MprF-mediated immune evasion is necessary for Lactiplantibacillus plantarum resilience in the Drosophila gut during inflammation

Multiple peptide resistance factor (MprF) confers resistance to cationic antimicrobial peptides (AMPs) in several pathogens, thereby enabling evasion of the host immune response. The role of MprF in commensals remains, however, uncharacterized. To close this knowledge gap, we used a common gut commensal of animals, Lactiplantibacillus plantarum, and its natural host, the fruit fly Drosophila melanogaster, as an experimental model to investigate the role of MprF in commensal-host interactions. The L. plantarum ΔmprF mutant that we generated exhibited deficiency in the synthesis of lysyl-phosphatidylglycerol (Lys-PG), resulting in increased negative cell surface charge and increased susceptibility to AMPs. Susceptibility to AMPs had no effect on ΔmprF mutant’s ability to colonize guts of uninfected flies. However, we observed significantly reduced abundance of the ΔmprF mutant after infection-induced inflammation in the guts of wild-type flies but not of flies lacking AMPs. Additionally, we found that the ΔmprF mutant compared to wild-type L. plantarum induces a stronger intestinal immune response in flies due to the increased release of immunostimulatory peptidoglycan fragments, indicating an important role of MprF in promoting host tolerance to commensals. Our further analysis suggests that MprF-mediated lipoteichoic acid modifications are involved in host immunomodulation. Overall, our results demonstrate that MprF, besides its well-characterized role in pathogen immune evasion and virulence, is also an important commensal resilience factor

Spectral Imaging within EDEN ISS for Plant Health and Productivity Assessment

EDEN ISS is a large multinational project, headed by the German Aerospace Center (DLR), to advance controlled environment agriculture and life support technologies. The project draws upon and extends lessons from the International Space Station (ISS), and places those lessons within a ground demonstration module deployed at the Neumayer III Antarctic station. EDEN ISS focusses on cultivation technologies for two exploration applications: spaceflight deployment within a module that allows simulation of safe food production on-board the ISS, and for utilization in future human space exploration vehicles and planetary outposts. These overall project goals connect the planetary surface analog concepts to near term operations of ISS and potential cis-lunar habitats. In addition to providing fresh produce to Neumayer Station III overwintering crews, the system will validate key plant growth system technologies and operational protocols for bioregenerative life support systems, while exploring science that may enhance these aims through remote monitoring of plant health, growth and development. Among the technologies to be developed and deployed will be imagers based on GoPro cameras modified to collect multi-wavelength data to equip the plant growth systems with the capacity to evaluate variations of Differential Vegetation Indices, a technology primarily restricted to the sunlight driven satellite-based uses of Normalized Differential Vegetation Index (NDVI). The use of NDVI-like approaches in systems like EDEN ISS that are highly controlled, especially for lighting inputs, is just beginning to be explored. The imaging project involves an integrated science backroom that will remotely assess the images and provide input to the plant production team on site. Project concepts and the development of NDVI like imaging with EDEN-ISS will be presented with a focus on the initial images that have examined differential pixel math specifically to anticipate stress responses prior to typical visible changes. The participation of ALP, JAC and RJF is supported by grants to RJF and ALP from NASA Space Life and Physical Sciences through KSC (including 17-NUP2017-0020) and the Florida Space Grant Consortium (UF NNX15 TO 016)

Analysis of Protein Complexes in Wheat Amyloplasts Reveals Functional Interactions among Starch Biosynthetic Enzymes1[C][W][OA]

Protein-protein interactions among enzymes of amylopectin biosynthesis were investigated in developing wheat (Triticum aestivum) endosperm. Physical interactions between starch branching enzymes (SBEs) and starch synthases (SSs) were identified from endosperm amyloplasts during the active phase of starch deposition in the developing grain using immunoprecipitation and cross-linking strategies. Coimmunoprecipitation experiments using peptide-specific antibodies indicate that at least two distinct complexes exist containing SSI, SSIIa, and either of SBEIIa or SBEIIb. Chemical cross linking was used to identify protein complexes containing SBEs and SSs from amyloplast extracts. Separation of extracts by gel filtration chromatography demonstrated the presence of SBE and SS forms in protein complexes of around 260 kD and that SBEII forms may also exist as homodimers. Analysis of cross-linked 260-kD aggregation products from amyloplast lysates by mass spectrometry confirmed SSI, SSIIa, and SBEII forms as components of one or more protein complexes in amyloplasts. In vitro phosphorylation experiments with γ-32P-ATP indicated that SSII and both forms of SBEII are phosphorylated. Treatment of the partially purified 260-kD SS-SBE complexes with alkaline phosphatase caused dissociation of the assembly into the respective monomeric proteins, indicating that formation of SS-SBE complexes is phosphorylation dependent. The 260-kD SS-SBEII protein complexes are formed around 10 to 15 d after pollination and were shown to be catalytically active with respect to both SS and SBE activities. Prior to this developmental stage, SSI, SSII, and SBEII forms were detectable only in monomeric form. High molecular weight forms of SBEII demonstrated a higher affinity for in vitro glucan substrates than monomers. These results provide direct evidence for the existence of protein complexes involved in amylopectin biosynthesis

High and Sustained Ex Vivo Frequency but Altered Phenotype of SARS-CoV-2-Specific CD4⁺ T-Cells in an Anti-CD20-Treated Patient with Prolonged COVID-19

Here, we longitudinally assessed the ex vivo frequency and phenotype of SARS-CoV-2 membrane protein (aa145–164) epitope-specific CD4+ T-cells of an anti-CD20-treated patient with prolonged viral positivity in direct comparison to an immunocompetent patient through an MHC class II DRB1*11:01 Tetramer analysis. We detected a high and stable SARS-CoV-2 membrane-specific CD4+ T-cell response in both patients, with higher frequencies of virus-specific CD4+ T-cells in the B-cell-depleted patient. However, we found an altered virus-specific CD4+ T-cell memory phenotype in the B-cell-depleted patient that was skewed towards late differentiated memory T-cells, as well as reduced frequencies of SARS-CoV-2-specific CD4+ T-cells with CD45RA− CXCR5+ PD-1+ circulating T follicular helper cell (cTFH) phenotype. Furthermore, we observed a delayed contraction of CD127− virus-specific effector cells. The expression of the co-inhibitory receptors TIGIT and LAG-3 fluctuated on the virus-specific CD4+ T-cells of the patient, but were associated with the inflammation markers IL-6 and CRP. Our findings indicate that, despite B-cell depletion and a lack of B-cell—T-cell interaction, a robust virus-specific CD4+ T-cell response can be primed that helps to control the viral replication, but which is not sufficient to fully abrogate the infection