Sorting of Arabidopsis NRAMP3 and NRAMP4 depends on adaptor protein complex AP4 and a dileucine-based motif.

Adaptor protein complexes mediate cargo selection and vesicle trafficking to different cellular membranes in all eukaryotic cells. Information on the role of AP4 in plants is still limited. Here, we present the analyses of Arabidopsis thaliana mutants lacking different subunits of AP4. These mutants show abnormalities in their development and in protein sorting. We found that growth of roots and etiolated hypocotyls, as well as male fertility and trichome morphology are disturbed in ap4. Analyses of GFP-fusions transiently expressed in mesophyll protoplasts demonstrated that the tonoplast (TP) proteins MOT2, NRAMP3 and NRAMP4, but not INT1, are partially sorted to the plasma membrane (PM) in the absence of a functional AP4 complex. Moreover, alanine mutagenesis revealed that in wild-type plants, sorting of NRAMP3 and NRAMP4 to the TP requires an N-terminal dileucine-based motif. The NRAMP3 or NRAMP4 N-terminal domain containing the dileucine motif was sufficient to redirect the PM localized INT4 protein to the TP and to confer AP4-dependency on sorting of INT1. Our data show that correct sorting of NRAMP3 and NRAMP4 depends on both, an N-terminal dileucine-based motif as well as AP4.

Detection and characterization of putative hypervirulent Klebsiella pneumoniae isolates in microbiological diagnostics.

Hypervirulent Klebsiella pneumoniae strains (hvKp) can cause invasive community-acquired infections in healthy patients of all ages. In this study, the prevalence of putative hvKp in a German tertiary center was investigated and hvKp were characterized by phenotypic and molecular assays. All K. pneumoniae isolates in routine microbiological diagnostics from a single center were screened by string-testing over a period of 6 months. String-test positive (≥ 0.5 mm) isolates were re-evaluated on different media and under various conditions (aerobe, anaerobe). For string-test positive isolates, genes (magA, iutA, rmpA and rmpA2) associated with hypermucoviscosity and hypervirulence were amplified by multiplex PCR. PCR-positive isolates were subjected to whole-genome sequencing and sedimentation and biofilm formation assays. From 1310 screened K. pneumoniae isolates in clinical routine 100 isolates (7.6%) were string test positive. From these, 9% (n = 9) were defined as putative hvKp (string-test+/PCR+). Highest rate of string-test-positive isolates was observed on MacConkey agar under aerobic conditions. Amongst these nine putative hvKp isolates, the international lineage ST23 carrying hvKp-plasmid pKpVP-1 was the most common, but also a rare ST86 with pKpVP-2 was identified. All nine isolates showed hypermucoviscosity and weak biofilm formation. In conclusion, 9% of string-positive, respectively 0.69% of all K. pneumoniae isolates from routine were defined as putative hypervirulent. MacConkey agar was the best medium for hvKp screening.

SARS-CoV-2 N gene dropout and N gene Ct value shift as indicator for the presence of B.1.1.7 lineage in a commercial multiplex PCR assay.

OBJECTIVES: Detection and surveillance of SARS-CoV-2 is of eminent importance, particularly due to the rapid emergence of variants of concern (VOCs). In this study we evaluated if a commercially available quantitative real-time PCR (qRT-PCR) assay can identify SARS-CoV-2 B.1.1.7 lineage samples by a specific N gene dropout or Ct value shift compared with the S or RdRp gene. METHODS: VOC B.1.1.7 and non-B.1.1.7 SARS-CoV-2-positive patient samples were identified via whole-genome sequencing and variant-specific PCR. Confirmed B.1.1.7 (n = 48) and non-B.1.1.7 samples (n = 58) were analysed using the Allplex™ SARS-CoV-2/FluA/FluB/RSV™ PCR assay for presence of SARS-CoV-2 S, RdRp and N genes. The N gene coding sequence of SARS-CoV-2 with and without the D3L mutation (specific for B.1.1.7) was cloned into pCR™II-TOPO™ vectors to validate polymorphism-dependent N gene dropout with the Allplex™ SARS-CoV-2/FluA/FluB/RSV™ PCR assay. RESULTS: All studied B.1.1.7-positive patient samples showed significantly higher Ct values in qRT-PCR (Δ6-10, N gene dropout on Ct values > 29) of N gene than the corresponding values of S (p ≤ 0.0001) and RdRp (p ≤ 0.0001) genes. The assay reliably discriminated B.1.1.7 and non-B.1.1.7 positive samples (area under the curve = 1) in a receiver operating characteristic curve analysis. Identical Ct value shifts (Δ7-10) were detected in reverse genetic experiments, using isolated plasmids containing N gene coding sequences corresponding to D3 or 3L variants. DISCUSSION: An N gene dropout or Ct value shift is shown for B.1.1.7-positive samples in the Allplex™ SARS-CoV-2/FluA/FluB/RSV™ PCR assay. This approach can be used as a rapid tool for B.1.1.7 detection in single assay high throughput diagnostics.