Toxoplasma gondii, a widespread parasite, has the ability to infect nearly any nucleated cell in warm-blooded vertebrates. It is estimated that around 2 billion people globally have been infected by this pathogen. Although most healthy individuals can effectively control parasite replication, certain parasites may evade the immune response, establishing cysts in the brain that are refractory to the immune system and resistance to available drugs. For its chronic persistence in the brain, the parasite relies on host cells\' nutrients, particularly amino acids and lipids. Therefore, understanding how latent parasites persist in the brain is crucial for identifying potential drug targets against chronic forms. While shielded within parasitophorous vacuoles (PVs) or cysts, Toxoplasma exploits the host endoplasmic reticulum (ER) metabolism to sustains its persistence in the brain, resulting in host neurological alterations. In this study, we demonstrate that T. gondii disrupts the host ER homeostasis, resulting in accumulation of unfolded protein with the host ER. The host counters this stress by initiating an autophagic pathway known as ER-phagy, which breaks down unfolded proteins into amino acids, promoting their recycling. Remarkably, the persistence of latent forms in cell culture as well as behavioral changes in mice caused by the latent infection could be successfully reversed by restricting the availability of various amino acids during T. gondi infection. Our findings unveil the underlying mechanisms employed by T. gondii to exploit host ER and lysosomal pathways, enhancing nutrient levels during infection. These insights provide new strategies for the treatment of toxoplasmosis. less

Bacteria have evolved complex transcriptional regulatory networks, as well as many diverse regulatory strategies at the RNA level, to enable more efficient use of metabolic resources and rapid response to changing conditions. However, most RNA-based regulatory mechanisms are not well conserved across different bacterial species despite controlling genes important for virulence or essential biosynthetic processes. Here, we characterize the activity of, and assess the fitness benefit conferred by, twelve cis-acting regulatory RNAs (including several riboswitches and a T-box), in the opportunistic pathogen Streptococcus pneumoniae TIGR4. By evaluating native locus mutants of each regulator that result in constitutively active and repressed expression, we establish that growth defects in planktonic culture are associated with constitutive repression of gene expression, while constitutive activation of gene expression is rarely deleterious. In contrast, in mouse nasal carriage and pneumonia models, strains with both constitutively active and repressed gene expression are significantly less fit than matched control strains. Furthermore, two RNA-regulated pathways, FMN synthesis/transport and pyrimidine synthesis/transport display the greatest sensitivity to mis-regulation or constitutive gene repression in both planktonic culture and in vivo environments. Our findings suggest that despite lack of an obvious phenotypes associated with gene over-expression in vitro, the fitness benefit conferred on bacteria via fine-tuned metabolic regulation through cis-acting regulatory RNAs is often substantial in vivo, and therefore easily sufficient to drive the evolution and maintenance of diverse RNA regulatory mechanisms. less

The Hippo kinases MST1 and MST2 initiate a highly conserved signaling cascade called the Hippo pathway that limits organ size and tumor formation in animals. Intriguingly, pathogens hijack this host pathway during infection, but the role of MST1/2 in innate immune cells against pathogens is unclear. In this study, we generated Mst1/2 knockout macrophages to investigate the regulatory activities of the Hippo kinases in immunity. Transcriptomic analyses identified differentially expressed genes (DEGs) that are enriched in biological pathways, such as systemic lupus erythematosus, tuberculosis, and apoptosis. Surprisingly, pharmacological inhibition of the downstream components LATS1/2 in the canonical Hippo pathway did not affect expression of a set of immune DEGs, suggesting that MST1/2 control these genes via alternative inflammatory Hippo signaling. Moreover, MST1/2 may affect immune communication by influencing the release of cytokines, such as TNF, CXCL10, and IL-1ra. Comparative analyses of the single- and double-knockout macrophages revealed that MST1 and MST2 differentially regulate TNF release and expression of the immune transcription factor, MAF, demonstrating that the two homologous Hippo kinases individually play a unique role in innate immunity. Notably, MST1 and MST2 are both required for macrophages to activate apoptosis. Lastly, we demonstrated that the Hippo kinases are critical factors in mammalian macrophages and single-cell amoebae to restrict infection by Legionella pneumophila, Escherichia coli, and Pseudomonas aeruginosa. Together, these results uncover non-canonical inflammatory Hippo signaling in macrophages and the evolutionarily conserved role of the Hippo kinases in anti-microbial defense of eukaryotic hosts. less

Louis Pasteur\'s experiments on tartaric acid laid the foundation for our understanding of molecular chirality, but major questions remain. By comparing the optical activity of naturally-occurring tartaric acid with chemically-synthesized paratartaric acid, Pasteur realized that naturally-occurring tartaric acid contained only L-tartaric acid while paratartaric acid consisted of a racemic mixture of D- and L-tartaric acid. Curiously, D-tartaric acid has no known natural source, yet several gut bacteria specifically degrade D-tartaric acid. Here, we investigated the oxidation of monosaccharides by inflammatory reactive oxygen and nitrogen species. We found that this reaction yields an array of alpha hydroxy carboxylic acids, including tartaric acid isomers. Utilization of inflammation-derived D- and L-tartaric acid enhanced colonization by Salmonella Typhimurium and E. coli in murine models of gut inflammation. Our findings suggest that byproducts of inflammatory radical metabolism, such as tartrate and other alpha hydroxy carboxylic acids, create transient nutrient niches for enteric pathogens and other potentially harmful bacteria. Furthermore, this work illustrates that inflammatory radicals generate a zoo of molecules, some of which may erroneously presumed to be xenobiotics. less

This study investigates acetone-butanol-ethanol (ABE) fermentation, a process using solventogenic Clostridium species to produce acetone, butanol, and ethanol. Recent biotechnological advancements, such as omics, systems biology, and metabolic engineering, have reignited interest in butanol production, responding to the increasing gasoline costs and the demand for sustainable energy systems. This study unravels the distinct physiological attributes of C. acetobutylicum (Cac) and C. pasteurianum (Cpa), significantly impacting sustainable bioenergy technologies. Employing response surface methodology (RSM), we embarked on a comprehensive statistical optimization journey in the co-culture system, Cac MTCC 11274 and Cpa MTCC 116, enhancing biobutanol production from mixed substrates. A spectrum of process parameters was scrutinized, encompassing the ratio of Cac and Cpa inoculum, sodium concentration, and the ratio of xylose to glucose. Statistical analysis revealed salt concentration's profound influence on biomass, total alcohol, and butanol production. The culmination of these endeavors yielded highly promising outcomes: a butanol concentration of 12.1 +/- 0.45 g L-1 (model prediction: 11.87 g L-1), biomass of 4.15 +/- 0.03 (model prediction: 4.06 OD600), and ABE concentration of 23.1 +/- 0.55 g L-1 (model prediction: 22.45 g L-1). These results represent a significant leap forward in bioenergy technologies, offering both practical insights and sustainable solutions for enhanced biofuel production. less

Extracellular vesicles (EVs) are now recognized as key players in the biology of numerous organisms, including pathogenic fungi. However, studying EVs in these organisms remains challenging. The recent implementation of new protocols to purify EVs in the pathogenic yeast Cryptococcus neoformans has resulted in a more detailed description of their structure and protein composition. Although a few publications describing RNA molecules associated with EVs have already been published, we reasoned that these new protocols would be beneficial for gaining a deeper understanding of the EV transcriptome. We thus purified EVs and confirmed that some RNAs were associated with these EV extracts. Iodixanol gradient analyses also revealed that these RNAs co-sedimented with EVs. We then sequenced these RNAs in parallel with RNAs extracted from the very cells producing these EVs using different types of sequencing libraries. Our data confirm the presence of siRNAs and tRFs associated with EVs, some of which are enriched. We also identified some snoRNAs, which in Cryptococcus are mostly borne by coding gene or lncRNA introns. less

Trypanosomatid parasites undergo developmental regulation to adapt to the different environments encountered during their life cycle. In Trypanosoma brucei, a genome wide selectional screen previously identified a regulator of the protein family ESAG9, which is highly expressed in stumpy forms, a morphologically distinct bloodstream stage adapted for tsetse transmission. This regulator, TbREG9.1, has an orthologue in Trypanosoma congolense, despite the absence of a stumpy morphotype in this parasite species, which is an important cause of livestock trypanosomosis. RNAi mediated gene silencing of TcREG9.1 in Trypanosoma congolense caused a loss of attachment of the parasites to a surface substrate in vitro, a key feature of the biology of these parasites that is distinct from T. brucei. This detachment was phenocopied by treatment of the parasites with a phosphodiesterase inhibitor, which also promotes detachment in the insect trypanosomatid Crithidia fasciculata. RNAseq analysis revealed that TcREG9.1 silencing caused the upregulation of mRNAs for several classes of surface molecules, including transferrin receptor-like molecules, immunodominant proteins, and molecules related to those associated with stumpy development in T. brucei. Depletion of TcREG9.1 in vivo also generated an enhanced level of parasites in the blood circulation consistent with reduced parasite attachment to the microvasculature. The morphological progression to insect forms of the parasite was also perturbed. We propose a model whereby TcREG9.1 acts as a regulator of attachment and development, with detached parasites being adapted for transmission. less

Human rhinovirus is a major driver of disease exacerbations for patients with asthma and chronic obstructive pulmonary disease. Importantly, the virus can disrupt immune cell functioning leading to secondary bacterial infections in these patients. MicroRNAs are important regulators of gene expression and control viral pathogenesis and immune cell functions. However, the role of different miRNAs during rhinovirus infections remains poorly defined. The aim of this study was to analyze the impact of RV16 on epithelial cell responses and identify miRNAs with altered expression levels in response to the viral infection and their target genes. HeLa-Ohio cells exposed to RV16 showed deficiencies in bacterial clearance and induced changes in the expression of several novel miRNAs identified using a custom panel of 88 different miRNAs and further analyzed using our newly developed computer program. Our experiments identified a panel of 9 differentially regulated miRNAs and our computer program narrowed this down to two that could represent biomarkers for RV16 infection. Our results also showed that upon upregulation of these miRNAs (miR-101-3p and miR-30b-5p) by RV16 there was a downregulation in EZH2, RARG and PTPN13 that could correlate with a productive viral infection and a worsened immune response. Taken together, our findings suggest that RV16 modifies the miRNA landscape in epithelial cells and that miR-101-3p and miR-30b-5p could represent key biomarkers for either early or late RV16 infection. less

In recent years, there has been a surge in metagenomic studies focused on identifying plasmids in environmental samples. While these studies have unearthed numerous novel plasmids, enriching our understanding of their environmental roles, a significant gap remains: the scarcity of information regarding the bacterial hosts of these newly discovered plasmids. Furthermore, even when plasmids are identified within bacterial isolates, the reported host is typically limited to the original isolate, with no insight into alternative hosts or the plasmid\'s potential host range. Given that plasmids depend on hosts for their existence, investigating plasmids without knowledge of potential hosts offers only a partial perspective. This study introduces a method for identifying potential hosts and host ranges for plasmids through alignment with CRISPR spacers. To validate the method, we compared the PLSDB plasmids database with the CRISPR spacers database, yielding host predictions for 46% of the plasmids. When compared to reported hosts, our predictions achieved an 84% concordance at the family level and 99% concordance at the phylum level. Moreover, the method frequently identified multiple potential hosts for a plasmid, thereby enabling predictions of alternative hosts and the host range. Notably, we found that CRISPR spacers predominantly target plasmid backbone genes while sparing functional genes, such as those linked to antibiotic resistance, aligning with our hypothesis that CRISPR spacers are acquired from plasmid-specific regions rather than insertion elements from diverse sources. Lastly, we illustrate the network of connections among different bacterial taxa through plasmids, revealing potential pathways for horizontal gene transfer. less

The increasing prevalence of dermatophyte resistance to terbinafine, a key drug in the treatment of dermatophytosis, represents a significant obstacle to treatment. Trichophyton rubrum is the most commonly isolated fungus in dermatophytosis. In T. rubrum, we identified TERG_07844, a gene encoding a previously uncharacterized putative protein kinase, as an ortholog of budding yeast Saccharomyces cerevisiae polyamine transport kinase 2 (Ptk2) and found that T. rubrum Ptk2 (TrPtk2) is involved in terbinafine tolerance. In both T. rubrum and S. cerevisiae, Ptk2 knockout strains were more sensitive to terbinafine compared to the wild types, suggesting that promotion of terbinafine tolerance is a conserved function of fungal Ptk2. The T. rubrum Ptk2 knockout strain ({Delta}TrPtk2) was sensitive to omeprazole, an inhibitor of plasma membrane proton pump Pma1, which is activated through phosphorylation by Ptk2 in S. cerevisiae. Overexpression of T. rubrum Pma1 (TrPma1) in {Delta}TrPtk2 suppressed terbinafine sensitivity, suggesting that the induction of terbinafine tolerance by TrPtk2 is mediated by TrPma1. Furthermore, omeprazole increased the terbinafine sensitivity of clinically isolated terbinafine-resistant strains. These findings suggest that, in dermatophytes, the TrPtk2-TrPma1 pathway plays a key role in promoting intrinsic terbinafine tolerance and may serve as a potential target for combinational antifungal therapy against terbinafine-resistant dermatophytes. less