Acquisition of a natural resistance gene renders a clinical strain of methicillin-resistant Staphylococcus aureus resistant to the synthetic antibiotic linezolid.

Linezolid, which targets the ribosome, is a new synthetic antibiotic that is used for treatment of infections caused by Gram-positive pathogens. Clinical resistance to linezolid, so far, has been developing only slowly and has involved exclusively target site mutations. We have discovered that linezolid resistance in a methicillin-resistant Staphylococcus aureus hospital strain from Colombia is determined by the presence of the cfr gene whose product, Cfr methyltransferase, modifies adenosine at position 2503 in 23S rRNA in the large ribosomal subunit. The molecular model of the linezolid-ribosome complex reveals localization of A2503 within the drug binding site. The natural function of cfr likely involves protection against natural antibiotics whose site of action overlaps that of linezolid. In the chromosome of the clinical strain, cfr is linked to ermB, a gene responsible for dimethylation of A2058 in 23S rRNA. Coexpression of these two genes confers resistance to all the clinically relevant antibiotics that target the large ribosomal subunit. The association of the ermB/cfr operon with transposon and plasmid genetic elements indicates its possible mobile nature. This is the first example of clinical resistance to the synthetic drug linezolid which involves a natural resistance gene with the capability of disseminating among Gram-positive pathogenic strains.

Enterococcus faecalis PcfC, a spatially localized substrate receptor for type IV secretion of the pCF10 transfer intermediate.

Upon sensing of peptide pheromone, Enterococcus faecalis efficiently transfers plasmid pCF10 through a type IV secretion (T4S) system to recipient cells. The PcfF accessory factor and PcfG relaxase initiate transfer by catalyzing strand-specific nicking at the pCF10 origin of transfer sequence (oriT). Here, we present evidence that PcfF and PcfG spatially coordinate docking of the pCF10 transfer intermediate with PcfC, a membrane-bound putative ATPase related to the coupling proteins of gram-negative T4S machines. PcfC and PcfG fractionated with the membrane and PcfF with the cytoplasm, yet all three proteins formed several punctate foci at the peripheries of pheromone-induced cells as monitored by immunofluorescence microscopy. A PcfC Walker A nucleoside triphosphate (NTP) binding site mutant (K156T) fractionated with the E. faecalis membrane and also formed foci, whereas PcfC deleted of its N-terminal putative transmembrane domain (PcfCDelta N103) distributed uniformly throughout the cytoplasm. Native PcfC and mutant proteins PcfCK156T and PcfCDelta N103 bound pCF10 but not pcfG or Delta oriT mutant plasmids as shown by transfer DNA immunoprecipitation, indicating that PcfC binds only the processed form of pCF10 in vivo. Finally, purified PcfCDelta N103 bound DNA substrates and interacted with purified PcfF and PcfG in vitro. Our findings support a model in which (i) PcfF recruits PcfG to oriT to catalyze T-strand nicking, (ii) PcfF and PcfG spatially position the relaxosome at the cell membrane to stimulate substrate docking with PcfC, and (iii) PcfC initiates substrate transfer through the pCF10 T4S channel by an NTP-dependent mechanism.

Transcriptional regulation of the Borrelia burgdorferi antigenically variable VlsE surface protein.

The Lyme disease agent Borrelia burgdorferi can persistently infect humans and other animals despite host active immune responses. This is facilitated, in part, by the vls locus, a complex system consisting of the vlsE expression site and an adjacent set of 11 to 15 silent vls cassettes. Segments of nonexpressed cassettes recombine with the vlsE region during infection of mammalian hosts, resulting in combinatorial antigenic variation of the VlsE outer surface protein. We now demonstrate that synthesis of VlsE is regulated during the natural mammal-tick infectious cycle, being activated in mammals but repressed during tick colonization. Examination of cultured B. burgdorferi cells indicated that the spirochete controls vlsE transcription levels in response to environmental cues. Analysis of PvlsE::gfp fusions in B. burgdorferi indicated that VlsE production is controlled at the level of transcriptional initiation, and regions of 5' DNA involved in the regulation were identified. Electrophoretic mobility shift assays detected qualitative and quantitative changes in patterns of protein-DNA complexes formed between the vlsE promoter and cytoplasmic proteins, suggesting the involvement of DNA-binding proteins in the regulation of vlsE, with at least one protein acting as a transcriptional activator.

Functional and physical associations of beta1 integrins in the activation of human T lymphocytes

Adhesion involves interactions between cells or cells with extracellular matrix components and is a fundamental process for all multicellular organisms as well as many pathogenic microbes. Integrins are heterodimeric transmembrane proteins that function as adhesion molecules and transduce signals between the extracellular environment and the intracellular cytoskeletal machinery. β1 integrin subfamily is highly expressed on T lymphocytes and mediates cell spreading, adhesion and coactivation. T lymphocytes have an important role in the regulation and homeostasis of the immune system therefore, the goals of this study were to first to investigate β1 integrin interaction with fibronectin binding protein A (FnbpA), a surface protein expressed on gram-negative bacteria Staphylococcus aureus. Second, characterize the association and function of a non-integrin surface protein, CD98, with β1 integrins on T lymphocytes. ^ FnbpA binds to fibronectin (FN), also a ligand for α5β1 and α4β1 integrins on T lymphocytes. Since both bacterial proteins FnbpA and T cell integrins utilize FN, it was of interest to determine the effects FnbpA on T cell activation. Results demonstrated that recombinant FnbpA (rFnbpA) coimmobilized with OKT3 mediated T cell coactivation in a soluble FN-dependent manner. Integrin α5β1 was identified as the main integrin utilized by Staphylococcus aureus FnbpA from studies using soluble antibodies to inhibit T cell proliferation and parallel plate flow chamber assays. The mechanism of rFnbpA-mediated coactivation was one that used soluble FN as a bridge between rFnbpA and integrin α5β1 on the T lymphocyte. ^ Since integrins are utilized by T lymphocytes and bacterial proteins, it was of interest to identify proteins involved in integrin regulation. Anti-CD98 mAb 80A10 was identified and characterized from a screen to identify surface proteins involved in integrin signaling and functions. CD98 is a non-integrin protein that was sensitive to integrin inhibition in human T lymphocyte aggregation and activation, thus suggested that CD98 shared a common signaling pathway with integrins. These results led to the question of whether CD98 physically associates with β1 integrins. Fluorescence microscopy and biochemical analysis determined that CD98 is specifically associated with β1 integrin on human T lymphocytes and may be part of a larger multimolecular signaling complex. ^

Screening for imprinted genes by allelic message display: Identification of a paternally expressed gene Impact on mouse chromosome 18

A systematic screen termed the allelic message display (AMD) was developed for the hunting of imprinted genes. In AMD, differential display PCR is adopted to image allelic expression status of multiple polymorphic transcripts in two parental mouse strains, reciprocal F1 hybrids and pooled backcross progenies. From the displayed patterns, paternally and maternally expressed transcripts can be unequivocally identified. The effectiveness of AMD screening was clearly demonstrated by the identification of a paternally expressed gene Impact on mouse chromosome 18, the predicted product of which belongs to the YCR59c/yigZ hypothetical protein family composed of yeast and bacterial proteins with currently unknown function. In contrast with previous screening methods necessitating positional cloning efforts or generation of parthenogenetic embryos, this approach requires nothing particular but appropriately crossed mice and can be readily applied to any tissues at various developmental stages. Hence, AMD would considerably accelerate the identification of imprinted genes playing pivotal roles in mammalian development and the pathogenesis of various diseases.

Special issue : The Human Intestinal Microbiota

Peer reviewed

A secreted Salmonella protein with homology to an avirulence determinant of plant pathogenic bacteria

Bacterial pathogens have evolved sophisticated mechanisms to interact with their hosts. A specialized type III protein secretion system capable of translocating bacterial proteins into host cells has emerged as a central factor in the interaction between a variety of mammalian and plant pathogenic bacteria with their hosts. Here we describe AvrA, a novel target of the centisome 63 type III protein secretion system of Salmonella enterica. AvrA shares sequence similarity with YopJ of the animal pathogen Yersinia pseudotuberculosis and AvrRxv of the plant pathogen Xanthomonas campestris pv. vesicatoria. These proteins are the first examples of putative targets of type III secretion systems in animal and plant pathogenic bacteria that share sequence similarity. They may therefore constitute a novel family of effector proteins with related functions in the cross-talk of these pathogens with their hosts.

Nuclear localization signal binding protein from Arabidopsis mediates nuclear import of Agrobacterium VirD2 protein

T-DNA nuclear import is a central event in genetic transformation of plant cells by Agrobacterium. Presumably, the T-DNA transport intermediate is a single-stranded DNA molecule associated with two bacterial proteins, VirD2 and VirE2, which most likely mediate the transport process. While VirE2 cooperatively coats the transported single-stranded DNA, VirD2 is covalently attached to its 5′ end. To better understand the mechanism of VirD2 action, a cellular receptor for VirD2 was identified and its encoding gene cloned from Arabidopsis. The identified protein, designated AtKAPα, specifically bound VirD2 in vivo and in vitro. VirD2–AtKAPα interaction was absolutely dependent on the carboxyl-terminal bipartite nuclear localization signal sequence of VirD2. The deduced amino acid sequence of AtKAPα was homologous to yeast and animal nuclear localization signal-binding proteins belonging to the karyopherin α family. Indeed, AtKAPα efficiently rescued a yeast mutant defective for nuclear import. Furthermore, AtKAPα specifically mediated transport of VirD2 into the nuclei of permeabilized yeast cells.

Molecular characterization and assembly of the needle complex of the Salmonella typhimurium type III protein secretion system

Many bacterial pathogens of plants and animals have evolved a specialized protein-secretion system termed type III to deliver bacterial proteins into host cells. These proteins stimulate or interfere with host cellular functions for the pathogen's benefit. The Salmonella typhimurium pathogenicity island 1 encodes one of these systems that mediates this bacterium's ability to enter nonphagocytic cells. Several components of this type III secretion system are organized in a supramolecular structure termed the needle complex. This structure is made of discrete substructures including a base that spans both membranes and a needle-like projection that extends outward from the bacterial surface. We demonstrate here that the type III secretion export apparatus is required for the assembly of the needle substructure but is dispensable for the assembly of the base. We show that the length of the needle segment is determined by the type III secretion associated protein InvJ. We report that InvG, PrgH, and PrgK constitute the base and that PrgI is the main component of the needle of the type III secretion complex. PrgI homologs are present in type III secretion systems from bacteria pathogenic for animals but are absent from bacteria pathogenic for plants. We hypothesize that the needle component may establish the specificity of type III secretion systems in delivering proteins into either plant or animal cells.

A framework for interpreting the leucine-rich repeats of the Listeria internalins

The surface protein InlB of the bacterial pathogen Listeria monocytogenes is required for inducing phagocytosis in various nonphagocytic mammalian cell types in vitro. InlB causes tyrosine phosphorylation of host cell adaptor proteins, activation of phosphoinositide 3-kinase, and rearrangements of the actin cytoskeleton. These events lead to phagocytic uptake of the bacterium by the host cell. InlB belongs to the internalin family of Listeria proteins, which also includes InlA, another surface protein involved in host cell invasion. The internalins are the largest class of bacterial proteins containing leucine-rich repeats (LRR), a motif associated with protein–protein interactions. The LRR motif is found in a functionally diverse array of proteins, including those involved in the plant immune system and in the mammalian innate immune response. Structural and functional interpretations of the sequences of internalin family members are presented in light of the recently determined x-ray crystal structure of the InlB LRR domain.

Exploitation of host cells by enteropathogenic Escherichia coli

Microbial pathogens have evolved many ingenious ways to infect their hosts and cause disease, including the subversion and exploitation of target host cells. One such subversive microbe is enteropathogenic Escherichia coli (EPEC). A major cause of infantile diarrhea in developing countries, EPEC poses a significant health threat to children worldwide. Central to EPEC-mediated disease is its colonization of the intestinal epithelium. After initial adherence, EPEC causes the localized effacement of microvilli and intimately attaches to the host cell surface, forming characteristic attaching and effacing (A/E) lesions. Considered the prototype for a family of A/E lesion-causing bacteria, recent in vitro studies of EPEC have revolutionized our understanding of how these pathogens infect their hosts and cause disease. Intimate attachment requires the type III-mediated secretion of bacterial proteins, several of which are translocated directly into the infected cell, including the bacteria's own receptor (Tir). Binding to this membrane-bound, pathogen-derived protein permits EPEC to intimately attach to mammalian cells. The translocated EPEC proteins also activate signaling pathways within the underlying cell, causing the reorganization of the host actin cytoskeleton and the formation of pedestal-like structures beneath the adherent bacteria. This review explores what is known about EPEC's subversion of mammalian cell functions and how this knowledge has provided novel insights into bacterial pathogenesis and microbe-host interactions. Future studies of A/E pathogens in animal models should provide further insights into how EPEC exploits not only epithelial cells but other host cells, including those of the immune system, to cause diarrheal disease.

Molecular cloning of the first metazoan beta-1,3 glucanase from eggs of the sea urchin Strongylocentrotus purpuratus.

We report the molecular cloning of the first beta-1,3 glucanase from animal tissue. Three peptide sequences were obtained from beta-1,3 glucanase that had been purified from eggs of the sea urchin Strongylocentrotus purpuratus and the gene was cloned by PCR using oligonucleotides deduced from the peptide sequences. The full-length cDNA shows a predicted enzyme structure of 499 aa with a hydrophobic signal sequence. A 3.2-kb message is present in eggs, during early embryogenesis, and in adult gut tissue. A polyclonal antibody to the native 68-kDa enzyme recognizes a single band during early embryogenesis that reappears in the adult gut, and recognizes a 57-kDa fusion protein made from a full-length cDNA clone for beta-1,3 glucanase. The identity of this molecule as beta-1,3 glucanase is confirmed by sequence homology, by the presence of all three peptide sequences in the deduced amino acid sequence, and by the recognition of the bacterial fusion protein by the antibody directed against the native enzyme. Data base searches show significant homology at the amino acid level to beta-1,3 glucanases from two species of bacteria and a clotting factor from the horseshoe crab. The homology with the bacteria is centered in a 304-aa region in which there are seven scattered regions of high homology between the four divergent species. These four species were also found to have two homologous regions in common with more distantly related plant, fungal, and bacterial proteins. A global phylogeny based on these regions strongly suggests that the glucanases are a very ancient family of genes. In particular, there is an especially deep split within genes taken from the bacterial genus Bacillus.

A Rapid FACS-Based Strategy to Isolate Human Gene Knockin and Knockout Clones

Gene targeting protocols for mammalian cells remain inefficient and labor intensive. Here we describe FASTarget, a rapid, fluorescent cell sorting based strategy to isolate rare gene targeting events in human somatic cells. A fluorescent protein is used as a means for direct selection of targeted clones obviating the need for selection and outgrowth of drug resistant clones. Importantly, the use of a promoter-less, ATG-less construct greatly facilitates the recovery of correctly targeted cells. Using this method we report successful gene targeting in up to 94% of recovered human somatic cell clones. We create functional EYFP-tagged knockin clones in both transformed and non-transformed human somatic cell lines providing a valuable tool for mammalian cell biology. We further demonstrate the use of this technology to create gene knockouts. Using this generally applicable strategy we can recover gene targeted clones within approximately one month from DNA construct delivery to obtaining targeted monoclonal cell lines.

The Trk Potassium Transporter Is Required for RsmB-Mediated Activation of Virulence in the Phytopathogen Pectobacterium wasabiae

Pectobacterium wasabiae (previously known as Erwinia carotovora) is an important plant pathogen that regulates the production of plant cell wall-degrading enzymes through an N-acyl homoserine lactone-based quorum sensing system and through the GacS/GacA two-component system (also known as ExpS/ExpA). At high cell density, activation of GacS/GacA induces the expression of RsmB, a noncoding RNA that is essential for the activation of virulence in this bacterium. A genetic screen to identify regulators of RsmB revealed that mutants defective in components of a putative Trk potassium transporter (trkH and trkA) had decreased rsmB expression. Further analysis of these mutants showed that changes in potassium concentration influenced rsmB expression and consequent tissue damage in potato tubers and that this regulation required an intact Trk system. Regulation of rsmB expression by potassium via the Trk system occurred even in the absence of the GacS/GacA system, demonstrating that these systems act independently and are both required for full activation of RsmB and for the downstream induction of virulence in potato infection assays. Overall, our results identified potassium as an essential environmental factor regulating the Rsm system, and the consequent induction of virulence, in the plant pathogen P. wasabiae.

Proteomic analysis of a noninvasive human model of acute inflammation and its resolution:the twenty-one day gingivitis model

The 21-day experimental gingivitis model, an established noninvasive model of inflammation in response to increasing bacterial accumulation in humans, is designed to enable the study of both the induction and resolution of inflammation. Here, we have analyzed gingival crevicular fluid, an oral fluid comprising a serum transudate and tissue exudates, by LC-MS/MS using Fourier transform ion cyclotron resonance mass spectrometry and iTRAQ isobaric mass tags, to establish meta-proteomic profiles of inflammation-induced changes in proteins in healthy young volunteers. Across the course of experimentally induced gingivitis, we identified 16 bacterial and 186 human proteins. Although abundances of the bacterial proteins identified did not vary temporally, Fusobacterium outer membrane proteins were detected. Fusobacterium species have previously been associated with periodontal health or disease. The human proteins identified spanned a wide range of compartments (both extracellular and intracellular) and functions, including serum proteins, proteins displaying antibacterial properties, and proteins with functions associated with cellular transcription, DNA binding, the cytoskeleton, cell adhesion, and cilia. PolySNAP3 clustering software was used in a multilayered analytical approach. Clusters of proteins that associated with changes to the clinical parameters included neuronal and synapse associated proteins.