Reconstitution of surface lipoprotein translocation reveals Slam as an outer membrane translocon in Gram-negative bacteria

Surface lipoproteins (SLPs) are peripherally attached to the outer leaflet of the outer membrane in many Gram-negative bacteria, playing significant roles in nutrient acquisition and immune evasion in the host. While the factors that are involved in the synthesis and delivery of SLPs in the inner membrane are well characterized, the molecular machineries required for the movement of SLPs to the surface are still not fully elucidated. In this study, we investigated the translocation of a surface lipoprotein TbpB through a Slam1-dependent pathway. Using purified components, we developed an in vitro translocation assay where unfolded TbpB is transported through Slam1 containing proteoliposomes, confirming Slam1 as an outer membrane translocon. While looking to identify factors to increase translocation efficiency, we discovered the periplasmic chaperone Skp interacted with TbpB in the periplasm of Escherichia coli. The presence of Skp was found to increase the translocation efficiency of TbpB in the reconstituted translocation assays. A knockout of Skp in Neisseria meningitidis revealed that Skp is essential for functional translocation of TbpB to the bacterial surface. Taken together, we propose a pathway for surface destined lipoproteins, where Skp acts as a holdase for Slam-mediated TbpB translocation across the outer membrane.

Thermodynamic Stability Is a Strong Predictor for the Delivery of DARPins to the Cytosol via Anthrax Toxin

Anthrax toxin has evolved to translocate its toxic cargo proteins to the cytosol of cells carrying its cognate receptor. Cargo molecules need to unfold to penetrate the narrow pore formed by its membrane-spanning subunit, protective antigen (PA). Various alternative cargo molecules have previously been tested, with some showing only limited translocation efficiency, and it may be assumed that these were too stable to be unfolded before passing through the anthrax pore. In this study, we systematically and quantitatively analyzed the correlation between the translocation of various designed ankyrin repeat proteins (DARPins) and their different sizes and thermodynamic stabilities. To measure cytosolic uptake, we used biotinylation of the cargo by cytosolic BirA, and we measured cargo equilibrium stability via denaturant-induced unfolding, monitored by circular dichroism (CD). Most of the tested DARPin cargoes, including target-binding ones, were translocated to the cytosol. Those DARPins, which remained trapped in the endosome, were confirmed by CD to show a high equilibrium stability. We could pinpoint a stability threshold up to which cargo DARPins still get translocated to the cytosol. These experiments have outlined the requirements for translocatable binding proteins, relevant stability measurements to assess translocatable candidates, and guidelines to further engineer this property if needed.

The HopQ-CEACAM Interaction Controls CagA Translocation, Phosphorylation, and Phagocytosis of Helicobacter pylori in Neutrophils

ABSTRACT The cag type IV secretion system (cag-T4SS) of Helicobacter pylori exploits specific cellular carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), such as CEACAM1, -3, -5, and -6, as cellular receptors for CagA translocation into human gastric epithelial cells. We studied the interaction of H. pylori with human CEACAM1, CEACAM3, and CEACAM6 receptors (hCEACAMs) expressed on myeloid cells from CEACAM-humanized mice. Human and CEACAM-humanized mouse polymorphonuclear neutrophils (PMNs) allowed a specific HopQ-dependent interaction strongly enhancing CagA translocation. Translocated CagA was tyrosine phosphorylated, which was not seen in wild-type (wt) murine neutrophils. In contrast, human or murine bone marrow-derived macrophages and dendritic cells (DCs) revealed a low hCEACAM expression and bacterial binding. CagA translocation and tyrosine-phosphorylation was low and independent of the HopQ-CEACAM interaction. Neutrophils, but not macrophages or DCs, from CEACAM-humanized mice, significantly upregulated the proinflammatory chemokine MIP-1α. However, macrophages showed a significantly reduced amount of CXCL1 (KC) and CCL2 (MCP-1) secretion in CEACAM-humanized versus wt cells. Thus, H. pylori, via the HopQ-CEACAM interaction, controls the production and secretion of chemokines differently in PMNs, macrophages, and DCs. We further show that upon H. pylori contact the oxidative burst of neutrophils and phagocytosis of H. pylori was strongly enhanced, but hCEACAM3/6 expression on neutrophils allowed the extended survival of H. pylori within neutrophils in a HopQ-dependent manner. Finally, we demonstrate that during a chronic mouse infection, H. pylori is able to systemically downregulate hCEACAM1 and hCEACAM6 receptor expression on neutrophils, probably to limit CagA translocation efficiency and most likely gastric pathology. IMPORTANCE Helicobacter pylori is highly adapted to humans and evades host immunity to allow its lifelong colonization. However, the H. pylori mouse model is artificial for H. pylori, and few adapted strains allow gastric colonization. Here, we show that human or CEACAM-humanized, but not mouse neutrophils are manipulated by the H. pylori HopQ-CEACAM interaction. Human CEACAMs are responsible for CagA phosphorylation, activation, and processing in neutrophils, whereas CagA translocation and tyrosine phosphorylation in DCs and macrophages is independent of the HopQ-CEACAM interaction. H. pylori affects the secretion of distinct chemokines in CEACAM-humanized neutrophils and macrophages. Most importantly, human CEACAMs on neutrophils enhance binding, oxidative burst, and phagocytosis of H. pylori and enhance bacterial survival in the phagosome. The H. pylori-CEACAM interaction modulates PMNs to reduce the H. pylori CagA translocation efficiency in vivo and to fine-tune the expression of CEACAM receptors on neutrophils to limit translocation of CagA and gastric pathology.

EFFECT OF NITROGEN ON THE ACCUMULATION AND REUTILIZATION OF DRY MASS IN GRAIN SORGHUM

Accumulation and reutilization of dry mass until anthesis and during a grain fillingperiod of sorghum in response to nitrogen fertilization in rates 0, 60, 120, 180, 240and 300 kg N.ha-1 was studied in a field experiment. Grain sorghum hybrid ECAlize was grown under not- irrigated conditions in the experimental field ofAgricultural University of Plovdiv, Bulgaria. The experimental design was arandomized, complete block design with four replications with a size ofexperimental plots of 20 m2 after wheat as predecessor. Standard farming practicesfor the region of Southern Bulgaria were applied. It was established that nitrogenfertilization significantly increased the amount of accumulated dry mass at anthesisand total above ground dry mass at maturity compared to N0. Not significant effectof higher rates (180, 240 and 300 kg N.ha-1) on the dry mass accumulation ofsorghum was found. Average post anthesis net dry mass accumulation was 3291kg.ha-1 and its amount increased in parallel with the nitrogen rate up to N180. Thehighest dry mass translocation, translocation efficiency, and contribution of preanthesisassimilations of the grain was established at nitrogen rate N120 with values2073 kg.ha-1, 25.0 % and 41,8 %, respectively. Growth of sorghum at highernitrogen rates N180, N240, N300 significantly decreased efficiency of dry masstranslocation and contribution of pre-anthesis assimilations of the grain. Nitrogenfertilization had very strong negative correlation with dry mass translocationefficiency (-0.860*) and contribution of pre-anthesis assimilations of the grain (-0.863*). Very strong positive correlation (0.988**) was found between dry masstranslocation efficiency and contribution of pre-anthesis assimilations of the grain.

Physiological and agronomic responses of maize (Zea maysL) cultivars to plant population and defoliation at post-anthesis in the humid rainforest

Variations in response pattern of maize (<em>Zea mays</em>)grown at plant populations, defoliated at post-anthesis in the rainforest were tested. Two field trials were conducted at Abeokuta, (Longitude 3⁰25’E, Latitude 7⁰15’N; 144 m a.s.l) and Ibadan (3⁰56’E, 7⁰33’N: 168 m a.s.l), Nigeria in 2015. The trials consisted of maize variety {2009 TZE-W DT STR [open pollinated variety (OPV)] and TZEI 124 × TZEI 25 (hybrid)]} in the main plot, plant population (71111, 80000 and 106666 plant ha^-1) in sub plot and defoliation (+ defoliation and – defoliation) as sub-sub plot. It was laid out in a split-split plot arrangement fitted into randomised complete block design with three replicates. OPV had significantly higher assimilatory surface, rate of current photosynthesis, reduced dry matter translocation efficiency, reduced days to 50 % anthesis and more 1000 grain massthan the hybrid maize, with similar grain yields. Both locations experienced increased leaf area index with increased plant population. Reduced 1000 grain massat both locations when maize was defoliated suggested a disruption in source:sink balance.

Effects of Manganese Nanoparticle Exposure on Nutrient Acquisition in Wheat (Triticum aestivum L.)

Nanoparticles are used in a variety of products, including fertilizer-nutrients and agro-pesticides. However, due to heightened reactivity of nano-scale materials, the effects of nanoparticle nutrients on crops can be more dramatic when compared to non nano-scale nutrients. This study evaluated the effect of nano manganese-(Mn) on wheat yield and nutrient acquisition, relative to bulk and ionic-Mn. Wheat was exposed to the Mn types in soil (6 mg/kg/plant), and nano-Mn was repeated in foliar application. Plant growth, grain yield, nutrient acquisition, and residual soil nutrients were assessed. When compared to the control, all Mn types significantly (p < 0.05) reduced shoot N by 9–18%. However, nano-Mn in soil exhibited other subtle effects on nutrient acquisition that were different from ionic or bulk-Mn, including reductions in shoot Mn (25%), P (33%), and K (7%) contents, and increase (30%) in soil residual nitrate-N. Despite lowering shoot Mn, nano-Mn resulted in a higher grain Mn translocation efficiency (22%), as compared to salt-Mn (20%), bulk-Mn (21%), and control (16%). When compared to soil, foliar exposure to nano-Mn exhibited significant differences: greater shoot (37%) and grain (12%) Mn contents; less (40%) soil nitrate-N; and, more soil (17%) and shoot (43%) P. These findings indicate that exposure to nano-scale Mn in soil could affect plants in subtle ways, differing from bulk or ionic-Mn, suggesting caution in its use in agriculture. Applying nano Mn as a foliar treatment could enable greater control on plant responses.