Antigen structure affects cellular routing through DC-SIGN.

Dendritic cell (DC) lectins mediate the recognition, uptake, and processing of antigens, but they can also be coopted by pathogens for infection. These distinct activities depend upon the routing of antigens within the cell. Antigens directed to endosomal compartments are degraded, and the peptides are presented on major histocompatibility complex class II molecules, thereby promoting immunity. Alternatively, HIV-1 can avoid degradation, as virus engagement with C-type lectin receptors (CLRs), such as DC-SIGN (DC-specific ICAM-3-grabbing nonintegrin) results in trafficking to surface-accessible invaginated pockets. This process appears to enable infection of T cells in trans We sought to explore whether antigen fate upon CLR-mediated internalization was affected by antigen physical properties. To this end, we employed the ring-opening metathesis polymerization to generate glycopolymers that each display multiple copies of mannoside ligand for DC-SIGN, yet differ in length and size. The rate and extent of glycopolymer internalization depended upon polymer structure-longer polymers were internalized more rapidly and more efficiently than were shorter polymers. The trafficking, however, did not differ, and both short and longer polymers colocalized with transferrin-labeled early endosomes. To explore how DC-SIGN directs larger particles, such as pathogens, we induced aggregation of the polymers to access particulate antigens. Strikingly, these particulate antigens were diverted to the invaginated pockets that harbor HIV-1. Thus, antigen structure has a dramatic effect on DC-SIGN-mediated uptake and trafficking. These findings have consequences for the design of synthetic vaccines. Additionally, the results suggest strategies for targeting DC reservoirs that harbor viral pathogens.

PoxA, yjeK, and elongation factor P coordinately modulate virulence and drug resistance in Salmonella enterica.

We report an interaction between poxA, encoding a paralog of lysyl tRNA-synthetase, and the closely linked yjeK gene, encoding a putative 2,3-beta-lysine aminomutase, that is critical for virulence and stress resistance in Salmonella enterica. Salmonella poxA and yjeK mutants share extensive phenotypic pleiotropy, including attenuated virulence in mice, an increased ability to respire under nutrient-limiting conditions, hypersusceptibility to a variety of diverse growth inhibitors, and altered expression of multiple proteins, including several encoded on the SPI-1 pathogenicity island. PoxA mediates posttranslational modification of bacterial elongation factor P (EF-P), analogous to the modification of the eukaryotic EF-P homolog, eIF5A, with hypusine. The modification of EF-P is a mechanism of regulation whereby PoxA acts as an aminoacyl-tRNA synthetase that attaches an amino acid to a protein resembling tRNA rather than to a tRNA.

The PhoQ histidine kinases of Salmonella and Pseudomonas spp. are structurally and functionally different: evidence that pH and antimicrobial peptide sensing contribute to mammalian pathogenesis.

The PhoQ sensor kinase is essential for Salmonella typhimurium virulence for animals, and a homologue exists in the environmental organism and opportunistic pathogen Pseudomonas aeruginosa. S. typhimurium PhoQ (ST-PhoQ) is repressed by millimolar concentrations of divalent cations and activated by antimicrobial peptides and at acidic pH. ST-PhoQ has a periplasmic Per-ARNT-Sim domain, a fold commonly employed for ligand binding. However, substrate binding is instead accomplished by an acidic patch in the periplasmic domain that interacts with the inner membrane through divalent cation bridges. The DNA sequence encoding this acidic patch is absent from Pseudomonas phoQ (PA-PhoQ). Here, we demonstrate that PA-PhoQ binds and is repressed by divalent cations, and can functionally complement a S. typhimurium phoQ-null mutant. Mutational analysis and NMR spectroscopy of the periplasmic domains of ST-PhoQ and PA-PhoQ indicate distinct mechanisms of binding divalent cation. The data are consistent with PA-PhoQ binding metal in a specific ligand-binding pocket. PA-PhoQ was partially activated by acidic pH but not by antimicrobial peptides. S. typhimurium expressing PA-PhoQ protein were attenuated for virulence in a mouse model, suggesting that the ability of Salmonella to sense host environments via antimicrobial peptides and acidic pH is an important contribution to pathogenesis.

The Salmonellae PhoQ sensor: mechanisms of detection of phagosome signals.

Sensor histidine kinases are widely used by prokaryotes to regulate gene expression in response to environmental signals, and such sensing is essential for virulence for plants and animals. PhoQ is one such sensor kinase that is conserved across a variety of Gram-negative pathogens and functions as part of a two-component system with its transcriptional regulator PhoP. Salmonella typhimurium PhoPQ is one of the most studied two-component systems and has been demonstrated to regulate hundreds of genes encoding the majority of virulence properties including intracellular survival, invasion, lipid A structure, resistance to antimicrobial peptides, and phagosome alteration. PhoQ is activated within acidified phagosomes and host tissues. PhoQ binds divalent cations to form bridges with the membrane that maintain the PhoQ repressed state. Antimicrobial peptides and acidic pH activate PhoQ and these signals, present within phagosomes and intestinal tissues, may serve as specific signatures of the host environment for Gram-negative pathogens. This review will focus on recent progress in the molecular details of PhoQ structure and sensing of defined signals. In addition, Pseudomonas aeruginosa and Salmonella typhimurium PhoQ are compared, providing evidence that a subset of PhoQ proteins of environmental organisms evolved more limited sensing capability using a different structural sensing mechanism.

Acidic pH and divalent cation sensing by PhoQ are dispensable for systemic salmonellae virulence.

Salmonella PhoQ is a histidine kinase with a periplasmic sensor domain (PD) that promotes virulence by detecting the macrophage phagosome. PhoQ activity is repressed by divalent cations and induced in environments of acidic pH, limited divalent cations, and cationic antimicrobial peptides (CAMP). Previously, it was unclear which signals are sensed by salmonellae to promote PhoQ-mediated virulence. We defined conformational changes produced in the PhoQ PD on exposure to acidic pH that indicate structural flexibility is induced in α-helices 4 and 5, suggesting this region contributes to pH sensing. Therefore, we engineered a disulfide bond between W104C and A128C in the PhoQ PD that restrains conformational flexibility in α-helices 4 and 5. PhoQ(W104C-A128C) is responsive to CAMP, but is inhibited for activation by acidic pH and divalent cation limitation. phoQ(W104C-A128C) Salmonella enterica Typhimurium is virulent in mice, indicating that acidic pH and divalent cation sensing by PhoQ are dispensable for virulence.

Quinoxalinone Inhibitors of the Lectin DC-SIGN.

The C-type lectin dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) can serve as a docking site for pathogens on the surface of dendritic cells. Pathogen binding to DC-SIGN can have diverse consequences for the host. DC-SIGN can facilitate HIV-1 dissemination, but the interaction of Mycobacterium tuberculosis with DC-SIGN is important for host immunity. The ability of pathogens to target DC-SIGN provides impetus to identify ligands that can perturb these interactions. Here, we describe the first stable small molecule inhibitors of DC-SIGN. These inhibitors were derived from a collection of quinoxalinones, which were assembled using a tandem cross metathesis-hydrogenation sequence. To assess the ability of these small molecules to block DC-SIGN-mediated glycan adhesion and internalization, we developed a sensitive flow cytometry assay. Our results reveal that the quinoxalinones are effective inhibitors of DC-SIGN-glycan interactions. These compounds block both glycan binding to cells and glycan internalization. We anticipate that these non-carbohydrate inhibitors can be used to elucidate the role of DC-SIGN in pathogenesis and immune function.

Noncarbohydrate glycomimetics and glycoprotein surrogates as DC-SIGN antagonists and agonists.

An understanding of the biological roles of lectins will be advanced by ligands that can inhibit or even recruit lectin function. To this end, glycomimetics, noncarbohydrate ligands that function analogously to endogenous carbohydrates, are being sought. The advantage of having such ligands is illustrated by the many roles of the protein DC-SIGN. DC-SIGN is a C-type lectin displayed on dendritic cells, where it binds to mannosides and fucosides to mediate interactions with other host cells or bacterial or viral pathogens. DC-SIGN engagement can modulate host immune responses (e.g., suppress autoimmunity) or benefit pathogens (e.g., promote HIV dissemination). DC-SIGN can bind to glycoconjugates, internalize glycosylated cargo for antigen processing, and transduce signals. DC-SIGN ligands can serve as inhibitors as well as probes of the lectin's function, so they are especially valuable for elucidating and controlling DC-SIGN's roles in immunity. We previously reported a small molecule that embodies key features of the carbohydrates that bind DC-SIGN. Here, we demonstrate that this noncarbohydrate ligand acts as a true glycomimetic. Using NMR HSQC experiments, we found that the compound mimics saccharide ligands: It occupies the same carbohydrate-binding site and interacts with the same amino acid residues on DC-SIGN. The glycomimetic also is functional. It had been shown previously to antagonize DC-SIGN function, but here we use it to generate DC-SIGN agonists. Specifically, appending this glycomimetic to a protein scaffold affords a conjugate that elicits key cellular signaling responses. Thus, the glycomimetic can give rise to functional glycoprotein surrogates that elicit lectin-mediated signaling.

Salmonella sensing of anti-microbial mechanisms to promote survival within macrophages.

Salmonella enterica is a facultative intracellular pathogen that replicates within macrophages. The interaction of this pathogen with mammalian cells is a complex process involving hundreds of bacterial products that are sensed by and alter mammalian hosts. Numerous bacterial genes and their protein products have been identified that are required for Salmonella to resist killing by host innate immunity and to modify host processes. Many of these genes are regulated by a specific bacterial sensor, the PhoQ protein, which responds to the acidified phagosome environment. PhoQ is a sensor histidine kinase, which when activated in vivo within acidified macrophage phagosomes, regulates cell surface modifications that promote resistance to antimicrobial peptides and oxidative stress, alter the phagosome to promote intracellular survival, and reduce innate immune recognition. In this review, we discuss mechanisms by which Salmonella interacts with macrophages and focus in detail on recent reports describing the role of antimicrobial peptides and pH in PhoQ activation.

Activation of the bacterial sensor kinase PhoQ by acidic pH.

The Salmonellae PhoQ sensor kinase senses the mammalian phagosome environment to activate a transcriptional program essential for virulence. The PhoQ periplasmic domain binds divalent cations, forming bridges with inner membrane phospholipids to maintain PhoQ repression. PhoQ also binds and is activated by cationic antimicrobial peptides. In this work, PhoQ is directly activated by exposure of the sensor domain to pH 5.5. NMR spectroscopy indicates that at acidic pH, the PhoQ periplasmic domain adopts a conformation different from that in the presence of divalent cations or antimicrobial peptides. The conformation is partially simulated by mutation of histidine 157, which is part of an interaction network that distinguishes the repressed conformation. The effects of antimicrobial peptides and pH on PhoQ activity are additive. We propose a model of activation by antimicrobial peptides via disruption of the cation bridges and/or by acidification of the periplasm through destabilization of the interaction network.