Myoferlin and dysferlin are members of the ferlin family of membrane proteins. Recent studies have shown that mutation or genetic disruption of myoferlin or dysferlin promotes muscular dystrophy-related phenotypes in mice, which are the result of impaired plasma membrane integrity. However, no biological functions have been ascribed to myoferlin in non-muscle tissues. Herein, using a proteomic analysis of endothelial cell (EC) caveolae/lipid raft microdomains we identified myoferlin in these domains and show that myoferlin is highly expressed in ECs and vascular tissues. The loss of myoferlin results in lack of proliferation, migration, and nitric oxide (NO) release in response to vascular endothelial growth factor (VEGF). Western blotting and surface biotinylation experiments show that loss of myoferlin reduces the expression level and autophosphorylation of VEGF receptor-2 (VEGFR-2) in native ECs. In a reconstituted cell system, transfection of myoferlin increases VEGFR-2 membrane expression and autophosphorylation in response to VEGF. In vivo, VEGFR-2 levels and VEGF-induced permeability are impaired in myoferlin-deficient mice. Mechanistically, myoferlin forms a complex with dynamin-2 and VEGFR-2, which prevents CBL-dependent VEGFR-2 polyubiquitination and proteasomal degradation. These data are the first to report novel biological activities for myoferlin and reveal the role of membrane integrity to VEGF signaling.

SsrA/SsrB is a primary two-component system that mediates the survival and replication of Salmonella within host cells. When activated, the SsrB response regulator directly promotes the transcription of multiple genes within Salmonella pathogenicity island 2 (SPI-2). As expression of the SsrB protein is promoted by several transcription factors, including SsrB itself, the expression of SPI-2 genes can increase to undesirable levels under activating conditions. Here, we report that Salmonella can avoid the hyperactivation of SPI-2 genes by using ptsN-encoded EIIA Ntr , a component of the nitrogen-metabolic phosphotransferase system. Under SPI-2-inducing conditions, the levels of SsrB-regulated gene transcription increased abnormally in a ptsN deletion mutant, whereas they decreased in a strain overexpressing EIIA Ntr . We found that EIIA Ntr controls SPI-2 genes by acting on the SsrB protein at the posttranscriptional level. EIIA Ntr interacted directly with SsrB, which prevented the SsrB protein from binding to its target promoter. Finally, the Salmonella strain, either lacking the ptsN gene or overexpressing EIIA Ntr , was unable to replicate within macrophages, and the ptsN deletion mutant was attenuated for virulence in mice. These results indicated that normal SPI-2 gene expression maintained by an EIIA Ntr -SsrB interaction is another determinant of Salmonella virulence.virulence gene regulation | nitrogen-metabolic phosphotransferase system (PTS) | protein-protein interaction

The need for SARS-CoV-2 next-generation vaccines has been highlighted by the rise of variants of concern (VoC) and the long-term threat of emerging coronaviruses. Here, we design and characterize four categories of engineered nanoparticle immunogens that recapitulate the structural and antigenic properties of prefusion SARS-CoV-2 Spike (S), S1 and RBD. These immunogens induce robust S-binding, ACE2-inhibition, and authentic and pseudovirus neutralizing antibodies against SARS-CoV-2. A Spike-ferritin nanoparticle (SpFN) vaccine elicits neutralizing titers (ID 50 > 10,000) following a single immunization, while RBD-Ferritin nanoparticle (RFN) immunogens elicit similar responses after two immunizations, and also show durable and potent neutralization against circulating VoC. Passive transfer of IgG purified from SpFN- or RFN-immunized mice protects K18-hACE2 transgenic mice from a lethal SARS-CoV-2 challenge. Furthermore, S-domain nanoparticle immunization elicits ACE2 blocking activity and ID50 neutralizing antibody titers >2,000 against SARS-CoV-1, highlighting the broad response elicited by these immunogens.

CD4+ T follicular helper cells (TFH) in germinal centers are required for maturation of B-cells. While the role of TFH-cells has been studied in blood and lymph nodes of HIV-1 infected individuals, its role in the mucosal tissues has not been investigated. We show that the gut and female reproductive tract (FRT) of humanized DRAG mice have a high level of human lymphocytes and a high frequency of TFH (CXCR5+PD-1++) and precursor-TFH (CXCR5+PD-1+) cells. The majority of TFH-cells expressed CCR5 and CXCR3 and are the most permissive to HIV-1 infection. A single low-dose intravaginal HIV-1 challenge of humanized DRAG mice results in 100% infectivity with accumulation of TFH-cells mainly in the Peyer’s patches and FRT. The novel finding of TFH-cells in the FRT may contribute to the high susceptibility of DRAG mice to HIV-1 infection. This mouse model thus provides new opportunities to study TFH-cells and to evaluate HIV-1 vaccines.