Protozoan predation enhances stress resistance and antibiotic tolerance in Burkholderia cenocepacia by triggering the SOS response.

Bacterivorous protists are thought to serve as training grounds for bacterial pathogens by subjecting them to the same hostile conditions that they will encounter in the human host. Bacteria that survive intracellular digestion exhibit enhanced virulence and stress resistance after successful passage through protozoa but the underlying mechanisms are unknown. Here we show that the opportunistic pathogen Burkholderia cenocepacia survives phagocytosis by ciliates found in domestic and hospital sink drains, and viable bacteria are expelled packaged in respirable membrane vesicles with enhanced resistance to oxidative stress, desiccation, and antibiotics, thereby contributing to pathogen dissemination in the environment. Reactive oxygen species generated within the protozoan phagosome promote the formation of persisters tolerant to ciprofloxacin by activating the bacterial SOS response. In addition, we show that genes encoding antioxidant enzymes are upregulated during passage through ciliates increasing bacterial resistance to oxidative radicals. We prove that suppression of the SOS response impairs bacterial intracellular survival and persister formation within protists. This study highlights the significance of protozoan food vacuoles as niches that foster bacterial adaptation in natural and built environments and suggests that persister switch within phagosomes may be a widespread phenomenon in bacteria surviving intracellular digestion.

End-binding protein 1 regulates the metabolic fate of CD4+ T lymphoblasts and Jurkat T cells and the organization of the mitochondrial network.

The organization of the mitochondrial network is relevant for the metabolic fate of T cells and their ability to respond to TCR stimulation. This arrangement depends on cytoskeleton dynamics in response to TCR and CD28 activation, which allows the polarization of the mitochondria through their change in shape, and their movement along the microtubules towards the immune synapse. This work focus on the role of End-binding protein 1 (EB1), a protein that regulates tubulin polymerization and has been previously identified as a regulator of intracellular transport of CD3-enriched vesicles. EB1-interferred cells showed defective intracellular organization and metabolic strength in activated T cells, pointing to a relevant connection of the cytoskeleton and metabolism in response to TCR stimulation, which leads to increased AICD. By unifying the organization of the tubulin cytoskeleton and mitochondria during CD4+ T cell activation, this work highlights the importance of this connection for critical cell asymmetry together with metabolic functions such as glycolysis, mitochondria respiration, and cell viability.

Activation kinetics of regulatory molecules during immunological synapse in T cells.

T cell activation through TCR stimulation leads to the formation of the immunological synapse (IS), a specialized adhesion organized between T lymphocytes and antigen presenting cells (APCs) in which a dynamic interaction among signaling molecules, the cytoskeleton and intracellular organelles achieves proper antigen-mediated stimulation and effector function. The kinetics of molecular reactions at the IS is essential to determine the quality of the response to the antigen stimulation. Herein, we describe methods based on biochemistry, flow cytometry and imaging in live and fixed cells to study the activation state and dynamics of regulatory molecules at the IS in the Jurkat T cell line CH7C17 and primary human and mouse CD4+ T lymphocytes stimulated by antigen presented by Raji and HOM2 B cell lines and human and mouse dendritic cells.

T cell activation and effector function in the human Jurkat T cell model.

In order to understand T cell function, it is necessary to completely decipher the molecular dynamics underlying T cell activation and effector function. In vitro easy-to-handle cellular models are valuable tools to study intracellular molecular mechanisms in live cells. The CD4 T cell line Jurkat (JK) has been widely employed to investigate intracellular signaling leading to T cell activation in response to T cell receptor (TCR) triggering. Here, we describe diverse, complementary protocols to evaluate the TCR- and costimulation-mediated T cell activation, as well as the immunological synapse assembly and cytokine production occurring as a consequence of successful early activation events. This in vitro model is extremely useful to address molecular mechanisms operating during T cell activation and effector function acting in diverse pathophysiological scenarios.

Autophagy and lipid droplets are a defense mechanism against toxic copper oxide nanotubes in the eukaryotic microbial model Tetrahymena thermophila.

The widespread use of inorganic nanomaterials of anthropogenic origin has significantly increased in the last decade, being now considered as emerging pollutants. This makes it necessary to carry out studies to further understand their toxicity and interactions with cells. In the present work we analyzed the toxicity of CuO nanotubes (CuONT) in the ciliate Tetrahymena thermophila, a eukaryotic unicellular model with animal biology. CuONT exposure rapidly induced ROS generation in the cell leading to oxidative stress and upregulation of genes encoding antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase), metal-chelating metallothioneins and cytochrome P450 monooxygenases. Comet assays and overexpression of genes involved in DNA repair confirmed oxidative DNA damage in CuONT-treated cells. Remarkably, both electron and fluorescent microscopy revealed numerous lipid droplets and autophagosomes containing CuONT aggregates and damaged mitochondria, indicating activation of macroautophagy, which was further confirmed by a dramatic upregulation of ATG (AuTophaGy related) genes. Treatment with autophagy inhibitors significantly increased CuONT toxicity, evidencing the protective role of autophagy towards CuONT-induced damage. Moreover, increased formation of lipid droplets appears as an additional mechanism of CuONT detoxification. Based on these results, we present a hypothetical scenario summarizing how T. thermophila responds to CuONT toxicity. This study corroborates the use of this ciliate as an excellent eukaryotic microbial model for analyzing the cellular response to stress caused by toxic metal nanoparticles.

Metallic Nanoparticles-Friends or Foes in the Battle against Antibiotic-Resistant Bacteria?

The rapid spread of antibiotic resistances among bacteria demands novel strategies for infection control, and metallic nanoparticles appear as promising tools because of their unique size and tunable properties that allow their antibacterial effects to be maximized. Furthermore, their diverse mechanisms of action towards multiple cell components have suggested that bacteria could not easily develop resistance against nanoparticles. However, research published over the last decade has proven that bacteria can indeed evolve stable resistance mechanisms upon continuous exposure to metallic nanoparticles. In this review, we summarize the currently known individual and collective strategies employed by bacteria to cope with metallic nanoparticles. Importantly, we also discuss the adverse side effects that bacterial exposure to nanoparticles may have on antibiotic resistance dissemination and that might constitute a challenge for the implementation of nanoparticles as antibacterial agents. Overall, studies discussed in this review point out that careful management of these very promising antimicrobials is necessary to preserve their efficacy for infection control.

PET/CT of COVID-19 as an Organizing Pneumonia.

An 85-year-old woman with history of melanoma is referred for a follow-up F-FDG PET/CT. F-FDG PET/CT scan showed bilateral and peripheral ground-glass opacities in upper and lower pulmonary lobes surrounded by consolidations of crescent shape with increased FDG uptake, findings compatible with organizing pneumonia. Following further inquiry, the patient reported low-grade fever, sore throat, and fatigue for the past 6 days. Because of the ongoing COVID-19 pandemic, the patient was tested for SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), which resulted positive.