Amanda Carroll-Portillo

Genome-wide analysis of the PreA/PreB (QseB/QseC) regulon of Salmonella enterica serovar Typhimurium

BackgroundThe Salmonella PreA/PreB two-component system (TCS) is an ortholog of the QseBC TCS of Escherichia coli. In both Salmonella and E. coli, this system has been shown to affect motility and virulence in response to quorum-sensing and hormonal signals, and to affect the transcription of the Salmonella enterica serovar Typhimurium (S. Typhimurium) pmrAB operon, which encodes an important virulence-associated TCS.ResultsTo determine the PreA/PreB regulon in S. Typhimurium, we performed DNA microarrays comparing the wild type strain and various preA and/or preB mutants in the presence of ectopically expressed preA (qseB). These data confirmed our previous findings of the negative effect of PreB on PreA gene regulation and identified candidate PreA-regulated genes. A proportion of the activated loci were previously identified as PmrA-activated genes (yibD, pmrAB, cptA, etc.) or were genes located in the local region around preA, including the preAB operon. The transcriptional units were defined in this local region by RT-PCR, suggesting three PreA activated operons composed of preA-preB, mdaB-ygiN, and ygiW-STM3175. Several putative virulence-related phenotypes were examined for preAB mutants, resulting in the observation of a host cell invasion and slight virulence defect of a preAB mutant. Contrary to previous reports on this TCS, we were unable to show a PreA/PreB-dependent effect of the quorum-sensing signal AI-2 or of epinephrine on S. Typhimurium with regard to bacterial motility.ConclusionThis work further characterizes this unorthadox OmpR/EnvZ class TCS and provides novel candidate regulated genes for further study. This first in-depth study of the PreA/PreB regulatory system phenotypes and regulation suggests significant comparative differences to the reported function of the orthologous QseB/QseC in E. coli.

Role of Salmonella enterica Serovar Typhimurium Two-Component System PreA/PreB in Modulating PmrA-Regulated Gene Transcription

The PmrA/PmrB two-component system encoded by the pmrCAB operon regulates the modification of Salmonella enterica serovar Typhimurium lipopolysaccharide leading to polymyxin B resistance. PmrA and PhoP are the only known activators of pmrCAB. A transposon mutagenesis screen for additional regulators of a pmrC::MudJ fusion led to the identification of a two-component system, termed PreA/PreB (pmrCAB regulators A and B), that controls the transcription of the pmrCAB operon in response to unknown signals. The initial observations indicated that insertions in, or a deletion of, the preB sensor, but not the preA response regulator, caused upregulation of pmrCAB. Interestingly, the expression of pmrCAB was not upregulated in a preAB mutant grown in LB broth, implicating PreA in the increased expression of pmrCAB in the preB strain. This was confirmed by overexpression of preA(+) in preAB or preB backgrounds, which resulted in significant upregulation or further upregulation of pmrCAB. No such effect was observed in any tested preB(+) backgrounds. Additionally, an ectopic construct expressing a preA[D51A] allele also failed to upregulate pmrC in any of the pre backgrounds tested, which implies that there is a need for phosphorylation in the activation of the target genes. The observed upregulation of pmrCAB occurred independently of the response regulators PmrA and PhoP. Although a preB mutation led to increased transcription of pmrCAB, this did not result in a measurable effect on polymyxin B resistance. Our genetic data support a model of regulation whereby, in response to unknown signals, the PreB sensor activates PreA, which in turn indirectly upregulates pmrCAB transcription.

Formation of a Mast Cell Synapse: FcεRI Membrane Dynamics upon Binding Mobile or Immobilized Ligands on Surfaces

FcεRI on mast cells form a synapse when presented with mobile, bilayer-incorporated Ag. In this study, we show that receptor reorganization within the contacting mast cell membrane is markedly different upon binding of mobile and immobilized ligands. Rat basophilic leukemia mast cells primed with fluorescent anti-DNP IgE were engaged by surfaces presenting either bilayer-incorporated, monovalent DNP-lipid (mobile ligand), or chemically cross-linked, multivalent DNP (immobilized ligand). Total internal reflection fluorescence imaging and electron microscopy methods were used to visualize receptor reorganization at the contact site. The spatial relationships of FcεRI to other cellular components at the synapse, such as actin, cholesterol, and linker for activation of T cells, were also analyzed. Stimulation of mast cells with immobilized polyvalent ligand resulted in typical levels of degranulation. Remarkably, degranulation also followed interaction of mast cells, with bilayers presenting mobile, monovalent ligand. Receptors engaged with mobile ligand coalesce into large, cholesterol-rich clusters that occupy the central portion of the contacting membrane. These data indicate that FcεRI cross-linking is not an obligatory step in triggering mast cell signaling and suggest that dense populations of mobile receptors are capable of initiating low-level degranulation upon ligand recognition.

Mast Cell Synapses and Exosomes: Membrane Contacts for Information Exchange

In addition to their central role in allergy, mast cells are involved in a wide variety of cellular interactions during homeostasis and disease. In this review, we discuss the ability of mast cells to extend their mechanisms for intercellular communication beyond the release of soluble mediators. These include formation of mast cell synapses on antigen presenting surfaces, as well as cell–cell contacts with dendritic cells and T cells. Release of membrane bound exosomes also provide for the transfer of antigen, mast cell proteins, and RNA to other leukocytes. With the recognition of the extended role mast cells have during immune modulation, further investigation of the processes in which mast cells are involved is necessary. This reopens mast cell research to exciting possibilities, demonstrating it to be an immunological frontier.

In vitro Capture, Transport, and Detection of Protein Analytes Using Kinesin‐Based Nanoharvesters

Miniaturization of lab-on-a-chip devices to nanoscale dimensions necessitates a level of systems integration currently found primarily in biological systems. Such devices will require new modes of transportingmacromolecularmaterials at nanometer length scales. In cells, efficient cytoplasmic transport is achieved by energy-consuming, active transport systems in which motor proteins transport cargo along cytoskeletal filaments. For example, the motor protein kinesin-1 carries cell organelles and macromolecules over considerable distances along microtubule filaments. Microtubules are hollow protein polymeric filaments with a diameter of 25 nm and tens of micrometers in length that form a 3D transportation network within the cell. Small groups of kinesin transport cargo at rates up to 12mms , with a catalytic efficiency (i.e., conversion of chemical energy into work) of 50%. Together, this transport system provides a highefficiency means of transporting macromolecular cargo through the highly viscous medium of cytoplasm. The intriguing and powerful properties of kinesin-based transport have spurred its application in hybrid nanoscale systems. Early work focused on applying microfabrication technologies and surface functionalization to guide the kinesinbased transport of molecular shuttles (i.e., stabilized microtubule filaments) and achieve directed transport ofmaterials at the nanoscale. In this mode of application, commonly referred to as the inverted or glidingmotility geometry, kinesin motor proteins are bound on a solid surface such that their catalytic and microtubule-binding domains extend into the solution. In the presence ofATP,microtubule filaments bind to

Mast cells and dendritic cells form synapses that facilitate antigen transfer for T cell activation

Mast cells (MCs) and dendritic cells (DCs) form synapses that are dependent on MC activation and integrin engagement, and these direct interactions stimulate changes in the secretion profile of select cytokines and facilitate transfer of endosomal contents from activated MCs to DCs.

Directed Attachment of Antibodies to Kinesin-Powered Molecular Shuttles

Biomolecular motors, such as kinesin, have been used to shuttle a range of biological and synthetic cargo in microfluidic architectures. A critical gap in this technology is the ability to controllably link macromolecular cargo on microtubule (MT) shuttles without forming extraneous byproducts that may potentially limit their application. Here we present a generalized approach for functionalizing MTs with antibodies in which covalent bonds are formed between the carbohydrate in Fc region of polyclonal antibodies and the positively charged amino acids on the MT surface using the crosslinker succinimidyl 4‐hydrazidoterephthalate hydrochloride (SHTH). Antibody‐functionalized MTs (Ab‐MTs) produced through this approach maintained motility characteristics and antigenic selectivity, and did not produce undesirable byproducts common to other approaches. We also demonstrate and characterize the application of these Ab‐MTs for capturing and transporting bacterial and viral antigens. While this approach cannot be applied to monoclonal antibodies, which lack a carbohydrate moiety, it may be used for selectively functionalizing MT shuttles with a variety of carbohydrate‐containing cargoes. Biotechnol. Bioeng. 2009; 104: 1182–1188.

A continuous network of lipid nanotubes fabricated from the gliding motility of kinesin powered microtubule filaments.

Synthetic interconnected lipid nanotube networks were fabricated on the millimeter scale based on the simple, cooperative interaction between phospholipid vesicles and kinesin-microtubule (MT) transport systems. More specifically, taxol-stabilized MTs, in constant 2D motion via surface absorbed kinesin, extracted and extended lipid nanotube networks from large Lα phase multilamellar liposomes (5-25 μm). Based on the properties of the inverted motility geometry, the total size of these nanofluidic networks was limited by MT surface density, molecular motor energy source (ATP), and total amount and physical properties of lipid source material. Interactions between MTs and extended lipid nanotubes resulted in bifurcation of the nanotubes and ultimately the generation of highly branched networks of fluidically connected nanotubes. The network bifurcation was easily tuned by changing the density of microtubules on the surface to increase or decrease the frequency of branching. The ability of these networks to capture nanomaterials at the membrane surface with high fidelity was subsequently demonstrated using quantum dots as a model system. The diffusive transport of quantum dots was also characterized with respect to using these nanotube networks for mass transport applications.

Distribution and Dynamics of Rat Basophilic Leukemia Immunoglobulin E Receptors (FcɛRI) on Planar Ligand-Presenting Surfaces

There is considerable interest in the signaling mechanisms of immunoreceptors, especially when triggered with membrane-bound ligands. We have quantified the spatiotemporal dynamics of the redistribution of immunoglobulin E-loaded receptors (IgE-FcepsilonRI) on rat basophilic leukemia-2H3 mast cells in contact with fluid and gel-phase membranes displaying ligands for immunoglobulin E, using total internal reflection fluorescence microscopy. To clearly separate the kinetics of receptor redistribution from cell spreading, and to precisely define the initial contact time (+/-50 ms), micropipette cell manipulation was used to bring individual cells into contact with surfaces. On ligand-free surfaces, there are micron-scale heterogeneities in fluorescence that likely reflect regions of the cell that are more closely apposed to the substrate. When ligands are present, receptor clusters form with this same size scale. The initial rate of accumulation of receptors into the clusters is consistent with diffusion-limited trapping with D approximately 10(-1) microm2/s. These results support the hypothesis that clusters form by diffusion to cell-surface contact regions. Over longer timescales (>10 s), individual clusters moved with both diffusive and directed motion components. The dynamics of the cluster motion is similar to the dynamics of membrane fluctuations of cells on ligand-free fluid membranes. Thus, the same cellular machinery may be responsible for both processes.

Correlation between the oral microbiome and brain resting state connectivity in smokers

Recent studies have shown a critical role for the gastrointestinal microbiome in brain and behavior via a complex gut–microbiome–brain axis, however, the influence of the oral microbiome in neurological processes is much less studied, especially in response to the stimuli in the oral microenvironment such as smoking. Additionally, given the complex structural and functional networks in brain system, our knowledge about the relationship between microbiome and brain functions on specific brain circuits is still very limited. In this pilot work, we leverage next generation microbial sequencing with functional MRI techniques to enable the delineation of microbiome-brain network links as well as their relations to cigarette smoking. Thirty smokers and 30 age- and sex-matched non-smokers were recruited for measuring both microbial community and brain functional networks. Statistical analyses were performed to demonstrate the influence of smoking on: the taxonomy and abundance of the constituents within the oral microbial community, brain functional network connectivity, and associations between microbial shifts and the brain signaling network. Among smokers, we found significant decrease in beta diversity (p = 6×10−3) and identified several classes (Betaproteobacteria, Spirochaetia, Synergistia, and Mollicutes) as having significant alterations in microbial abundance. Metagenomic analyses demonstrate that the microbiota with altered abundance are mainly involved in pathways related to cell processes, DNA repair, immune system, and neurotransmitters signaling. One brain functional network connectivity component with marginal difference between smokers and nonsmokers (p = 0.033) consists of connectivity between default network and other task-positive networks (i.e., executive control network). This brain functional component was also significantly associated with some smoking- and immune- related oral microbiota, suggesting potential influence of smoking-induced oral microbiome dysbiosis in regulating brain functional connectivity, possibly through immunological or neurotransmitter signaling pathways. This work is the first attempt to link oral microbiome and brain functional networks, and provides the support for future work in characterizing the role of oral microbiome in mediating smoking effects on brain activity.