Measuring the Adhesion Forces for the Multivalent Binding of Vancomycin-Conjugated Dendrimer to Bacterial Cell-Wall Peptide

Multivalent ligand–receptor interaction provides the fundamental basis for the hypothetical notion that high binding avidity relates to the strong force of adhesion. Despite its increasing importance in the design of targeted nanoconjugates, an understanding of the physical forces underlying the multivalent interaction remains a subject of urgent investigation. In this study, we designed three vancomycin (Van)-conjugated dendrimers G5­(Van)_<i>n</i> (<i>n</i> = mean valency = 0, 1, 4) for bacterial targeting with generation 5 (G5) poly­(amidoamine) dendrimer as a multivalent scaffold and evaluated both their binding avidity and physical force of adhesion to a bacterial model surface by employing surface plasmon resonance (SPR) spectroscopy and atomic force microscopy. The SPR experiment for these conjugates was performed in a biosensor chip surface immobilized with a bacterial cell-wall peptide Lys-d-Ala-d-Ala. Of these, G5­(Van)₄ bound most tightly with a <i>K</i>_D of 0.34 nM, which represents an increase in avidity by 2 or 3 orders of magnitude relative to a monovalent conjugate G5­(Van)₁ or free vancomycin, respectively. By single-molecule force spectroscopy, we measured the adhesion force between G5­(Van)_<i>n</i> and the same cell-wall peptide immobilized on the surface. The distribution of adhesion forces increased in proportion to vancomycin valency with the mean force of 134 pN at <i>n</i> = 4 greater than 96 pN at <i>n</i> = 1 at a loading rate of 5200 pN/s. In summary, our results are strongly supportive of the positive correlation between the avidity and adhesion force in the multivalent interaction of vancomycin nanoconjugates

MicroRNA and mRNA Transcriptome Profiling in Primary Human Astrocytes Infected with <i>Borrelia burgdorferi</i>

<div><p>Lyme disease is caused by infection with the bacterium <i>Borrelia burgdorferi (Bb)</i>, which is transmitted to humans by deer ticks. The infection manifests usually as a rash and minor systemic symptoms; however, the bacteria can spread to other tissues, causing joint pain, carditis, and neurological symptoms. Lyme neuroborreliosis presents itself in several ways, such as Bell’s palsy, meningitis, and encephalitis. The molecular basis for neuroborreliosis is poorly understood. Analysis of the changes in the expression levels of messenger RNAs and non-coding RNAs, including microRNAs, following <i>Bb</i> infection could therefore provide vital information on the pathogenesis and clinical symptoms of neuroborreliosis. To this end, we used cultured primary human astrocytes, key responders to CNS infection and important components of the blood-brain barrier, as a model system to study RNA and microRNA changes in the CNS caused by <i>Bb</i>. Using whole transcriptome RNA-seq, we found significant changes in 38 microRNAs and 275 mRNAs at 24 and 48 hours following <i>Bb</i> infection. Several of the RNA changes affect pathways involved in immune response, development, chromatin assembly (including histones) and cell adhesion. Further, several of the microRNA predicted target mRNAs were also differentially regulated. Overall, our results indicate that exposure to <i>Bb</i> causes significant changes to the transcriptome and microRNA profile of astrocytes, which has implications in the pathogenesis, and hence potential treatment strategies to combat this disease.</p></div

Selected long non-coding RNAs altered in response to <i>Bb</i>.

<p>Selected long non-coding RNAs altered in response to <i>Bb</i>.</p

MicroRNA differential expression pathway analysis.

<p>We identified pathways targeted by differentially expressed miRNAs using the microT-CDS algorithm in DIANA-miRPath. Heat map of microRNAs vs Pathways, where microRNAs and pathways are clustered using Euclidean distances and complete linkage of binary values (0 = non-significant p-value and 1 = significant p-value). Red squares signify a significant p-value and light yellow signify a non-significant p-value.</p

The microRNA-only isolation method for sequencing resulted in a higher percentage of unique reads that mapped to microRNAs.

<p>Astrocytes treated with <i>Bb</i> were lysed, and either total RNA and microRNA, or the microRNA-only fractions were isolated using procedures according to the miRNeasy kit (A). The microRNA from both preparations and libraries made using the Illumina TruSeq small RNA library kit. (B). Libraries were size-selected using the Illumina custom RNA ladder for size selection of the ~145-160bp band, and sequenced using the MiSeq. Lane 1: small RNA marker; lanes 2–3: duplicates of total RNA+miRNA preps; lanes 4–5: duplicates of miRNA only preps (C). The total number of reads, the percent alignments and number of unique reads were comparable in both conditions, but the microRNA-only fraction had a significantly higher percentage of unique reads that mapped to microRNAs relative to the total RNA+microRNA fraction (48% vs 18%). See text for details.</p

Differential expression of microRNAs following exposure to <i>Bb</i>.

<p>MicroRNAs were isolated from astrocytes treated with <i>Bb</i> for 24h (N = 3) and 48h (N = 3), using the microRNA-only procedure at the same time as RNAs, as described in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170961#pone.0170961.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170961#pone.0170961.g003" target="_blank">3</a>. Heat map analysis (A) showing changes in a subset of microRNAs (B) following 48h of <i>Bb</i> treatment. (C) Validation of microRNA changes were performed on 4 microRNAs (see text for details), and revealed upregulation of miR143, miR146b-1, miR199a1 and miR376a2. T-test (* = p-value<0.05; *** = p-value <0.0001).</p

Selected inflammation and immune function genes altered in response to <i>Bb</i>.

<p>Selected inflammation and immune function genes altered in response to <i>Bb</i>.</p

Effects of geohelminth infections on hemoglobin, anemia, and mid upper arm circumference by type of infection and gravida group among pregnant women in Gem, July 2003.

<p>Abbreviations: AOR: adjusted odds ratio; CI: confidence interval; MUAC: mid upper arm circumference. Significant differences or odds ratios are printed in bold.</p>*<p>Models adjusted for malaria, marital status, treatment of water and a report of soil eating and other geohelminths unless indicated otherwise. Malaria, marital status, treatment of water and a report of soil eating were significantly associated with anemia and hemoglobin level in the model for all women, and for uniformity kept in the models by gravidity, even when not significant.</p>†<p>Model adjusted for malaria, water treatment and other geohelminths.</p>‡<p>Model only adjusted for other geohelminths.</p

Prevalence of malaria by geohelminth infection and gravidity group among 390 pregnant women in Gem, July 2003.

<p>Prevalence of malaria by geohelminth infection and gravidity group among 390 pregnant women in Gem, July 2003.</p

Characteristics of the participating pregnant women by presence of a stool sample result, Gem, July 2003.

<p>Abbreviations: SES: socio-economic status; MUAC: mid-upper arm circumference.</p>*<p>Chi-square test <i>P</i><0.05 comparing characteristic of women with vs. without a stool sample.</p>†<p>Trimester of pregnancy missing for 3 women; SES missing for 156 women (23.2%); years of education missing for 110 women (16.3%); water source and treatment missing for 133 women (19.8%); body mass index missing for 3 women.</p