Laura Tomás-Gallardo

Salmonella enterica has evolved an arsenal of effector proteins secreted via type III secretion systems (T3SS) to manipulate host cell functions. Among these, the NEL family E3 ubiquitin ligases (SlrP, SspH1, and SspH2) are known to modulate immune signaling, but the breadth of their impact on the host ubiquitylome remains unexplored. In this study, we have performed a global proteomic analysis to identify host proteins ubiquitylated in response to expression of these three effectors in human cells. Using enrichment strategies combined with mass spectrometry under conditions where the proteasome is active or not, we identified 214 putative substrates of ubiquitylation. Gene ontology and KEGG pathway analysis revealed enrichment in pathways related to RNA processing, ribosome biogenesis, cytoskeleton organization, chromatin remodeling, and vesicular trafficking. In vitro ubiquitylation assays validated five novel substrates and revealed differential substrate specificity and patterns of ubiquitin chain topology among the effectors. Notably, expression of SspH1 in Saccharomyces cerevisiae disrupted polysome profiles in a ligase activity dependent manner, indicating a direct impact of the bacterial effector on translation of eukaryotic cells. Comparison with previously published global interactomes and ubiquitylomes supports a model in which Salmonella NEL effectors subvert a broader range of host pathways through targeted ubiquitylation. Our findings uncover new roles for NEL ubiquitin ligases in host manipulation and provide a holistic analysis of their effects on the ubiquitylome of the host cell, constituting a valuable resource for the study of bacterial pathogenesis and infection biology.

High-throughput screening studies provide an additional approach to discovering repurposed drugs for antimicrobial treatments. In this work, we report the identification of ENOblock, an anticancer drug, as an antimicrobial agent. We computationally and experimentally validated that ENOblock synergizes with colistin, the last resort antibiotic. Additionally, we identified enolase as the potential bacterial target for ENOblock. The in silico and in vitro antibacterial activity of ENOblock translated into potent in vivo efficacy in an animal infection model. Collectively, the preclinical data support the selection of ENOblock as a promising candidate for antimicrobial development, with the potential to address the urgent threat of infections caused by Acinetobacter baumannii .

CRISPR-Cas13 RNA-targeting systems are widely used in basic and applied sciences. However, its application has recently generated controversy due to collateral activity in mammalian cells and mouse models. Moreover, its competence could be improved in vivo. Here, we optimized transient formulations as ribonucleoprotein complexes or mRNA-gRNA combinations to enhance the CRISPR-RfxCas13d system in zebrafish. We i) use chemically modified gRNAs to allow more penetrant loss-of-function phenotypes, ii) improve nuclear RNA targeting, and iii) compare different computational models and determine the most accurate to predict gRNA activity in vivo. Furthermore, we demonstrate that transient CRISPR-RfxCas13d can effectively deplete endogenous mRNAs in zebrafish embryos without inducing collateral effects, except when targeting extremely abundant and ectopic RNAs. Finally, we implement alternative RNA-targeting CRISPR-Cas systems such as CRISPR-Cas7-11 and CRISPR-DjCas13d. Altogether, these findings contribute to CRISPR-Cas technology optimization for RNA targeting in zebrafish through transient approaches and assist in the progression of in vivo applications.

Summary: The appressorium of phytopathogenic fungi is a specific structure with a crucial role in plant cuticle penetration. Pathogens with melanized appressoria break the cuticle through cell wall melanization and intracellular turgor pressure. However, in fungi with nonmelanized appressorium, the mechanisms governing cuticle penetration are poorly understood. Here we characterize Row1, a previously uncharacterized appressoria‐specific protein of Ustilago maydis that localizes to membrane and secretory vesicles. Deletion of row1 decreases appressoria formation and plant penetration, thereby reducing virulence. Specifically, the Δrow1 mutant has a thicker cell wall that is more resistant to glucanase degradation. We also observed that the Δrow1 mutant has secretion defects. We show that Row1 is functionally conserved at least among Ustilaginaceae and belongs to the Row family, which consists of five other proteins that are highly conserved among Basidiomycota fungi and are involved in U. maydis virulence. We observed similarities in localization between Row1 and Row2, which is also involved in cell wall remodelling and secretion, suggesting similar molecular functions for members of this protein family. Our data suggest that Row1 could modify the chitin‐glucan matrix of the fungal cell wall and may be involved in unconventional protein secretion, thereby promoting both appressoria maturation and penetration.

Bacteria must be provided with a battery of tools integrated into regulatory networks, in order to respond and, consequently, adapt their physiology to changing environments. Within these networks, transcription factors finely orchestrate the expression of genes in response to a variety of signals, by recognizing specific DNA sequences at their promoter regions. Rhizobia are host-interacting soil bacteria that face severe changes to adapt their physiology from free-living conditions to the nitrogen-fixing endosymbiotic state inside root nodules associated with leguminous plants. One of these cues is the low partial pressure of oxygen within root nodules. Surface plasmon resonance (SPR) constitutes a technique that allows to measure molecular interactions dynamics at real time by detecting changes in the refractive index of a surface. Here, we implemented the SPR methodology to analyze the discriminatory determinants of transcription factors for specific interaction with their target genes. We focused on FixK₂, a CRP/FNR-type protein with a central role in the complex oxygen-responsive regulatory network in the soybean endosymbiont Bradyrhizobium diazoefficiens. Our study unveiled relevant residues for protein-DNA interaction as well as allowed us to monitor kinetics and stability protein-DNA complex. We believe that this approach can be employed for the characterization of other relevant transcription factors which can assist to the better understanding of the adaptation of bacteria with agronomic or human interest to their different modes of life. Key words CRP/FNR-type proteins Microoxia Nitrogen-fixing symbiosis Promoter Protein-DNA interaction Rhizobia SPR Transcription factors

Summary: The appressorium of phytopathogenic fungi is a specific structure with a crucial role in plant cuticle penetration. Pathogens with melanized appressoria break the cuticle through cell wall melanization and intracellular turgor pressure. However, in fungi with non-melanized appressorium, the mechanisms governing cuticle penetration are poorly understood. Here we characterize Row1, a previously uncharacterized appressoria-specific protein of Ustilago maydis that localizes to membrane and secretory vesicles. Deletion of row1 decrease appressoria formation and plant penetration, thereby reducing virulence. Specifically, the Δ row1 mutant has a thicker cell wall that is more resistant to glucanase degradation. We also observed that the Δ row1 mutant has secretion defects. Our data suggest that Row1 could modify the glucans that form the fungal cell wall and may be involved in unconventional protein secretion, thereby promoting both appressoria maturation and penetration. We show that Row1 is functionally conserved at least among Ustilaginaceae and belongs to the Row family, which consists of five other proteins that are highly conserved among Basidiomycota fungi and are involved in U. maydis virulence. We observed similarities in localization between Row1 and Row2, which is also involved in cell wall remodelling and secretion, suggesting similar molecular functions for members of this protein family.

FixK₂ is a CRP/FNR-type transcription factor that plays a central role in a sophisticated regulatory network for the anoxic, microoxic and symbiotic lifestyles of the soybean endosymbiont Bradyrhizobium diazoefficiens. Aside from the balanced expression of the fixK₂ gene under microoxic conditions (induced by the two-component regulatory system FixLJ and negatively auto-repressed), FixK₂ activity is posttranslationally controlled by proteolysis, and by the oxidation of a singular cysteine residue (C183) near its DNA-binding domain. To simulate the permanent oxidation of FixK₂, we replaced C183 for aspartic acid. Purified C183D FixK₂ protein showed both low DNA binding and in vitro transcriptional activation from the promoter of the fixNOQP operon, required for respiration under symbiosis. However, in a B. diazoefficiens strain coding for C183D FixK₂, expression of a fixNOQP’-‘lacZ fusion was similar to that in the wild type, when both strains were grown microoxically. The C183D FixK₂ encoding strain also showed a wild-type phenotype in symbiosis with soybeans, and increased fixK₂ gene expression levels and FixK₂ protein abundance in cells. These two latter observations, together with the global transcriptional profile of the microoxically cultured C183D FixK₂ encoding strain, suggest the existence of a finely tuned regulatory strategy to counterbalance the oxidation-mediated inactivation of FixK₂ in vivo.

Summary: The FixK ₂ protein plays a pivotal role in a complex regulatory network, which controls genes for microoxic, denitrifying, and symbiotic nitrogen‐fixing lifestyles in Bradyrhizobium diazoefficiens . Among the microoxic‐responsive FixK ₂ ‐activated genes are the fixNOQP operon, indispensable for respiration in symbiosis, and the nnrR regulatory gene needed for the nitric‐oxide dependent induction of the norCBQD genes encoding the denitrifying nitric oxide reductase. FixK ₂ is a CRP/FNR‐type transcription factor, which recognizes a 14 bp‐palindrome (FixK ₂ box) at the regulated promoters through three residues (L195, E196, and R200) within a C‐terminal helix‐turn‐helix motif. Here, we mapped the determinants for discriminatory FixK ₂ ‐mediated regulation. While R200 was essential for DNA binding and activity of FixK ₂ , L195 was involved in protein–DNA complex stability. Mutation at positions 1, 3, or 11 in the genuine FixK ₂ box at the fixNOQP promoter impaired transcription activation by FixK ₂ , which was residual when a second mutation affecting the box palindromy was introduced. The substitution of nucleotide 11 within the NnrR box at the norCBQD promoter allowed FixK ₂ ‐mediated activation in response to microoxia. Thus, position 11 within the FixK ₂ /NnrR boxes constitutes a key element that changes FixK ₂ targets specificity, and consequently, it might modulate B. diazoefficiens lifestyle as nitrogen fixer or as denitrifier.