Supplement 1: Pupil function engineering for enhanced nanoparticle visibility in wide-field interferometric microscopy

Originally published in Optica on 20 February 2017 (optica-4-2-247

A High-Throughput Method to Examine Protein-Nucleotide Interactions Identifies Targets of the Bacterial Transcriptional Regulatory Protein Fur

<div><p>The <u>F</u>erric <u>u</u>ptake <u>r</u>egulatory protein (Fur) is a transcriptional regulatory protein that functions to control gene transcription in response to iron in a number of pathogenic bacteria. In this study, we applied a label-free, quantitative and high-throughput analysis method, <u>I</u>nterferometric <u>R</u>eflectance <u>I</u>maging <u>S</u>ensor (IRIS), to rapidly characterize Fur-DNA interactions <i>in vitro</i> with predicted Fur binding sequences in the genome of <i>Neisseria gonorrhoeae</i>, the causative agent of the sexually transmitted disease gonorrhea. IRIS can easily be applied to examine multiple protein-protein, protein-nucleotide and nucleotide-nucleotide complexes simultaneously and demonstrated here that seventy percent of the predicted Fur boxes in promoter regions of iron-induced genes bound to Fur <i>in vitro</i> with a range of affinities as observed using this microarray screening technology. Combining binding data with mRNA expression levels in a gonococcal <i>fur</i> mutant strain allowed us to identify five new gonococcal genes under Fur-mediated direct regulation.</p></div

Predicted Fur binding sequences within the promoter regions of <i>N. gonorrhoeae</i> iron-induced genes.¹

1<p>Iron regulation as previously determined by microarray analyses of <i>N. gonorrhoeae</i> grown in defined medium CDM (-Fe) or CDM with 10 µM ferric nitrate (+Fe) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096832#pone.0096832-Ducey1" target="_blank">[50]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096832#pone.0096832-Jackson1" target="_blank">[51]</a>.</p>2<p>Fur regulation was determined by comparing mRNA levels in <i>N. gonorrhoeae</i> WT, <i>fur</i> mutant and <i>fur</i> complemented strains grown under iron-replete and iron-deplete conditions at 1h after addition of iron and desferal using qRT-PCR. NR; not regulated by Fur. Activated; mRNA level of the gene was decreased in the <i>fur</i> mutant strain under either iron-replete or iron-deplete conditions compared to that in the wild type strain. Repressed; mRNA level of the gene was increased in the <i>fur</i> mutant strain under either iron-replete or iron-deplete conditions compared to that in the wild type strain. NE, not examined; transcriptional regulation was not tested in this study.</p>3<p>Fur regulation of the genes as previously determined <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096832#pone.0096832-Yu1" target="_blank">[8]</a>.</p>4<p><i>In vitro</i> Fur binding to the 500 bp upstream sequence of ATG of these genes was determined using EMSA and foot printing in previous studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096832#pone.0096832-Yu1" target="_blank">[8]</a>.</p

Transcriptional regulation patterns of genes determined by quantitative real-time PCR.

<p>The RNA samples were purified from cultures of the wild-type (WT) and <i>fur</i> mutant and <i>fur</i> complemented strains under iron-replete (+Fe, grey bars) or iron-deplete (-Fe, white bars) conditions 1 h after addition of 100 µM iron or 150 µM desferal. The mRNA levels of <i>fbpA</i> and <i>norB</i>, genes that were repressed and activated by iron-bound Fur respectively, were used as controls for iron and Fur regulation in <i>N. gonorrhoeae</i>. The mRNA levels observed for the five conditions (WT strain under −Fe conditions, <i>fur</i> mutant strain under +Fe and −Fe conditions, and <i>fur</i> complemented strain under +Fe and −Fe conditions) were compared to the value of WT strain under +Fe conditions. The final results were represented as mean ± standard deviation. A * indicates significantly different compared to the mRNA level of WT+Fe. A ** indicates significantly different compared to the mRNA level of WT-Fe. The gene designations of <i>N. gonorrhoeae</i> F62 were assigned according to their homologues in <i>N. gonorrhoeae</i> FA1090.</p

Identification of Fur binding to predicted Fur boxes by label-free IRIS screening.

<p>A set of ∼50 bp dsDNA probes containing the predicted Fur box in the middle of the probes were designed for IRIS screening (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096832#pone.0096832.s006" target="_blank">Table S4</a></b>). Each dsDNA probe was immobilized on a chip to produce a spot with a diameter of approximately 100 µm. Three concentrations of Fur protein, 200 nM (red bars), 400 nM (blue bars) and 800 nM (orange bars), were incubated with the individually prepared arrays (in addition to a 0 nM control incubation (black bars) and the binding of Fur protein to dsDNA spots was measured. A mass increase for each spot was represented as a differential spot height (DSH). The known Fur boxes in <i>fur</i>, <i>norB</i> and <i>nspA</i> promoter regions were used as positive controls, and the <i>aniA</i> promoter region was used as negative control in this IRIS assay. (<b>A</b>) Differential spot height for each dsDNA probe after incubation with 0 nM, 200 nM, 400 nM and 800 nM Fur protein, respectively. (<b>B</b>) The number of Fur dimers bound per dsDNA molecule of each probe as calculated from initial and post-incubation mass density measurements. The gene designations of <i>N. gonorrhoeae</i> F62 were assigned according to their homologues in <i>N. gonorrhoeae</i> FA1090.</p

Fur binding to the predicted Fur boxes. (A) Cold competition assay for the specificity of Fur binding to the predicted Fur boxes.

<p>³²P-labeled ∼50 bp dsDNAs were analyzed after incubation with Fur and unlabeled dsDNA probes (cold probes). Two types of cold competitor probes were used: <i>fur</i>, which contains a Fur box, and <i>rmp</i>, which does not bind to Fur. When the unlabeled probes <i>fur</i> compete out binding of labeled probes, but the unlabeled <i>rmp</i> cannot, the binding of Fur to the predicted Fur box was considered specific. Lane 1, free ³²P-labeled DNA; Lane 2 through Lane 10 contained gonococcal Fur. For the <i>fur</i> and NGO0073 Fur box, Fur protein was added at a concentration of 100 nM and for the NGO0101 Fur box, 400 nM of Fur protein was added. The fold excess of the cold probes was increased from Lane 3 to Lane 6 and Lane 7 to Lane 10 ranging from 50 fold, 500 fold to 1000 fold (indicated by the triangles). <b>(B) Fur binding affinities determined by EMSA.</b> The ³²P labeled dsDNA probes were incubated with a gradient of concentrations of purified gonococcal Fur protein (Lane 1 to Lane 10, 0 nM, 5 nM, 25 nM, 50 nM, 100 nM, 200 nM, 400 nM, 600 nM, 800 nM and 1000 nM, respectively). Arrows indicate the shift of Fur-bound probes. The apparent binding affinities (K_D) were calculated using GraphPad Prism and were represented as mean ± standard error. The gene designations of <i>N. gonorrhoeae</i> F62 were assigned according to their homologues in <i>N. gonorrhoeae</i> FA1090.</p

Schematic depicting Fur mediated control mechanisms in <i>N. gonorrhoeae</i> as revealed by IRIS.

<p>Solid lines with arrowheads indicate direct activation of transcription via Fur binding to promoter regions of gonococcal genes. Solid lines with bars indicate direct repression of transcription via binding of Fur to promoter regions of gonococcal genes. Ellipsoids indicate transcriptional regulatory proteins other than Fur. Transcriptional repression of NGO0641, encoding a methyltransferase (triangle), results in alteration of DNA methylation (*) and subsequently alters transcription of a subset of genes. Transcriptional repression of NGO0155, encoding a putative transcriptional regulator, results in alteration of transcription of NGO0155 target genes. A subset of genes contain a Fur box in their putative promoter regions but were not regulated by Fur under the used growth conditions in this study are termed silent Fur binding.</p

Enhanced light microscopy visualization of virus particles from Zika virus to filamentous ebolaviruses

<div><p>Light microscopy is a powerful tool in the detection and analysis of parasites, fungi, and prokaryotes, but has been challenging to use for the detection of individual virus particles. Unlabeled virus particles are too small to be visualized using standard visible light microscopy. Characterization of virus particles is typically performed using higher resolution approaches such as electron microscopy or atomic force microscopy. These approaches require purification of virions away from their normal millieu, requiring significant levels of expertise, and can only enumerate small numbers of particles per field of view. Here, we utilize a visible light imaging approach called Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS) that allows automated counting and sizing of thousands of individual virions. Virions are captured directly from complex solutions onto a silicon chip and then detected using a reflectance interference imaging modality. We show that the use of different imaging wavelengths allows the visualization of a multitude of virus particles. Using Violet/UV illumination, the SP-IRIS technique is able to detect individual flavivirus particles (~40 nm), while green light illumination is capable of identifying and discriminating between vesicular stomatitis virus and vaccinia virus (~360 nm). Strikingly, the technology allows the clear identification of filamentous infectious ebolavirus particles and virus-like particles. The ability to differentiate and quantify unlabeled virus particles extends the usefulness of traditional light microscopy and can be embodied in a straightforward benchtop approach allowing widespread applications ranging from rapid detection in biological fluids to analysis of virus-like particles for vaccine development and production.</p></div

Measured particle distribution of virus preparation measured by SP-IRIS.

<p>The contrast of the individual detected particle is converted to particle diameter using the response curve created with polystyrene bead standard and refractive index of the virus.</p

SP-IRIS real-time detection and characterization of Ebola virus in-liquid.

<p>a) SP-IRIS image of captured Ebola virus with identified particles and filaments color-coded at eighteen minutes. Scale bar 5 micron b) A zoomed portion of Ebola antibody spot highlighted by square box in 7a. Scale bar 1 micron. c) Plot of the number of small particle and medium (blue) and large (red) filamentous Ebola virions as a function of time.</p