Preparing for patients with high-consequence infectious diseases: Example of a high-level isolation unit.

INTRODUCTION: Patients with high-consequence infectious diseases (HCID) are rare in Western Europe. However, high-level isolation units (HLIU) must always be prepared for patient admission. Case fatality rates of HCID can be reduced by providing optimal intensive care management. We here describe a single centre's preparation, its embedding in the national context and the challenges we faced during the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) pandemic. METHODS: Ten team leaders organize monthly whole day trainings for a team of doctors and nurses from the HLIU focusing on intensive care medicine. Impact and relevance of training are assessed by a questionnaire and a perception survey, respectively. Furthermore, yearly exercises with several partner institutions are performed to cover different real-life scenarios. Exercises are evaluated by internal and external observers. Both training sessions and exercises are accompanied by intense feedback. RESULTS: From May 2017 monthly training sessions were held with a two-month and a seven-month break due to the first and second wave of the SARS-CoV-2 pandemic, respectively. Agreement with the statements of the questionnaire was higher after training compared to before training indicating a positive effect of training sessions on competence. Participants rated joint trainings for nurses and doctors at regular intervals as important. Numerous issues with potential for improvement were identified during post processing of exercises. Action plans for their improvement were drafted and as of now mostly implemented. The network of the permanent working group of competence and treatment centres for HCID (Ständiger Arbeitskreis der Kompetenz- und Behandlungszentren für Krankheiten durch hochpathogene Erreger (STAKOB)) at the Robert Koch-Institute (RKI) was strengthened throughout the SARS-CoV-2 pandemic. DISCUSSION: Adequate preparation for the admission of patients with HCID is challenging. We show that joint regular trainings of doctors and nurses are appreciated and that training sessions may improve perceived skills. We also show that real-life scenario exercises may reveal additional deficits, which cannot be easily disclosed in training sessions. Although the SARS-CoV-2 pandemic interfered with our activities the enhanced cooperation among German HLIU during the pandemic ensured constant readiness for the admission of HCID patients to our or to collaborating HLIU. This is a single centre's experience, which may not be generalized to other centres. However, we believe that our work may address aspects that should be considered when preparing a unit for the admission of patients with HCID. These may then be adapted to the local situations.

The Arabidopsis NIMIN proteins affect NPR1 differentially.

NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) is the central regulator of the pathogen defense reaction systemic acquired resistance (SAR). NPR1 acts by sensing the SAR signal molecule salicylic acid (SA) to induce expression of PATHOGENESIS-RELATED (PR) genes. Mechanistically, NPR1 is the core of a transcription complex interacting with TGA transcription factors and NIM1-INTERACTING (NIMIN) proteins. Arabidopsis NIMIN1 has been shown to suppress NPR1 activity in transgenic plants. The Arabidopsis NIMIN family comprises four structurally related, yet distinct members. Here, we show that NIMIN1, NIMIN2, and NIMIN3 are expressed differentially, and that the encoded proteins affect expression of the SAR marker PR-1 differentially. NIMIN3 is expressed constitutively at a low level, but NIMIN2 and NIMIN1 are both responsive to SA. While NIMIN2 is an immediate early SA-induced and NPR1-independent gene, NIMIN1 is activated after NIMIN2, but clearly before PR-1. Notably, NIMIN1, like PR-1, depends on NPR1. In a transient assay system, NIMIN3 suppresses SA-induced PR-1 expression, albeit to a lesser extent than NIMIN1, whereas NIMIN2 does not negatively affect PR-1 gene activation. Furthermore, although binding to the same domain in the C-terminus, NIMIN1 and NIMIN2 interact differentially with NPR1, thus providing a molecular basis for their opposing effects on NPR1. Together, our data suggest that the Arabidopsis NIMIN proteins are regulators of the SAR response. We propose that NIMINs act in a strictly consecutive and SA-regulated manner on the SA sensor protein NPR1, enabling NPR1 to monitor progressing threat by pathogens and to promote appropriate defense gene activation at distinct stages of SAR. In this scenario, the defense gene PR-1 is repressed at the onset of SAR by SA-induced, yet instable NIMIN1.

NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1) and some NPR1-related proteins are sensitive to salicylic acid.

NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1; also known as NIM1) is a master regulator of systemic acquired resistance (SAR). SAR is induced by salicylic acid (SA), leading to the expression of PATHOGENESIS-RELATED (PR) genes. Current evidence suggests that NPR1 is part of a transcription complex tethered to activation sequence-1 (as-1)-like cis-acting elements in PR-1 gene promoters through TGA transcription factors, and that SA-dependent PR-1 gene expression is regulated by NIM1-INTERACTING (NIMIN) proteins. In Arabidopsis, NPR1 is active only after SA induction. Regulation of Arabidopsis NPR1 activity has been proposed to comprise cysteine-156 (Cys-156), mediating SA-induced cytoplasmic oligomer-nuclear monomer exchange, and Cys-521 and Cys-529, mediating SA-dependent transcriptional activation. Tobacco NPR1 does not harbour these residues. To understand the function of tobacco NPR1, we analysed its biochemical capabilities in a heterologous system: yeast. Tobacco NPR1 differs from Arabidopsis NPR1 in its subcellular localization and its transactivation potential. Yet, both tobacco and Arabidopsis NPR1, as well as tobacco NIM1-like1, alter some of their biochemical activities in response to SA. Whereas the addition of SA to yeast growth medium induces transcriptional activity in tobacco NPR1, its interaction with NIMIN2-type proteins is suppressed. The effects of SA are specific, sensitive and occur coordinately. They are abolished completely by mutation of the arginine residue within the invariable penta-amino acid motif LENRV, as present in the nonfunctional Arabidopsis nim1-4 allele. Furthermore, NPR1 proteins with the LENRV domain coincidently harbour a broad and strongly conserved NIMIN1/NIMIN2 binding site. Our data suggest that NPR1 and some NPR1-like proteins are sensitive to the plant hormone SA, altering some of their biochemical capabilities to enable stimulus-dependent gene expression. The sensitivity of NPR1 proteins to SA, together with their differential interaction with diverse NIMIN proteins, seems a plausible molecular basis for the timely and coordinated activation of PR genes during SAR.

Tobacco NIMIN2 proteins control PR gene induction through transient repression early in systemic acquired resistance.

SUMMARY: NPR1 (for Nonexpressor of PR genes; also known as NIM1) is a positive regulator of systemic acquired resistance (SAR) in Arabidopsis, which controls the induction of Pathogenesis-Related (PR) genes by salicylic acid (SA). NPR1 interacts with members of two protein families, TGA transcription factors and NIMIN (for NIM1-interacting) proteins. In Arabidopsis, NIMIN1, NIMIN2 and NIMIN3 constitute a small gene family of structurally related, yet distinct members. To unravel the biological significance of NIMIN interaction with NPR1, we searched a tobacco yeast two-hybrid cDNA library for NPR1- and NIMIN2-binding proteins. One NPR1 cDNA clone and three clones encoding NIMIN proteins were isolated. Although clearly similar to At NPR1, Nt NPR1 does not interact with At NIMIN3. Furthermore, all Nt NIMIN proteins identified are structurally related to At NIMIN2, thus forming a small NIMIN2 subfamily in tobacco. cDNA clones encoding At NIMIN1 or At NIMIN3 homologues were not identified. The function of NIMIN2 proteins was studied by expression of Nt NIMIN2a chimeric genes in tobacco. While constitutive NIMIN2a over-expression delayed PR-1 protein induction, suppression of NIMIN2 transcripts enhanced the accumulation of PR-1 proteins. In both cases, the effects of altered NIMIN2 transcript levels became evident foremost early in SAR. Notably, Nt NIMIN2 gene expression is elevated prior to the induction of the PR-1a gene. Together, the data suggest that, in tobacco, NIMIN2 proteins control PR-1 gene expression, and that NIMIN2-mediated control is exerted through transient PR-1 repression before SAR has fully developed. Furthermore, although sharing conserved domains and functions, tobacco and Arabidopsis NPR1 and NIMIN proteins are clearly distinct.

Interaction of NIMIN1 with NPR1 modulates PR gene expression in Arabidopsis.

The Arabidopsis thaliana NONEXPRESSER OF PR GENES1 (NPR1, also known as NIM1) protein is an essential positive regulator of salicylic acid (SA)-induced PATHOGENESIS-RELATED (PR) gene expression and systemic acquired resistance (SAR). PR gene activity is regulated at the level of redox-dependent nuclear transport of NPR1. NPR1 interacts with members of the TGA family of transcription factors that are known to bind to SA-responsive elements in the PR-1 promoter. In an attempt to identify proteins involved in SA-mediated signal transduction, we previously described the isolation of three novel genes encoding distinct albeit structurally related proteins designated NIMIN1 (for NIM1-INTERACTING1), NIMIN2, and NIMIN3 that interact with NPR1 in the yeast two-hybrid system. Here, we show that NIMIN1 and NPR1 can be copurified from plant extracts, providing biochemical evidence for their interaction. We provide functional evidence for this interaction by describing transgenic plants constitutively expressing high amounts of NIMIN1. These plants show reduced SA-mediated PR gene induction and a compromised SAR, thus mimicking the described phenotype conferred by npr1. Moreover, they showed reduced RESISTANCE gene-mediated protection. These effects were dependent on the ability of NIMIN1 to interact with NPR1. Mutant plants with a T-DNA insertion in NIMIN1 as well as transgenic plants with reduced NIMIN1 mRNA levels showed hyperactivation of PR-1 gene expression after SA treatment but no effect on the disease resistance phenotype. Our results strongly suggest that NIMIN1 negatively regulates distinct functions of NPR1, providing a mechanism to modulate specific features of SAR.

Salicylic acid (SA)-dependent gene activation can be uncoupled from cell death-mediated gene activation: the SA-inducible NIMIN-1 and NIMIN-2 promoters, unlike the PR-1a promoter, do not respond to cell death signals in tobacco.

SUMMARY Tobacco pathogenesis-related (PR) genes of group 1 are induced during pathogen defence (hypersensitive response, HR, and systemic acquired resistance, SAR), after exogenous application of salicylic acid (SA), and by developmental cues. Likewise, SA enhances transcripts for Arabidopsis NIMIN-1 and NIMIN-2, which interact with NPR1/NIM1, a key regulator of SAR. To further illuminate gene activation during pathogen defence, reporter gene expression from the NIMIN-1 and NIMIN-2 promoters was analysed in transgenic tobacco plants in direct comparison to PR-1 gene expression. NIMIN[GUS] chimeric genes were highly sensitive to SA, whereas NIMIN[GUS], unlike PR1a[GUS], expression was only weak in necrotic tissue exhibiting HR. Furthermore, PR-1a, but not NIMIN, promoter constructs were activated systemically in response to local cell death elicited by expression of the proapoptotic Bax gene. Conversely, NIMIN-1[GUS] expression was completely suppressed during pathogen defence in plants depleted from SA, whereas PR-1 proteins still accumulated in necrotic tissue. These findings demonstrate that SA-dependent gene activation can be uncoupled from cell death-induced gene activation. Whereas PR-1a induction during the HR and SAR responses is mediated by HR-associated signals and SA, activation of the NIMIN-1 and NIMIN-2 promoters in infected tobacco relies on SA, but not on cell death signals.

Salicylic acid and the hypersensitive response initiate distinct signal transduction pathways in tobacco that converge on the as-1-like element of the PR-1a promoter.

Tobacco pathogenesis-related protein 1a (PR-1a) is induced in plants during the hypersensitive response (HR) after exposure of plants to salicylic acid (SA) and by developmental cues. Gene activation by these diverse stimuli is mediated via an as-1-like element in the PR-1a upstream region. To further analyze the significance of this cis-acting sequence, an authentic as-1 element from the cauliflower mosaic virus 35S RNA promoter was inserted into the PR-1a promoter in place of the as-1-like motif. Reporter gene analysis in transgenic tobacco plants demonstrated that as-1 can functionally replace the as-1-like element in the PR-1a promoter in response to all stimuli. However, reporter gene induction from the as-1 carrying promoter was enhanced in response to SA compared to the wild-type promoter, and the ratio of reporter gene activities in SA treated leaf tissue to tissue exhibiting the HR increased with the as-1 promoter construct. Our findings support a model where PR-1a gene expression relies on at least two distinct signal transduction pathways initiated by SA and by a yet unknown signal produced during the HR, that promote different, albeit related, transcription complexes on the PR-1a as-1-like element. Analysis of PR-1 proteins in plants expressing salicylate hydroxylase yielded additional evidence that an HR dependent pathway leads to high level PR-1 gene induction in tobacco.