The Genome of a Pathogenic Rhodococcus: Cooptive Virulence Underpinned by Key Gene Acquisitions

We report the genome of the facultative intracellular parasite Rhodococcus equi, the only animal pathogen within the biotechnologically important actinobacterial genus Rhodococcus. The 5.0-Mb R. equi 103S genome is significantly smaller than those of environmental rhodococci. This is due to genome expansion in nonpathogenic species, via a linear gain of paralogous genes and an accelerated genetic flux, rather than reductive evolution in R. equi. The 103S genome lacks the extensive catabolic and secondary metabolic complement of environmental rhodococci, and it displays unique adaptations for host colonization and competition in the short-chain fatty acid–rich intestine and manure of herbivores—two main R. equi reservoirs. Except for a few horizontally acquired (HGT) pathogenicity loci, including a cytoadhesive pilus determinant (rpl) and the virulence plasmid vap pathogenicity island (PAI) required for intramacrophage survival, most of the potential virulence-associated genes identified in R. equi are conserved in environmental rhodococci or have homologs in nonpathogenic Actinobacteria. This suggests a mechanism of virulence evolution based on the cooption of existing core actinobacterial traits, triggered by key host niche–adaptive HGT events. We tested this hypothesis by investigating R. equi virulence plasmid-chromosome crosstalk, by global transcription profiling and expression network analysis. Two chromosomal genes conserved in environmental rhodococci, encoding putative chorismate mutase and anthranilate synthase enzymes involved in aromatic amino acid biosynthesis, were strongly coregulated with vap PAI virulence genes and required for optimal proliferation in macrophages. The regulatory integration of chromosomal metabolic genes under the control of the HGT–acquired plasmid PAI is thus an important element in the cooptive virulence of R. equi

3D Porous Chitosan-Alginate Scaffolds As An In Vitro Model For Evaluating Nanoparticle-Mediated Tumor Targeting And Gene Delivery To Prostate Cancer

Cationic nanoparticles (NPs) for targeted gene delivery are conventionally evaluated using 2D in vitro cultures. However, this does not translate well to corresponding in vivo studies because of the marked difference in NP behavior in the presence of the tumor microenvironment. In this study, we investigated whether prostate cancer (PCa) cells cultured in three-dimensional (3D) chitosan-alginate (CA) porous scaffolds could model cationic NP-mediated gene targeted delivery to tumors in vitro. We assessed in vitro tumor cell proliferation, formation of tumor spheroids, and expression of marker genes that promote tumor malignancy in CA scaffolds. The efficacy of NP-targeted gene delivery was evaluated in PCa cells in 2D cultures, PCa tumor spheroids grown in CA scaffolds, and PCa tumors in a mouse TRAMP-C2 flank tumor model. PCa cells cultured in CA scaffolds grew into tumor spheroids and displayed characteristics of higher malignancy as compared to those in 2D cultures. Significantly, targeted gene delivery was only observed in cells cultured in CA scaffolds, whereas cells cultured on 2D plates showed no difference in gene delivery between targeted and nontarget control NPs. In vivo NP evaluation confirmed targeted gene delivery, indicating that only CA scaffolds correctly modeled NP-mediated targeted delivery in vivo. These findings suggest that CA scaffolds serve as a better in vitro platform than 2D cultures for evaluation of NP-mediated targeted gene delivery to PCa

A Bioinformatics 3D Cellular Morphotyping Strategy For Assessing Biomaterial Scaffold Niches

Many biomaterial scaffolds have been advanced to provide synthetic cell niches for tissue engineering and drug screening applications; however, current methods for comparing scaffold niches focus on cell functional outcomes or attempt to normalize materials properties between different scaffold formats. We demonstrate a three-dimensional (3D) cellular morphotyping strategy for comparing biomaterial scaffold cell niches between different biomaterial scaffold formats. Primary human bone marrow stromal cells (hBMSCs) were cultured on 8 different biomaterial scaffolds, including fibrous scaffolds, hydrogels, and porous sponges, in 10 treatment groups to compare a variety of biomaterial scaffolds and cell morphologies. A bioinformatics approach was used to determine the 3D cellular morphotype for each treatment group by using 82 shape metrics to analyze approximately 1000 cells. We found that hBMSCs cultured on planar substrates yielded planar cell morphotypes, while those cultured in 3D scaffolds had elongated or equiaxial cellular morphotypes with greater height. Multivariate analysis was effective at distinguishing mean shapes of cells in flat substrates from cells in scaffolds, as was the metric L1-depth (the cell height along its shortest axis after aligning cells with a characteristic ellipsoid). The 3D cellular morphotyping technique enables direct comparison of cellular microenvironments between widely different types of scaffolds and design of scaffolds based on cell structure-function relationships

Modeling, Validation And Verification Of Three-Dimensional Cell-Scaffold Contacts From Terabyte-Sized Images

Background: Cell-scaffold contact measurements are derived from pairs of co-registered volumetric fluorescent confocal laser scanning microscopy (CLSM) images (z-stacks) of stained cells and three types of scaffolds (i.e., spun coat, large microfiber, and medium microfiber). Our analysis of the acquired terabyte-sized collection is motivated by the need to understand the nature of the shape dimensionality (1D vs 2D vs 3D) of cell-scaffold interactions relevant to tissue engineers that grow cells on biomaterial scaffolds. Results: We designed five statistical and three geometrical contact models, and then down-selected them to one from each category using a validation approach based on physically orthogonal measurements to CLSM. The two selected models were applied to 414 z-stacks with three scaffold types and all contact results were visually verified. A planar geometrical model for the spun coat scaffold type was validated from atomic force microscopy images by computing surface roughness of 52.35 nm ±31.76 nm which was 2 to 8 times smaller than the CLSM resolution. A cylindrical model for fiber scaffolds was validated from multi-view 2D scanning electron microscopy (SEM) images. The fiber scaffold segmentation error was assessed by comparing fiber diameters from SEM and CLSM to be between 0.46% to 3.8% of the SEM reference values. For contact verification, we constructed a web-based visual verification system with 414 pairs of images with cells and their segmentation results, and with 4968 movies with animated cell, scaffold, and contact overlays. Based on visual verification by three experts, we report the accuracy of cell segmentation to be 96.4% with 94.3% precision, and the accuracy of cell-scaffold contact for a statistical model to be 62.6% with 76.7% precision and for a geometrical model to be 93.5% with 87.6% precision. Conclusions: The novelty of our approach lies in (1) representing cell-scaffold contact sites with statistical intensity and geometrical shape models, (2) designing a methodology for validating 3D geometrical contact models and (3) devising a mechanism for visual verification of hundreds of 3D measurements. The raw and processed data are publicly available from https://isg.nist.gov/deepzoomweb/data/together with the web -based verification system

Effect Of The Scaffold Microenvironment On Cell Polarizability And Capacitance Determined By Probabilistic Computations

In living systems, it is frequently stated that form follows function by virtue of evolutionary pressures on organism development, but in the study of how functions emerge at the cellular level, function often follows form. We study this chicken versus egg problem of emergent structure-property relationships in living systems in the context of primary human bone marrow stromal cells cultured in a variety of microenvironments that have been shown to cause distinct patterns of cell function and differentiation. Through analysis of a publicly available catalog of three-dimensional (3D) cell shape data, we introduce a family of metrics to characterize the \u27form\u27 of the cell populations that emerge from a variety of diverse microenvironments. In particular, measures of form are considered that are expected to have direct significance for cell function, signaling and metabolic activity: dimensionality, polarizability and capacitance. Dimensionality was assessed by an intrinsic measure of cell shape obtained from the polarizability tensor. This tensor defines ellipsoids for arbitrary cell shapes and the thinnest dimension of these ellipsoids, P 1 , defines a reference minimal scale for cells cultured in a 3D microenvironment. Polarizability governs the electric field generated by a cell, and determines the cell\u27s ability to detect electric fields. Capacitance controls the shape dependence of the rate at which diffusing molecules contact the surface of the cell, and this has great significance for inter-cellular signaling. These results invite new approaches for designing scaffolds which explicitly direct cell dimensionality, polarizability and capacitance to guide the emergence of new cell functions derived from the acquired form

3D Porous Chitosan-Alginate Scaffolds Promote Proliferation And Enrichment Of Cancer Stem-Like Cells

Cancer stem cells are increasingly becoming a primary target for new cancer treatment development. The ability to study their transient behavior in vitro will provide the opportunity for high-throughput testing of more effective therapies. We have previously demonstrated the use of 3D porous chitosan-alginate (CA) scaffolds to promote cancer stem-like cell (CSC) proliferation and enrichment in glioblastoma. Here we use 3D porous CA scaffolds to promote cancer stem-like cell enrichment in cell lines from prostate, liver, and breast cancers, and investigate the proliferation, morphology, and gene expressions of cells cultured in CA scaffolds as compared to 2D controls. The 3D CA scaffold cultures for all three cancer types showed reduced proliferation, formation of tumor spheroids, and increased expression of CSC associated mark genes (CD133 and NANOG), as opposed to monolayers. Additionally, we present a putative mechanism for the cancer stem-like cell enrichment on CA scaffolds. This study demonstrates that the cancer stem-like cell enrichment in CA scaffolds is a robust process that is not restricted to particular cancer types

3D Porous Chitosan–Alginate Scaffolds as an In Vitro Model for Evaluating Nanoparticle-Mediated Tumor Targeting and Gene Delivery to Prostate Cancer

The cationic nanoparticles (NPs) for targeted gene delivery is conventionally evaluated using 2D in vitro cultures. However, this does not translate well to corresponding in vivo studies because of the marked difference in NP behavior in the presence of the tumor microenvironment. In this study, we investigated if prostate cancer (PCa) cells cultured in three–dimensional (3D) chitosan-alginate (CA) porous scaffolds could model cationic nanoparticle-mediated gene targeted delivery to tumors in vitro. We assessed in vitro tumor cell proliferation, formation of tumor spheroids, and expression of marker genes that promote tumor malignancy in CA scaffolds. The efficacy of NP targeted gene delivery was evaluated in PCa cells in 2D cultures, PCa tumor spheroids grown in CA scaffolds, and PCa tumors in a mouse TRAMP-C2 flank tumor model. PCa cells cultured in CA scaffolds grew into tumor spheroids and displayed characteristics of higher malignancy as compared to those in 2D cultures. Significantly, targeted gene delivery was only observed in cells cultured in CA scaffolds whereas cells cultured on 2D plates showed no difference in gene delivery between targeted and non-target control NPs. In vivo NP evaluation confirmed targeted gene delivery indicating that only CA scaffolds correctly modeled NP-mediated targeted delivery in vivo. These findings suggest that CA scaffolds serve as a better in vitro platform than 2D cultures for evaluation of NP-mediated targeted gene delivery to PCa

Inhibition of Staphylococcus aureus cysteine proteases by human serpin potentially limits staphylococcal virulence

Bacterial proteases are considered virulence factors and it is presumed that by abrogating their activity, host endogenous protease inhibitors play a role in host defense against invading pathogens. Here we present data showing that Staphylococcus aureus cysteine proteases (staphopains) are efficiently inhibited by Squamous Cell Carcinoma Antigen 1 (SCCA1), an epithelial-derived serpin. The high association rate constant (k(ass)) for inhibitory complex formation (1.9×10(4) m/s and 5.8×10(4) m/s for staphopain A and stapho-pain B interaction with SCCA1, respectively), strongly suggests that SCCA1 can regulate staphopain activity in vivo at epithelial surfaces infected/colonized by S. aureus. The mechanism of staphopain inhibition by SCCA1 is apparently the same for serpin interaction with target serine proteases whereby the formation of a covalent complex result in cleavage of the inhibitory reactive site peptide bond and associated release of the C-terminal serpin fragment. Interestingly, the SCCA1 reactive site closely resembles a motif in the reactive site loop of native S. aureus-derived inhibitors of the staphopains (staphostatins). Given that S. aureus is a major pathogen of epithelial surfaces, we suggest that SCCA1 functions to temper the virulence of this bacterium by inhibiting the staphopains