sugar transport ͉ phosphorylation ͉ x-ray crystallography T he phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS) (1) catalyzes the synchronized uptake and phosphorylation of a number of carbohydrates in eubacteria (group translocation) (2, 3). With some variations, the PTS comprises three proteins. In the cytoplasm, PEP phosphorylates enzyme I (EI), which then transfers the phosphoryl group to the histidine phosphocarrier protein, HPr. From HPr, the phosphoryl group is transferred to various sugar-specific membrane associated transporters [enzyme II (EII)], each comprising two cytoplasmic domains, EIIA and EIIB, and an integral membrane domain EIIC. Within EII, EIIA accepts the phosphoryl group from HPr and donates it to EIIB, whereupon EIIC mediates sugar translocation. In addition to controlling sugar translocation, the phosphorylation state of PTS proteins is associated with regulation of metabolic pathways and signaling in bacterial cells (4-8).The Ϸ64-kDa EI is a homodimer, which is more tightly associated at the phosphorylated state than the unphosphorylated state (9-14). The phosphorylation by PEP requires Mg 2ϩ and targets the N atom of His-189 (numbering scheme of EI from Escherichia coli) (15). The dimer association rate constant is two to three orders of magnitude slower than typical rates measured for other dimeric proteins, suggesting that oligomerization is accompanied by major conformational rearrangements (13,16,17). The monomer-dimer equilibrium has been studied in vitro by various methods (18-21), and it has been proposed that the transition plays a regulatory role in the PEP:sugar phosphotransferase system. Yet, transient kinetic studies indicated that the EI dimer phosphorylates HPr without dissociating into monomers (17).Proteolytic cleavage of EI produces two domains (22, 23). The EI N-terminal domain (EIN, residues 1-230) contains the residue that transfers the phosphoryl group, 24) and the HPr-binding domain, whereas the EI C-terminal domain (EIC, residues 261-575) binds PEP in the presence of Mg 2ϩ (the PEP-binding domain) (22,25) and mediates dimerization (26,27). Site-directed mutagenesis showed that Cys-502, located on EIC, is essential for phosphorylation of His-189 by PEP (28). The structure of EIN from E. coli has been determined by x-ray crystallography (29) and NMR spectroscopy (30).

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A guiding principle for HIV vaccine design has been that cellular and humoral immunity work together to provide the strongest degree of efficacy. However, three efficacy trials of Ad5-vectored HIV vaccines showed no protection. Transmission was increased in two of the trials, suggesting that this vaccine strategy elicited CD4+ T-cell responses that provide more targets for infection, attenuating protection or increasing transmission. The degree to which this problem extends to other HIV vaccine candidates is not known. Here, we show that a gp120-CD4 chimeric subunit protein vaccine (full-length single chain) elicits heterologous protection against simian-human immunodeficiency virus (SHIV) or simian immunodeficiency virus (SIV) acquisition in three independent rhesus macaque repeated low-dose rectal challenge studies with SHIV162P3 or SIVmac251. Protection against acquisition was observed with multiple formulations and challenges. In each study, protection correlated with antibody-dependent cellular cytotoxicity specific for CD4-induced epitopes, provided that the concurrent antivaccine T-cell responses were minimal. Protection was lost in instances when T-cell responses were high or when the requisite antibody titers had declined. Our studies suggest that balance between a protective antibody response and antigen-specific T-cell activation is the critical element to vaccine-mediated protection against HIV. Achieving and sustaining such a balance, while enhancing antibody durability, is the major challenge for HIV vaccine development, regardless of the immunogen or vaccine formulation.

Candida albicans, the major invasive fungal pathogen of humans, can cause both debilitating mucosal infections and fatal invasive infections. Understanding the complex nature of the host-pathogen interaction in each of these contexts is essential to developing desperately needed therapies to treat fungal infections. RNA-seq enables a systems-level understanding of infection by facilitating comprehensive analysis of transcriptomes from multiple species (e.g., host and pathogen) simultaneously. We used RNA-seq to characterize the transcriptomes of both C. albicans and human endothelial cells or oral epithelial cells during in vitro infection. Network analysis of the differentially expressed genes identified the activation of several signaling pathways that have not previously been associated with the host response to fungal pathogens. Using an siRNA knockdown approach, we demonstrate that two of these pathways-platelet-derived growth factor BB (PDGF BB) and neural precursor-cell-expressed developmentally down-regulated protein 9 (NEDD9)-govern the host-pathogen interaction by regulating the uptake of C. albicans by host cells. Using RNA-seq analysis of a mouse model of hematogenously disseminated candidiasis (HDC) and episodes of vulvovaginal candidiasis (VVC) in humans, we found evidence that many of the same signaling pathways are activated during mucosal (VVC) and/or disseminated (HDC) infections in vivo. Our analyses have uncovered several signaling pathways at the interface between C. albicans and host cells in various contexts of infection, and suggest that PDGF BB and NEDD9 play important roles in this interaction. In addition, these data provide a valuable community resource for better understanding host-fungal pathogen interactions.

Given the lethality of H5N1 avian influenza viruses (AIV) and the recurring spread from poultry to humans, an effective vaccine against H5N1 viruses may be needed to prevent a pandemic. We generated experimental vaccine vectors based on recombinant vesicular stomatitis virus (VSV) expressing the H5 hemagglutinin (HA) from an H5N1 virus isolated in 1997. The HA gene was expressed either from an attenuated wild-type VSV vector or from a single-cycle vector containing a deletion of the VSV G gene. We found that all of the vectors induced potent neutralizing antibody titers against the homologous and antigenically heterologous H5N1 viruses isolated in 2004 and 2005. Vaccination of mice with any combination of prime or prime/boost vectors provided long-lasting protection (>7 months) against challenge with AIV, even in animals receiving a single dose of single-cycle vaccine. Our data indicate that these recombinants are promising vaccine candidates for pandemic influenza.