Recent advances in therapeutic targets identification and development of treatment strategies towards Pseudomonas aeruginosa infections.

The opportunistic human pathogen Pseudomonas aeruginosa is the causal agent of a wide variety of infections. This non-fermentative Gram-negative bacillus can colonize zones where the skin barrier is weakened, such as wounds or burns. It also causes infections of the urinary tract, respiratory system or bloodstream. P. aeruginosa infections are common in hospitalized patients for which multidrug-resistant, respectively extensively drug-resistant isolates can be a strong contributor to a high rate of in-hospital mortality. Moreover, chronic respiratory system infections of cystic fibrosis patients are especially concerning, since very tedious to treat. P. aeruginosa exploits diverse cell-associated and secreted virulence factors, which play essential roles in its pathogenesis. Those factors encompass carbohydrate-binding proteins, quorum sensing that monitor the production of extracellular products, genes conferring extensive drug resistance, and a secretion system to deliver effectors to kill competitors or subvert host essential functions. In this article, we highlight recent advances in the understanding of P. aeruginosa pathogenicity and virulence as well as efforts for the identification of new drug targets and the development of new therapeutic strategies against P. aeruginosa infections. These recent advances provide innovative and promising strategies to circumvent infection caused by this important human pathogen.

A unique, newly discovered four-member protein family involved in extracellular fatty acid binding in Yarrowia lipolytica.

BACKGROUND: Yarrowia lipolytica, a nonconventional oleaginous yeast species, has attracted attention due to its high lipid degradation and accumulation capacities. Y. lipolytica is used as a chassis for the production of usual and unusual lipids and lipid derivatives. While the genes involved in the intracellular transport and activation of fatty acids in different cellular compartments have been characterized, no genes involved in fatty acid transport from the extracellular medium into the cell have been identified thus far. In this study, we identified secreted proteins involved in extracellular fatty acid binding. RESULTS: Recent analysis of the Y. lipolytica secretome led to the identification of a multigene family that encodes four secreted proteins, preliminarily named UP1 to UP4. These proteins were efficiently overexpressed individually in wild-type and multideletant strain (Q4: Δup1Δup2Δup3Δup4) backgrounds. Phenotypic analysis demonstrated the involvement of these proteins in the binding of extracellular fatty acids. Additionally, gene deletion and overexpression prevented and promoted sensitivity to octanoic acid (C8) toxicity, respectively. The results suggested binding is dependent on aliphatic chain length and fatty acid concentration. 3D structure modeling supports the proteins' role in fatty acid assimilation at the molecular level. CONCLUSIONS: We discovered a family of extracellular-fatty-acid-binding proteins in Y. lipolytica and have proposed to name its members eFbp1 to eFbp4. The exact mode of eFbps action remains to be deciphered individually and synergistically; nevertheless, it is expected that the proteins will have applications in lipid biotechnology, such as improving fatty acid production and/or bioconversion.

New roles for Yarrowia lipolytica in molecules synthesis and biocontrol.

Reprogramming of host metabolism is a common strategy for improving desired compounds in host cells and is essential to generate overproducing strains in biotechnology. As a promising feedstock converter, Yarrowia lipolytica has been engineered to extend its bioproduction ability related to the synthesis of new value-added molecules relevant to human food and disease treatment. New synthetic tools have been reported and new enzymes with biotechnological importance are recovered. Additionally, metabolic events occurring during substrate utilization and recombinant protein production have been elucidated. Its contributions as feed and in controlling disease in the food industry have also been provided. Likewise, the recent abilities of Yarrowia lipolytica in the bioconversion of food waste into single-cell protein have been reported. These aforementioned events made the novelty of this review compared to the existing ones on this oleaginous yeast. KEY POINTS: • The production of biolipids by the heterotrophic yeast Yarrowia lipolytica is examined. • A Summary of information concerning new value-added molecules has been highlighted. • Special focus on the importance of Yarrowia lipolytica in regulating the immune system has been provided.

A review of approaches to control bacterial leaf blight in rice.

The Gram-negative bacteria Xanthomonas oryzae pv. oryzae, the causative agent of bacterial leaf blight (BLB), received attention for being an economically damaging pathogen of rice worldwide. This damage prompted efforts to better understand the molecular mechanisms governing BLB disease progression. This research revealed numerous virulence factors that are employed by this vascular pathogen to invade the host, outcompete host defence mechanisms, and cause disease. In this review, we emphasize the virulence factors and molecular mechanisms that X. oryzae pv. oryzae uses to impair host defences, recent insights into the cellular and molecular mechanisms underlying host-pathogen interactions and components of pathogenicity, methods for developing X. oryzae pv. oryzae-resistant rice cultivars, strategies to mitigate disease outbreaks, and newly discovered genes and tools for disease management. We conclude that the implementation and application of cutting-edge technologies and tools are crucial to avoid yield losses from BLB and ensure food security.

Yeasts of the Blastobotrys genus are promising platform for lipid-based fuels and oleochemicals production.

Strains of the yeast genus Blastobotrys (subphylum Saccharomycotina) represent a valuable biotechnological resource for basic biochemistry research, single-cell protein, and heterologous protein production processes. Species of this genus are dimorphic, non-pathogenic, thermotolerant, and can assimilate a variety of hydrophilic and hydrophobic substrates. These can constitute a single-cell oil platform in an emerging bio-based economy as oleaginous traits have been discovered recently. However, the regulatory network of lipogenesis in these yeasts is poorly understood. To keep pace with the growing market demands for lipid-derived products, it is critical to understand the lipid biosynthesis in these unconventional yeasts to pinpoint what governs the preferential channelling of carbon flux into lipids instead of the competing pathways. This review summarizes information relevant to the regulation of lipid metabolic pathways and prospects of metabolic engineering in Blastobotrys yeasts for their application in food, feed, and beyond, particularly for fatty acid-based fuels and oleochemicals. KEY POINTS: • The production of biolipids by heterotrophic yeasts is reviewed. • Summary of information concerning lipid metabolism regulation is highlighted. • Special focus on the importance of diacylglycerol acyltransferases encoding genes in improving lipid production is made.

The native acyltransferase-coding genes DGA1 and DGA2 affect lipid accumulation in Blastobotrys raffinosifermentans differently when overexpressed.

Blastobotrys raffinosifermentans is an ascomycetous yeast with biotechnological applications, recently shown to be an oleaginous yeast accumulating lipids under nitrogen limitation. Diacylglycerol acyltransferases (DGATs) act in the lipid storage pathway, in the last step of triacylglycerol biosynthesis. Two DGAT families are widespread in eukaryotes. We first checked that B. raffinosifermentans strain LS3 possessed both types of DGAT, and we then overexpressed the native DGAT-encoding genes, DGA1 and DGA2, separately or together. DGA2 (from the DGAT1 family) overexpression was sufficient to increase lipid content significantly in LS3, to up to 26.5% of dry cell weight (DCW), 1.6 times the lipid content of the parental strain (16.90% of DCW) in glucose medium under nitrogen limitation. By contrast, DGA1 (of the DGAT2 type) overexpression led to a large increase (up to 140-fold) in the amount of the corresponding transcript, but had no effect on overall lipid content relative to the parental strain. Analysis of the expression of the native genes over time in the parental strain revealed that DGA2 transcript levels quadrupled between 8 and 24 h in the N-limited lipogenic medium, whereas DGA1 transcript levels remained stable. This survey highlights the predominant role of the DGAT1 family in lipid accumulation and demonstrates the suitability of B. raffinosifermentans for engineering for lipid production.

Elucidating the response of Kluyveromyces lactis to arsenite and peroxide stress and the role of the transcription factor KlYap8.

All organisms need to sense and respond to a range of stress conditions. In this study, we used transcriptional profiling to identify genes and cellular processes that are responsive during arsenite and tert-butyl hydroperoxide exposure in Kluyveromyces lactis. Many arsenite-responsive genes encode proteins involved in redox processes, protein folding and stabilization, and transmembrane transport. The majority of peroxide-responsive genes encode functions related to transcription, translation, redox processes, metabolism and transport. A substantial number of these stress-regulated genes contain binding motifs for the AP-1 like transcription factors KlYap1 and KlYap8. We demonstrate that KlYap8 binds to and regulates gene expression through a 13 base-pair promoter motif, and that KlYap8 provides protection against arsenite, antimonite, cadmium and peroxide toxicity. Direct transport assays show that Klyap8Δ cells accumulate more arsenic and cadmium than wild type cells and that the Klyap8Δ mutant is defective in arsenic and cadmium export. KlYap8 regulates gene expression in response to both arsenite and peroxide, and might cooperate with KlYap1 in regulation of specific gene targets. Comparison of KlYap8 with its Saccharomyces cerevisiae orthologue ScYap8 indicates that KlYap8 senses and responds to multiple stress signals whereas ScYap8 is only involved in the response to arsenite and antimonite. Thus, our data suggest that functional specialization of ScYap8 has occurred after the whole genome duplication event. This is the first genome-wide stress response analysis in K. lactis and the first demonstration of KlYap8 function.

Quantification of yeast and bacterial gene transcripts in retail cheeses by reverse transcription-quantitative PCR.

The cheese microbiota contributes to a large extent to the development of the typical color, flavor, and texture of the final product. Its composition is not well defined in most cases and varies from one cheese to another. The aim of the present study was to establish procedures for gene transcript quantification in cheeses by reverse transcription-quantitative PCR. Total RNA was extracted from five smear-ripened cheeses purchased on the retail market, using a method that does not involve prior separation of microbial cells. 16S rRNA and malate:quinone oxidoreductase gene transcripts of Corynebacterium casei, Brevibacterium aurantiacum, and Arthrobacter arilaitensis and 26S rRNA and beta tubulin gene transcripts of Geotrichum candidum and Debaryomyces hansenii could be detected and quantified in most of the samples. Three types of normalization were applied: against total RNA, against the amount of cheese, and against a reference gene. For the first two types of normalization, differences of reverse transcription efficiencies from one sample to another were taken into account by analysis of exogenous control mRNA. No good correlation was found between the abundances of target mRNA or rRNA transcripts and the viable cell concentration of the corresponding species. However, in most cases, no mRNA transcripts were detected for species that did not belong to the dominant species. The applications of gene expression measurement in cheeses containing an undefined microbiota, as well as issues concerning the strategy of normalization and the assessment of amplification specificity, are discussed.

Roles of non-coding RNAs in the metabolism and pathogenesis of bladder cancer.

Bladder cancer (BC) is featured as the second most common malignancy of the urinary tract worldwide with few treatments leading to high incidence and mortality. It stayed a virtually intractable disease, and efforts to identify innovative and effective therapies are urgently needed. At present, more and more evidence shows the importance of non-coding RNA (ncRNA) for disease-related study, diagnosis, and treatment of diverse types of malignancies. Recent evidence suggests that dysregulated functions of ncRNAs are closely associated with the pathogenesis of numerous cancers including BC. The detailed mechanisms underlying the dysregulated role of ncRNAs in cancer progression are still not fully understood. This review mainly summarizes recent findings on regulatory mechanisms of the ncRNAs, long non-coding RNAs, microRNAs, and circular RNAs, in cancer progression or suppression and focuses on the predictive values of ncRNAs-related signatures in BC clinical outcomes. A deeper understanding of the ncRNA interactive network could be compelling framework for developing biomarker-guided clinical trials.

Roles of RNA-binding proteins in neurological disorders, COVID-19, and cancer.

RNA-binding proteins (RBPs) have emerged as important players in multiple biological processes including transcription regulation, splicing, R-loop homeostasis, DNA rearrangement, miRNA function, biogenesis, and ribosome biogenesis. A large number of RBPs had already been identified by different approaches in various organisms and exhibited regulatory functions on RNAs' fate. RBPs can either directly or indirectly interact with their target RNAs or mRNAs to assume a key biological function whose outcome may trigger disease or normal biological events. They also exert distinct functions related to their canonical and non-canonical forms. This review summarizes the current understanding of a wide range of RBPs' functions and highlights their emerging roles in the regulation of diverse pathways, different physiological processes, and their molecular links with diseases. Various types of diseases, encompassing colorectal carcinoma, non-small cell lung carcinoma, amyotrophic lateral sclerosis, and Severe acute respiratory syndrome coronavirus 2, aberrantly express RBPs. We also highlight some recent advances in the field that could prompt the development of RBPs-based therapeutic interventions.

A Yarrowia lipolytica Strain Engineered for Pyomelanin Production.

The yeast Yarrowia lipolytica naturally produces pyomelanin. This pigment accumulates in the extracellular environment following the autoxidation and polymerization of homogentisic acid, a metabolite derived from aromatic amino acids. In this study, we used a chassis strain optimized to produce aromatic amino acids for the de novo overproduction of pyomelanin. The gene 4HPPD, which encodes an enzyme involved in homogentisic acid synthesis (4-hydroxyphenylpyruvic acid dioxygenase), was characterized and overexpressed in the chassis strain with up to three copies, leading to pyomelanin yields of 4.5 g/L. Homogentisic acid is derived from tyrosine. When engineered strains were grown in a phenylalanine-supplemented medium, pyomelanin production increased, revealing that the yeast could convert phenylalanine to tyrosine, or that the homogentisic acid pathway is strongly induced by phenylalanine.

Exon junction complex components Y14 and Mago still play a role in budding yeast.

Since their divergence from Pezizomycotina, the mRNA metabolism of budding yeasts have undergone regressive evolution. With the dramatic loss of introns, a number of quality control mechanisms have been simplified or lost during evolution, such as the exon junction complex (EJC). We report the identification of the core EJC components, Mago, Y14, and eIF4A3, in at least seven Saccharomycotina species, including Yarrowia lipolytica. Peripheral factors that join EJC, either to mediate its assembly (Ibp160 or Cwc22), or trigger downstream processes, are present in the same species, forming an evolutionary package. Co-immunoprecipitation studies in Y. lipolytica showed that Mago and Y14 have retained the capacity to form heterodimers, which successively bind to the peripheral factors Upf3, Aly/REF, and Pym. Phenotypes and RNA-Seq analysis of EJC mutants showed evidence of Y14 and Mago involvement in mRNA metabolism. Differences in unspliced mRNA levels suggest that Y14 binding either interferes with pre-mRNA splicing or retains mRNA in the nucleus before their export and translation. These findings indicate that yeast could be a relevant model for understanding EJC function.

Transcriptomic response of Debaryomyces hansenii during mixed culture in a liquid model cheese medium with Yarrowia lipolytica.

Yeasts play a crucial role in cheese ripening. They contribute to the curd deacidification, the establishment of acid-sensitive bacterial communities, and flavour compounds production via proteolysis and catabolism of amino acids (AA). Negative yeast-yeast interaction was observed between the yeast Yarrowia lipolytica 1E07 (YL1E07) and the yeast Debaryomyces hansenii 1L25 (DH1L25) in a model cheese but need elucidation. YL1E07 and DH1L25 were cultivated in mono and co-cultures in a liquid synthetic medium (SM) mimicking the cheese environment and the growth inhibition of DH1L25 in the presence of YL1E07 was reproduced. We carried out microbiological, biochemical (lactose, lactate, AA consumption and ammonia production) and transcriptomic analyses by microarray technology to highlight the interaction mechanisms. We showed that the DH1L25 growth inhibition in the presence of YL1E07 was neither due to the ammonia production nor to the nutritional competition for the medium carbon sources between the two yeasts. The transcriptomic study was the key toward the comprehension of yeast-yeast interaction, and revealed that the inhibition of DH1L25 in co-culture is due to a decrease of the mitochondrial respiratory chain functioning.

Multiple Parameters Drive the Efficiency of CRISPR/Cas9-Induced Gene Modifications in Yarrowia lipolytica.

Yarrowia lipolytica is an oleaginous yeast of growing industrial interest for biotechnological applications. In the last few years, genome edition has become an easier and more accessible prospect with the world wild spread development of CRISPR/Cas9 technology. In this study, we focused our attention on the production of the two key elements of the CRISPR-Cas9 ribonucleic acid protein complex in this non-conventional yeast. The efficiency of NHEJ-induced knockout was measured by time-course monitoring using multiple parameters flow cytometry, as well as phenotypic and genotypic observations, and linked to nuclease production levels showing that its strong overexpression is unnecessary. Thus, the limiting factor for the generation of a functional ribonucleic acid protein complex clearly resides in guide expression, which was probed by testing different linker lengths between the transfer RNA promoter and the sgRNA. The results highlight a clear deleterious effect of mismatching bases at the 5' end of the target sequence. For the first time in yeast, an investigation of its maturation from the primary transcript was undertaken by sequencing multiple sgRNAs extracted from the host. These data provide insights into of the yeast small RNA processing, from synthesis to maturation, and suggests a pathway for their degradation in Y. lipolytica. Subsequently, a whole-genome sequencing of a modified strain detected no abnormal modification due to off-target effects, confirming CRISPR/Cas9 as a safe strategy for editing Y. lipolytica genome. Finally, the optimized system was used to promote in vivo directed mutagenesis via homology-directed repair with a ssDNA oligonucleotide.

Differential gene retention as an evolutionary mechanism to generate biodiversity and adaptation in yeasts.

The evolutionary history of the characters underlying the adaptation of microorganisms to food and biotechnological uses is poorly understood. We undertook comparative genomics to investigate evolutionary relationships of the dairy yeast Geotrichum candidum within Saccharomycotina. Surprisingly, a remarkable proportion of genes showed discordant phylogenies, clustering with the filamentous fungus subphylum (Pezizomycotina), rather than the yeast subphylum (Saccharomycotina), of the Ascomycota. These genes appear not to be the result of Horizontal Gene Transfer (HGT), but to have been specifically retained by G. candidum after the filamentous fungi-yeasts split concomitant with the yeasts' genome contraction. We refer to these genes as SRAGs (Specifically Retained Ancestral Genes), having been lost by all or nearly all other yeasts, and thus contributing to the phenotypic specificity of lineages. SRAG functions include lipases consistent with a role in cheese making and novel endoglucanases associated with degradation of plant material. Similar gene retention was observed in three other distantly related yeasts representative of this ecologically diverse subphylum. The phenomenon thus appears to be widespread in the Saccharomycotina and argues that, alongside neo-functionalization following gene duplication and HGT, specific gene retention must be recognized as an important mechanism for generation of biodiversity and adaptation in yeasts.

Overview of a surface-ripened cheese community functioning by meta-omics analyses.

Cheese ripening is a complex biochemical process driven by microbial communities composed of both eukaryotes and prokaryotes. Surface-ripened cheeses are widely consumed all over the world and are appreciated for their characteristic flavor. Microbial community composition has been studied for a long time on surface-ripened cheeses, but only limited knowledge has been acquired about its in situ metabolic activities. We applied metagenomic, metatranscriptomic and biochemical analyses to an experimental surface-ripened cheese composed of nine microbial species during four weeks of ripening. By combining all of the data, we were able to obtain an overview of the cheese maturation process and to better understand the metabolic activities of the different community members and their possible interactions. Furthermore, differential expression analysis was used to select a set of biomarker genes, providing a valuable tool that can be used to monitor the cheese-making process.

Chemical sensing of DNT by engineered olfactory yeast strain.

With the increasing threat of environmental toxicants including biological and chemical warfare agents, fabricating innovative biomimetic systems to detect these harmful agents is critically important. With the broad objective of developing such a biosensor, here we report the construction of a Saccharomyces cerevisiae strain containing the primary components of the mammalian olfactory signaling pathway. In this engineered yeast strain, WIF-1alpha, olfactory receptor signaling is coupled to green fluorescent protein expression. Using this 'olfactory yeast', we screened for olfactory receptors that could report the presence of the odorant 2,4-dinitrotoluene, an explosive residue mimic. With this approach, we have identified the novel rat olfactory receptor Olfr226, which is closely related to the mouse olfactory receptors Olfr2 and MOR226-1, as a 2,4-dinitrotoluene-responsive receptor.

Galpha13 stimulates cell migration through cortactin-interacting protein Hax-1.

Galpha13, the alpha-subunit of the heterotrimeric G protein G13, has been shown to stimulate cell migration in addition to inducing oncogenic transformation. Cta, a Drosophila ortholog of G13, has been shown to be critical for cell migration leading to the ventral furrow formation in Drosophila embryos. Loss of Galpha13 has been shown to disrupt cell migration associated with angiogenesis in developing mouse embryos. Whereas these observations point to the vital role of G13-orthologs in regulating cell migration, widely across the species barrier, the mechanism by which Galpha13 couples to cytoskeleton and cell migration is largely unknown. Here we show that Galpha13 physically interacts with Hax-1, a cytoskeleton-associated, cortactin-interacting intracellular protein, and this interaction is required for Galpha13-stimulated cell migration. Hax-1 interaction is specific to Galpha13, and this interaction is more pronounced with the mutationally or functionally activated form of Galpha13 as compared with the wild-type Galpha13. Expression of Hax-1 reduces the formation of actin stress fibers and focal adhesion complexes in Galpha13-expressing NIH3T3 cells. Coexpression of Hax-1 also attenuates Galpha(13)-stimulated activity of Rho while potentiating Galpha13-stimulated activity of Rac. The presence of a quadnary complex consisting of Galpha13, Hax-1, Rac, and cortactin indicates the role of Hax-1 in tethering Galpha13 to the cytoskeletal component(s) involved in cell movement. Whereas the expression of Hax-1 potentiates Galpha13-mediated cell movement, silencing of endogenous Hax-1 with Hax-1-specific small interfering RNAs drastically reduces Galpha13-mediated cell migration. These findings, along with the observation that Hax-1 is overexpressed in metastatic tumors and tumor cell lines, suggest a novel role for the association of oncogenic Galpha13 and Hax-1 in tumor metastasis.

JLP: A scaffolding protein that tethers JNK/p38MAPK signaling modules and transcription factors.

Extracellular signals are transduced into cells through mitogen-activated protein kinases (MAPKs), which are activated by their upstream kinases. Recently, families of scaffolding proteins have been identified to tether specific combinations of these kinases along specific signaling pathways. Here we describe a protein, JLP (c-Jun NH2-terminal kinase-associated leucine zipper protein), which acts as a scaffolding protein to bring together Max and c-Myc along with JNK (c-Jun NH2-terminal kinase) and p38MAPK, as well as their upstream kinases MKK4 (MAPK kinase 4) and MEKK3 (MAPK kinase kinase 3). Thus, JLP defines a family of scaffolding proteins that bring MAPKs and their target transcription factors together for the execution of specific signaling pathways.