhanced lipid uptake through cargo non-selective (macropinocytosis, tunneling nanotubes) and cargo-selective mechanisms (lipid transporters), whereas de novo lipid synthesis was strongly reduced. Enzalutamide induced extensive lipid remodeling of all major phospholipid classes at the expense of storage lipids, leading to increased desaturation and acyl chain length of membrane lipids. The rise in membrane PUFA levels enhanced membrane fluidity and lipid peroxidation, causing hypersensitivity to glutathione peroxidase (GPX4) inhibition and ferroptosis. Combination treatments against AR and fatty acid desaturation, lipase activities or growth medium supplementation with antioxidants or PUFAs altered GPX4 dependence. Despite multidrug tolerance, PCa cells displayed an enhanced sensitivity to inhibition of lysosomal processing of exogenous lipids, highlighting an increased dependence on lipid uptake in the therapy-tolerant state.ConclusionsOur work provides mechanistic insight into processes of lipid metabolism that underpin the acquisition of therapy-induced GPX4 dependence and ferroptosis hypersensitivity to standard of care therapies in PCa. It demonstrated novel strategies to suppress the therapy-tolerant state that may have potential to delay and combat resistance to androgen receptor-targeted therapies, a currently unmet clinical challenge of advanced PCa. Since enhanced GPX4 dependence is an adaptive phenotype shared by several types of cancer in response to different therapies, our work might have universal implications for our understanding of metabolic events that underpin resistance to cancer therapies.

Clinical Mutations That Partially Activate the Stringent Response Confer Multidrug Tolerance in Staphylococcus aureus

ABSTRACT Antibiotic tolerance is an underappreciated antibiotic escape strategy that is associated with recurrent and relapsing infections, as well as acting as a precursor to resistance. Tolerance describes the ability of a bacterial population to survive transient exposure to an otherwise lethal concentration of antibiotic without exhibiting an elevated MIC. It is detected in time-kill assays as a lower rate of killing than a susceptible strain and can be quantified by the metric minimum duration for killing (MDK). The molecular mechanisms behind tolerance are varied, but activation of the stringent response (SR) via gene knockouts and/or chemical induction has long been associated with tolerance. More recently, two Gram-positive clinical isolates from persistent bacteremias were found to bear mutations in the SR controller, Rel, that caused elevated levels of the alarmone (p)ppGpp. Here, we show that introduction of either of these mutations into Staphylococcus aureus confers tolerance to five different classes of antibiotic as a result of (p)ppGpp-mediated growth defects (longer lag time and/or lower growth rate). The degree of tolerance is related to the severity of the growth defect and ranges from a 1.5- to 3.1-fold increase in MDK. Two classes of proposed SR inhibitor were unable to reverse or reduce this tolerance. Our findings reveal the significance of SR-activating mutations in terms of tolerance and clinical treatment failures. The panel of strains reported here provide a clinically relevant model of tolerance for further investigation of its link to resistance development, as well as potential validation of high-throughput tolerance screens.