Distinct roles for Arabidopsis SUMO protease ESD4 and its closest homolog ELS1

SUMO conjugation affects a broad range of processes in Arabidopsis thaliana, including flower initiation, pathogen defense, and responses to cold, drought and salt stress. We investigated two sequence-related SUMO-specific proteases that are both widely expressed and show that they differ significantly in their properties. The closest homolog of SUMO protease ESD4, ESD4-LIKE SUMO PROTEASE 1 (ELS1, alternatively called AtULP1a) has SUMO-specific proteolytic activity, but is functionally distinct from ESD4, as shown by intracellular localization, mutant phenotype and heterologous expression in yeast mutants. Furthermore, we show that the growth defects caused by loss of ESD4 function are not due to increased synthesis of the stress signal salicylic acid, as was previously shown for a SUMO ligase, indicating that impairment of the SUMO system affects plant growth in different ways. Our results demonstrate that two A. thaliana SUMO proteases showing close sequence similarity have distinct in vivo functions

Two Acinetobacter baumannii Isolates Obtained From a Fatal Necrotizing Fasciitis Infection Display Distinct Genomic and Phenotypic Characteristics in Comparison to Type Strains

Acinetobacter baumannii has been recognized as a critical pathogen that causes severe infections worldwide not only because of the emergence of extensively drug-resistant (XDR) derivatives, but also because of its ability to persist in medical environments and colonize compromised patients. While there are numerous reports describing the mechanisms by which this pathogen acquires resistance genes, little is known regarding A. baumannii's virulence functions associated with rare manifestations of infection such as necrotizing fasciitis, making the determination and implementation of alternative therapeutic targets problematic. To address this knowledge gap, this report describes the analysis of the NFAb-1 and NFAb-2 XDR isolates, which were obtained at two time points during a fatal case of necrotizing fasciitis, at the genomic and functional levels. The comparative genomic analysis of these isolates with the ATCC 19606T and ATCC 17978 strains showed that the NFAb-1 and NFAb-2 isolates are genetically different from each other as well as different from the ATCC 19606T and ATCC 17978 clinical isolates. These genomic differences could be reflected in phenotypic differences observed in these NFAb isolates. Biofilm, cell viability and flow cytometry assays indicate that all tested strains caused significant decreases in A549 human alveolar epithelial cell viability with ATCC 17978, NFAb-1 and NFAb-2 producing significantly less biofilm and significantly more hemolysis and capacity for intracellular invasion than ATCC 19606T. NFAb-1 and NFAb-2 also demonstrated negligible surface motility but significant twitching motility compared to ATCC 19606T and ATCC 17978, likely due to the presence of pili exceeding 2 µm in length, which are significantly longer and different from those previously described in the ATCC 19606T and ATCC 17978 strains. Interestingly, infection with cells of the NFAb-1 isolate, which were obtained from a premortem blood sample, lead to significantly higher mortality rates than NFAb-2 bacteria, which were obtained from postmortem tissue samples, when tested using the Galleria mellonella in vivo infection model. These observations suggest potential changes in the virulence phenotype of the A. baumannii necrotizing fasciitis isolates over the course of infection by mechanisms and cell processes that remain to be identified

Arabidopsis thaliana SPF1 and SPF2 are nuclear-located ULP2-like SUMO proteases that act downstream of SIZ1 in plant development

Post-translational modifiers such as the small ubiquitin-like modifier (SUMO) peptide act as fast and reversible protein regulators. Functional characterization of the sumoylation machinery has determined the key regulatory role that SUMO plays in plant development. Unlike components of the SUMO conjugation pathway, SUMO proteases (ULPs) are encoded by a relatively large gene family and are potential sources of specificity within the pathway. This study reports a thorough comparative genomics and phylogenetic characterization of plant ULPs, revealing the presence of one ULP1-like and three ULP2-like SUMO protease subgroups within plant genomes. As representatives of an under-studied subgroup, Arabidopsis SPF1 and SPF2 were subjected to functional characterization. Loss-of-function mutants implicated both proteins with vegetative growth, flowering time, and seed size and yield. Mutants constitutively accumulated SUMO conjugates, and yeast complementation assays associated these proteins with the function of ScUlp2 but not ScUlp1. Fluorescence imaging placed both proteins in the plant cell nucleoplasm. Transcriptomics analysis indicated strong regulatory involvement in secondary metabolism, cell wall remodelling, and nitrate assimilation. Furthermore, developmental defects of the spf1-1 spf2-2 (spf1/2) double-mutant opposed those of the major E3 ligase siz1 mutant and, most significantly, developmental and transcriptomic characterization of the siz1 spf1/2 triple-mutant placed SIZ1 as epistatic to SPF1 and SPF2.We thank Mark Hochstrasser (Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, USA) for kindly providing the ulp1-ts yeast mutant strain. This research was funded by FEDER (through COMPETE), and by Fundacao para a Ciencia e Tecnologia (FCT), within the scope of project SUMOdulator (FCOMP-01-0124-FEDER-028459 and PTDC/BIA-PLA/3850/2012). PHC was supported by FCT (SFRH/BD/44484/2008). HA and FF were supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) (NORTE-01-0145-FEDER-000007 and Norte-01-0145-FEDER-000008, respectively). The work was supported by FEDER through the COMPETE 2020-Operacional Programme for Competitiveness and Internationalisation (POCI), Portugal 2020, and by Portuguese funds through FCT, within the framework of projects 'Rede de Investigacao em Biodiversidade e Biologia Evolutiva' (POCI-01-0145-FEDER-006821) and 'Institute for Research and Innovation in Health Sciences' (POCI-01-0145-FEDER-007274). This research was also supported by a grant from the Spanish Ministerio de Ciencia y Tecnologia (AGL2016-75819-C2-1-R) and FEDER (PCQ, AGC, ERB)

Detection and Functional Characterization of a 215 Amino Acid N-Terminal Extension in the Xanthomonas Type III Effector XopD

During evolution, pathogens have developed a variety of strategies to suppress plant-triggered immunity and promote successful infection. In Gram-negative phytopathogenic bacteria, the so-called type III protein secretion system works as a molecular syringe to inject type III effectors (T3Es) into plant cells. The XopD T3E from the strain 85-10 of Xanthomonas campestris pathovar vesicatoria (Xcv) delays the onset of symptom development and alters basal defence responses to promote pathogen growth in infected tomato leaves. XopD was previously described as a modular protein that contains (i) an N-terminal DNA-binding domain (DBD), (ii) two tandemly repeated EAR (ERF-associated amphiphillic repression) motifs involved in transcriptional repression, and (iii) a C-terminal cysteine protease domain, involved in release of SUMO (small ubiquitin-like modifier) from SUMO-modified proteins. Here, we show that the XopD protein that is produced and secreted by Xcv presents an additional N-terminal extension of 215 amino acids. Closer analysis of this newly identified N-terminal domain shows a low complexity region rich in lysine, alanine and glutamic acid residues (KAE-rich) with high propensity to form coiled-coil structures that confers to XopD the ability to form dimers when expressed in E. coli. The full length XopD protein identified in this study (XopD1-760) displays stronger repression of the XopD plant target promoter PR1, as compared to the XopD version annotated in the public databases (XopD216-760). Furthermore, the N-terminal extension of XopD, which is absent in XopD216-760, is essential for XopD type III-dependent secretion and, therefore, for complementation of an Xcv mutant strain deleted from XopD in its ability to delay symptom development in tomato susceptible cultivars. The identification of the complete sequence of XopD opens new perspectives for future studies on the XopD protein and its virulence-associated functions in planta

Patient engagement in fertility research: bench research, ethics, and social justice

Abstract Background Patient and Public Involvement (PPI) in research is increasingly being utilized to better connect patients and researchers. The Patient Engagement Studio (PES) supports PPI in research by working directly with researchers throughout various stages of their projects. Recently, two researchers presented to the PES for assistance with their project, Embryo+™. The purpose of Embryo+™ is to decrease miscarriage rates using RNA sequencing technology that screens for the most viable embryos. To date, no examples of PPI directly in the planning or implementation of bench research concerning in vitro fertilization and embryo transfer have been identified. Main body Embryo+™ researchers met in-person with the PES two times (fall 2019; each meeting had 9 PES members in attendance) for initial feedback and protocol development. After these meetings, PES leadership and Embryo+™ researchers decided that the unique nature of the project merited a PPI evaluation. Subsequent evaluation of engagement efforts occurred by reviewing the PES reports for the Embryo+™ researchers, conducting two recorded web-based discussion meetings with the PES (summer 2020; meeting 1 n = 7; meeting 2 n = 6), and a brief survey (n = 13). The discussion meetings provided an opportunity for the PES members to define engagement themes through consensus via verbal agreement to the studio director’s periodic summaries during the discussions. Combining survey results and PES themes allowed for a broad discussion for meaningful engagement. The Embryo+™ researchers established trust with the patients by changing some of their language in response to patient suggestions, allowing for unintended ethical conversations, and implementing the patient developed protocols. Overall, the patient experts thought this project was very meaningful and valuable, quantified by a mean loyalty score 89.43 (s.d. 10.29). Conclusion Bench science researchers may need additional PPI training prior to engaging with patient groups. PPI in this project was successful in large part due to this training, where the director emphasized the importance of gaining trust with the patients. The researchers applied what they learned and several examples of how to develop trust with patients are discussed. If trust is established, PPI in an ethically charged, basic science research study can be both valuable and successful

Genome-scale epigenetic reprogramming during epithelial-to-mesenchymal transition

Epithelial to mesenchymal transition (EMT) is an extreme example of cell plasticity, important for normal development, injury repair, and malignant progression. Widespread epigenetic reprogramming occurs during stem cell differentiation and malignant transformation, but EMT-related epigenetic reprogramming is poorly understood. Here we investigated epigenetic modifications during TGF-β-mediated EMT. While DNA methylation was unchanged during EMT, we found global reduction of the heterochromatin mark H3-lys9 dimethylation (H3K9Me2), increase of the euchromatin mark H3-lys4 trimethylation (H3K4Me3), and increase of the transcriptional mark H3-lys36 trimethylation (H3K36Me3). These changes were largely dependent on lysine-specific deaminase-1 (Lsd1), and Lsd1 loss-of-function experiments showed marked effects on EMT-driven cell migration and chemoresistance. Genome-scale mapping revealed that chromatin changes were largely specific to large organized heterochromatin K9-modifications (LOCKs), suggesting that EMT is characterized by reprogramming of specific chromatin domains across the genome