Ariel - Volume 8 Number 4

Executive Editor James W. Lockard Jr. Issues Editor Neeraj K. Kanwal Business Manager Neeraj K. Kanwal University News Martin Trichtinger World News Doug Hiller Opinions Elizabeth A. McGuire Features Patrick P. Sokas Sports Desk Shahab S. Minassian Managing Editor Edward H. Jasper Managing Associate Brenda Peterson Photography Editor Robert D. Lehman, Jr. Graphics Christine M. Kuhnl

Display of Cell Surface Sites for Fibronectin Assembly Is Modulated by Cell Adherence to 1F3 and C-Terminal Modules of Fibronectin

BACKGROUND: Fibronectin-null cells assemble soluble fibronectin shortly after adherence to a substrate coated with intact fibronectin but not when adherent to the cell-binding domain of fibronectin (modules (7)F3-(10)F3). Interactions of adherent cells with regions of adsorbed fibronectin other than modules (7)F3-(10)F3, therefore, are required for early display of the cell surface sites that initiate and direct fibronectin assembly. METHODOLOGY/PRINCIPAL FINDINGS: To identify these regions, coatings of proteolytically derived or recombinant pieces of fibronectin containing modules in addition to (7)F3-(10)F3 were tested for effects on fibronectin assembly by adherent fibronectin-null fibroblasts. Pieces as large as one comprising modules (2)F3-(14)F3, which include the heparin-binding and cell adhesion domains, were not effective in supporting fibronectin assembly. Addition of module (1)F3 or the C-terminal modules to modules (2)F3-(14)F3 resulted in some activity, and addition of both (1)F3 and the C-terminal modules resulted in a construct, (1)F3-C, that best mimicked the activity of a coating of intact fibronectin. Constructs (1)F3-C V0, (1)F3-C V64, and (1)F3-C Delta(V(15)F3(10)F1) were all able to support fibronectin assembly, suggesting that (1)F3 through (11)F1 and/or (12)F1 were important for activity. Coatings in which the active parts of (1)F3-C were present in different proteins were much less active than intact (1)F3-C. CONCLUSIONS: These results suggest that (1)F3 acts together with C-terminal modules to induce display of fibronectin assembly sites on adherent cells

Sexual Transmission of a Plant Pathogenic Bacterium, Candidatus Liberibacter asiaticus, between Conspecific Insect Vectors during Mating

Candidatus Liberibacter asiaticus is a fastidious, phloem-inhabiting, gram-negative bacterium transmitted by Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae). The bacterium is the presumed causal agent of huanglongbing (HLB), one of the most destructive and economically important diseases of citrus. We investigated whether Las is transmitted between infected and uninfected D. citri adults during courtship. Our results indicate that Las was sexually transmitted from Las-infected male D. citri to uninfected females at a low rate (<4%) during mating. Sexual transmission was not observed following mating of infected females and uninfected males or among adult pairs of the same sex. Las was detected in genitalia of both sexes and also in eggs of infected females. A latent period of 7 days or more was required to detect the bacterium in recipient females. Rod shaped as well as spherical structures resembling Las were observed in ovaries of Las-infected females with transmission electron microscopy, but were absent in ovaries from uninfected D. citri females. The size of the rod shaped structures varied from 0.39 to 0.67 µm in length and 0.19 to 0.39 µm in width. The spherical structures measured from 0.61 to 0.80 µm in diameter. This investigation provides convincing evidence that a plant pathogenic bacterium is sexually transmitted from male to female insects during courtship and established evidence that bacteria persist in reproductive organs. Moreover, these findings provide an alternative sexually horizontal mechanism for the spread of Las within populations of D. citri, even in the absence of infected host trees

Fibronectin Matrix Assembly Suppresses Dispersal of Glioblastoma Cells

Glioblastoma (GBM), the most aggressive and most common form of primary brain tumor, has a median survival of 12–15 months. Surgical excision, radiation and chemotherapy are rarely curative since tumor cells broadly disperse within the brain. Preventing dispersal could be of therapeutic benefit. Previous studies have reported that increased cell-cell cohesion can markedly reduce invasion by discouraging cell detachment from the tumor mass. We have previously reported that α5β1 integrin-fibronectin interaction is a powerful mediator of indirect cell-cell cohesion and that the process of fibronectin matrix assembly (FNMA) is crucial to establishing strong bonds between cells in 3D tumor-like spheroids. Here, we explore a potential role for FNMA in preventing dispersal of GBM cells from a tumor-like mass. Using a series of GBM-derived cell lines we developed an in vitro assay to measure the dispersal velocity of aggregates on a solid substrate. Despite their similar pathologic grade, aggregates from these lines spread at markedly different rates. Spreading velocity is inversely proportional to capacity for FNMA and restoring FNMA in GBM cells markedly reduces spreading velocity by keeping cells more connected. Blocking FNMA using the 70 KDa fibronectin fragment in FNMA-restored cells rescues spreading velocity, establishing a functional role for FNMA in mediating dispersal. Collectively, the data support a functional causation between restoration of FNMA and decreased dispersal velocity. This is a first demonstration that FNMA can play a suppressive role in GBM dispersal

Conservation of Gene Order and Content in the Circular Chromosomes of ‘Candidatus Liberibacter asiaticus’ and Other Rhizobiales

‘Ca. Liberibacter asiaticus,’ an insect-vectored, obligate intracellular bacterium associated with citrus-greening disease, also called “HLB," is a member of the Rhizobiales along with nitrogen-fixing microsymbionts Sinorhizobium meliloti and Bradyrhizobium japonicum, plant pathogen Agrobacterium tumefaciens and facultative intracellular mammalian pathogen Bartonella henselae. Comparative analyses of their circular chromosomes identified 514 orthologous genes shared among all five species. Shared among all five species are 50 identical blocks of microsyntenous orthologous genes (MOGs), containing a total of 283 genes. While retaining highly conserved genomic blocks of microsynteny, divergent evolution, horizontal gene transfer and niche specialization have disrupted macrosynteny among the five circular chromosomes compared. Highly conserved microsyntenous gene clusters help define the Rhizobiales, an order previously defined by 16S RNA gene similarity and herein represented by the three families: Bartonellaceae, Bradyrhizobiaceae and Rhizobiaceae. Genes without orthologs in the other four species help define individual species. The circular chromosomes of each of the five Rhizobiales species examined had genes lacking orthologs in the other four species. For example, 63 proteins are encoded by genes of ‘Ca. Liberibacter asiaticus’ not shared with other members of the Rhizobiales. Of these 63 proteins, 17 have predicted functions related to DNA replication or RNA transcription, and some of these may have roles related to low genomic GC content. An additional 17 proteins have predicted functions relevant to cellular processes, particularly modifications of the cell surface. Seventeen unshared proteins have specific metabolic functions including a pathway to synthesize cholesterol encoded by a seven-gene operon. The remaining 12 proteins encoded by ‘Ca. Liberibacter asiaticus’ genes not shared with other Rhizobiales are of bacteriophage origin. ‘Ca. Liberibacter asiaticus’ shares 11 genes with only Sinorhizobium meliloti and 12 genes are shared with only Bartonella henselae

Transcriptome Profiling of Citrus Fruit Response to Huanglongbing Disease

Huanglongbing (HLB) or “citrus greening” is the most destructive citrus disease worldwide. In this work, we studied host responses of citrus to infection with Candidatus Liberibacter asiaticus (CaLas) using next-generation sequencing technologies. A deep mRNA profile was obtained from peel of healthy and HLB-affected fruit. It was followed by pathway and protein-protein network analysis and quantitative real time PCR analysis of highly regulated genes. We identified differentially regulated pathways and constructed networks that provide a deep insight into the metabolism of affected fruit. Data mining revealed that HLB enhanced transcription of genes involved in the light reactions of photosynthesis and in ATP synthesis. Activation of protein degradation and misfolding processes were observed at the transcriptomic level. Transcripts for heat shock proteins were down-regulated at all disease stages, resulting in further protein misfolding. HLB strongly affected pathways involved in source-sink communication, including sucrose and starch metabolism and hormone synthesis and signaling. Transcription of several genes involved in the synthesis and signal transduction of cytokinins and gibberellins was repressed while that of genes involved in ethylene pathways was induced. CaLas infection triggered a response via both the salicylic acid and jasmonic acid pathways and increased the transcript abundance of several members of the WRKY family of transcription factors. Findings focused on the fruit provide valuable insight to understanding the mechanisms of the HLB-induced fruit disorder and eventually developing methods based on small molecule applications to mitigate its devastating effects on fruit production

Fibronectin Unfolding Revisited: Modeling Cell Traction-Mediated Unfolding of the Tenth Type-III Repeat

Fibronectin polymerization is essential for the development and repair of the extracellular matrix. Consequently, deciphering the mechanism of fibronectin fibril formation is of immense interest. Fibronectin fibrillogenesis is driven by cell-traction forces that mechanically unfold particular modules within fibronectin. Previously, mechanical unfolding of fibronectin has been modeled by applying tensile forces at the N- and C-termini of fibronectin domains; however, physiological loading is likely focused on the solvent-exposed RGD loop in the 10th type-III repeat of fibronectin (10FNIII), which mediates binding to cell-surface integrin receptors. In this work we used steered molecular dynamics to study the mechanical unfolding of 10FNIII under tensile force applied at this RGD site. We demonstrate that mechanically unfolding 10FNIII by pulling at the RGD site requires less work than unfolding by pulling at the N- and C- termini. Moreover, pulling at the N- and C-termini leads to 10FNIII unfolding along several pathways while pulling on the RGD site leads to a single exclusive unfolding pathway that includes a partially unfolded intermediate with exposed hydrophobic N-terminal β-strands – residues that may facilitate fibronectin self-association. Additional mechanical unfolding triggers an essential arginine residue, which is required for high affinity binding to integrins, to move to a position far from the integrin binding site. This cell traction-induced conformational change may promote cell detachment after important partially unfolded kinetic intermediates are formed. These data suggest a novel mechanism that explains how cell-mediated forces promote fibronectin fibrillogenesis and how cell surface integrins detach from newly forming fibrils. This process enables cells to bind and unfold additional fibronectin modules – a method that propagates matrix assembly

Comparison of the ‘Ca. Liberibacter asiaticus’ Genome Adapted for an Intracellular Lifestyle with Other Members of the Rhizobiales

An intracellular plant pathogen ‘Candidatus Liberibacter asiaticus,’ a member of the Rhizobiales, is related to Sinorhizobium meliloti, Bradyrhizobium japonicum, nitrogen fixing endosymbionts, Agrobacterium tumefaciens, a plant pathogen, and Bartonella henselae, an intracellular mammalian pathogen. Whole chromosome comparisons identified at least 50 clusters of conserved orthologous genes found on the chromosomes of all five metabolically diverse species. The intracellular pathogens ‘Ca. Liberibacter asiaticus’ and Bartonella henselae have genomes drastically reduced in gene content and size as well as a relatively low content of guanine and cytosine. Codon and amino acid preferences that emphasize low guanosine and cytosine usage are globally employed in these genomes, including within regions of microsynteny and within signature sequences of orthologous proteins. The length of orthologous proteins is generally conserved, but not their isoelectric points, consistent with extensive amino acid substitutions to accommodate selection for low GC content. The ‘Ca. Liberibacter asiaticus’ genome apparently has all of the genes required for DNA replication present in Sinorhizobium meliloti except it has only two, rather than three RNaseH genes. The gene set required for DNA repair has only one rather than ten DNA ligases found in Sinorhizobium meliloti, and the DNA PolI of ‘Ca. Liberibacter asiaticus’ lacks domains needed for excision repair. Thus the ability of ‘Ca. Liberibacter asiaticus’ to repair mutations in its genome may be impaired. Both ‘Ca. Liberibacter asiaticus and Bartonella henselae lack enzymes needed for the metabolism of purines and pyrimidines, which must therefore be obtained from the host. The ‘Ca. Liberibacter asiaticus’ genome also has a greatly reduced set of sigma factors used to control transcription, and lacks sigma factors 24, 28 and 38. The ‘Ca. Liberibacter asiaticus’ genome has all of the hallmarks of a reduced genome of a pathogen adapted to an intracellular lifestyle

Plasma and cellular fibronectin: distinct and independent functions during tissue repair

Fibronectin (FN) is a ubiquitous extracellular matrix (ECM) glycoprotein that plays vital roles during tissue repair. The plasma form of FN circulates in the blood, and upon tissue injury, is incorporated into fibrin clots to exert effects on platelet function and to mediate hemostasis. Cellular FN is then synthesized and assembled by cells as they migrate into the clot to reconstitute damaged tissue. The assembly of FN into a complex three-dimensional matrix during physiological repair plays a key role not only as a structural scaffold, but also as a regulator of cell function during this stage of tissue repair. FN fibrillogenesis is a complex, stepwise process that is strictly regulated by a multitude of factors. During fibrosis, there is excessive deposition of ECM, of which FN is one of the major components. Aberrant FN-matrix assembly is a major contributing factor to the switch from normal tissue repair to misregulated fibrosis. Understanding the mechanisms involved in FN assembly and how these interplay with cellular, fibrotic and immune responses may reveal targets for the future development of therapies to regulate aberrant tissue-repair processes