Mutually dependent secretion of proteins required for mycobacterial virulence.

The ESX-1 locus is a region critical for full virulence in Mycobacterium tuberculosis, which encodes two secreted proteins as well as other genes involved in their secretion. The mechanism of secretion of the two proteins, ESAT-6 and CFP-10, and their function remain unknown. Using proteomic methods to search for additional proteins secreted by the ESX-1 locus, we discovered that a protein encoded by a chromosomally unlinked gene, espA, is also secreted by strains that contain the ESX-1 locus but not by strains with ESX-1 deletions. Mutations in individual ESX-1 genes, including those that encode ESAT-6 and CFP-10, were found to block EspA secretion. Surprisingly, mutants that lack espA reciprocally failed to secrete ESAT-6 and CFP-10 and were as attenuated as ESX-1 mutants in virulence assays. The results indicate that secretion of these proteins, which are each critical for virulence of pathogenic mycobacteria, is mutually dependent. The results further suggest that discerning the nature of the interaction and the structure of macromolecular complexes will provide insights into both an alternative mechanism of protein secretion and mycobacterial virulence.

Comprehensive identification of conditionally essential genes in mycobacteria.

An increasing number of microbial genomes have been completely sequenced, and the identified genes are categorized based on their homology to genes of known function. However, the function of a large number of genes cannot be determined on this basis alone. Here, we describe a technique, transposon site hybridization (TraSH), which allows rapid functional characterization by identifying the complete set of genes required for growth under different conditions. TraSH combines high-density insertional mutagenesis with microarray mapping of pools of mutants. We have made large pools of independent transposon mutants in mycobacteria by using a mariner-based transposon and efficient phage transduction. By using TraSH, we have defined the set of genes required for growth of Mycobacterium bovis bacillus Calmette-Guérin on minimal but not rich medium. Genes of both known and unknown functions were identified. Of the genes with known functions, nearly all were involved in amino acid biosynthesis. TraSH is a powerful method for categorizing gene function that should be applicable to a variety of microorganisms.

Mast cells migrate, but do not degranulate, in response to fractalkine, a membrane-bound chemokine expressed constitutively in diverse cells of the skin.

Mast cells (MC) are anatomically located near nerves and blood vessels in skin and the gastrointestinal tract and tend to localize within certain cutaneous tumors such as neurofibromas. However, the molecular mechanisms by which MC home to these sites are not well characterized. Fractalkine (FK) is a membrane-bound CX3C chemokine that displays constitutive expression in dendritic cells as well as in non-hematopoietic tissues including mammalian brain. Here we show that FK is constitutively expressed by skin endothelial cells, dermal dendrocytes and cells within neurofibromas. By reverse transcription-PCR, FK receptor, CX3CR1, is expressed by cultured murine bone marrow-derived MC (BMMC) of both connective tissue and mucosal phenotypes. Non-activated human dermal MC isolated from neonatal foreskin similarly demonstrated CX3CR1 expression. In chemotaxis assays, FK attracted MC with maximal migration occurring between 25 - 125 ng / ml. BMMC were not stimulated to release proinflammatory mediators in the presence of FK as measured by granule-associated beta-hexosaminidase release. Thus, CX3CR1 is expressed by MC and effectively mediates chemotaxis without inducing degranulation. We propose that the constitutive expression of FK on certain cells in the skin may be a factor in the tissue-specific homing of MC.

Identification of endoglycan, a member of the CD34/podocalyxin family of sialomucins.

CD34 and podocalyxin are structurally related sialomucins, which are expressed in multiple tissues including vascular endothelium and hematopoietic progenitors. These glycoproteins have been proposed to be involved in processes as diverse as glomerular filtration, inhibition of stem cell differentiation, and leukocyte-endothelial adhesion. Using homologies present in the cytoplasmic tails of these proteins, we have identified a novel member of this family, which we designate endoglycan. This protein shares a similar overall domain structure with the other family members including a sialomucin domain, but also possesses an extremely acidic amino-terminal region. In addition, endoglycan contains several potential glycosaminoglycan attachment sites and is modified with chondroitin sulfate. Endoglycan mRNA and protein were detected in both endothelial cells and CD34(+) bone marrow cells. Thus, CD34, podocalyxin, and endoglycan comprise a family of sialomucins sharing both structural similarity and sequence homology, which are expressed by both endothelium and multipotent hematopoietic progenitors. While the members of this family may perform overlapping functions at these sites, the unique structural features of endoglycan suggest distinct functions for this molecule.

Fractalkine, a CX3C chemokine, is expressed by dendritic cells and is up-regulated upon dendritic cell maturation.

The lone CX3C chemokine, fractalkine (FK), is expressed in a membrane-bound form on activated endothelial cells and mediates attachment and firm adhesion of T cells, monocytes and NK cells. We now show that FK is associated with dendritic cells (DC) in epidermis and lymphoid organs. In normal human skin, dual-color fluorescence microscopy co-localized FK expression with Langerhans cells expressing CD1a. In tonsil, FK-positive DC expressed CD83, a marker for mature DC. Human and murine cultured DC up-regulated FK mRNA expression with maturation. Furthermore, CD40 ligation, but not TNF-alpha or lipopolysaccharide treatment, of activated, migratory DC that had migrated from skin explants resulted in a 2.5-fold increase of surface expression of FK without significant alterations of expression of CD80, CD86, CD54 or MHC class II. Since FK mediates adhesion of T cells to activated endothelial cells, the increased expression of FK during DC maturation (and particularly by CD40 ligation) may play a role in the ability of T cells and mature DC to form conjugates and engage in cell-cell communication.

Identification of podocalyxin-like protein as a high endothelial venule ligand for L-selectin: parallels to CD34.

The leukocyte adhesion molecule, L-selectin, mediates the recruitment of lymphocytes to secondary lymphoid organs via interactions with specific ligands presented on high endothelial venules (HEV). Although the HEV-derived ligands for L-selectin are still incompletely defined, they share a common sialomucin-like structure which is thought to present clustered oligosaccharides to the lectin domain of L-selectin. Podocalyxin-like protein (PCLP) is a transmembrane sialomucin that is similar in structure to the well-characterized L-selectin ligand CD34. PCLP has been shown previously to be expressed on the foot processes of podocytes in the kidney glomerulus as well as on vascular endothelium at some sites. We have determined that PCLP is present on HEV, where it binds to both recombinant L-selectin and the HEV-specific monoclonal antibody MECA-79. Furthermore, purified HEV-derived PCLP is able to support the tethering and rolling of lymphocytes under physiological flow conditions in vitro. These results suggest a novel function for PCLP as an adhesion molecule and allow the definition of conserved structural features in PCLP and CD34, which may be important for L-selectin ligand function.

RNA secondary structure and binding sites for gag gene products in the 5' packaging signal of human immunodeficiency virus type 1.

The selective encapsidation of retroviral RNA requires sequences in the Gag protein, as well as a cis-acting RNA packaging signal (psi site) near the 5' end of the genomic transcript. Gag protein of human immunodeficiency virus type 1 (HIV-1) has recently been found to bind specifically to the HIV-1 psi element in vitro. Here we report studies aimed at mapping features within the genetically defined psi locus that are required for binding of HIV-1 Gag or of its processed nucleocapsid derivative. The full-length HIV-1 Gag (p55) and nucleocapsid (p15) sequences were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. In a gel shift assay containing excess competitor tRNA, affinity-purified GST-p15 and GST-p55 proteins bound to a 206-nucleotide psi RNA element spanning the major splice donor and gag start codons but did not bind to antisense psi transcripts. Quantitative filter-binding assays revealed that both GST-p55 and GST-p15 bound to this RNA sequence with identical affinities (apparent Kd congruent to 5 x 10(-8) M), indicating that all major determinants of psi binding affinity reside within the nucleocapsid portion of Gag. Chemical and RNase accessibility mapping, coupled with computerized sequence analysis, suggested a model for psi RNA structure comprising four independent stem-loops. Filter-binding studies revealed that RNAs corresponding to three of these hypothetical stem-loops can each function as a independent Gag binding site and that each is bound with approximately fourfold-lower apparent affinity than the full-length psi locus. Interaction of Gag with these regions is likely to play a major role in directing HIV-1 RNA encapsidation in vivo.