Construction of Minitransposons for Constitutive and Inducible Expression of Pertussis Toxin in Bvg-Negative Bordetella-Bronchiseptica

Appropriately detoxified pertussis toxin (PT) of Bordetella pertussis is considered to be an essential component of new-generation whooping cough vaccines, but the development of a procedure to obtain high levels of purified toxin has been and continues to be a major difficulty. To produce a system enabling the biological separation of PT from other virulence determinants of B. pertussis and the attainment of high yields of the toxin, minitransposons containing the PT operon were constructed and stably integrated into the chromosome of Bordetella virulence regulatory gene (bvg)-negative Bordetella bronchiseptica ATCC 10580. Since the minitransposons introduced into Bordetella spp. lack the cognate transposase function, they are unable to undergo further transposition events or mediate gene deletions and rearrangements that lead to strain instability. The TnPtacPT minitransposon contains the PT operon under the control of the tac promoter and directs IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible expression of PT in B. bronchiseptica ATCC 10580. The level of IPTG-induced PT expression was, however, lower than that found for the wild-type B. pertussis Tohama I strain. The TnfusPT minitransposon contains a promoterless PT operon which is only expressed after insertion of the transposon downstream of an appropriately oriented indigenous promoter. After "promoter probing" of B. bronchiseptica with the transposon, clones were screened for PT production by immunoblotting with specific monoclonal antibodies. One clone, designated B. bronchiseptica 10580:: TnfusPT1, expresses significantly higher levels of PT than does B. pertussis Tohama I. The recombinant toxin produced was biologically active in the Chinese hamster ovary cell-clustering assay. High-level expression of PT from a B. bronchiseptica host promoter should provide better yields of the toxin from bacteria not producing other bvg-regulated pathogenesis factors that may play a role in the undesired side effects of current pertussis vaccine preparations

Identifications of novel mechanisms in breast cancer cells involving duct-like multicellular spheroid formation after exposure to the Random Positioning Machine

Many cell types form three-dimensional aggregates (MCS; multicellular spheroids), when they are cultured under microgravity. MCS often resemble the organ, from which the cells have been derived. In this study we investigated human MCF-7 breast cancer cells after a 2 h-, 4 h-, 16 h-, 24 h-and 5d-exposure to a Random Positioning Machine (RPM) simulating microgravity. At 24 h few small compact MCS were detectable, whereas after 5d many MCS were floating in the supernatant above the cells, remaining adherently (AD). The MCS resembled the ducts formed in vivo by human epithelial breast cells. In order to clarify the underlying mechanisms, we harvested MCS and AD cells separately from each RPM-culture and measured the expression of 29 selected genes with a known involvement in MCS formation. qPCR analyses indicated that cytoskeletal genes were unaltered in short-term samples. IL8, VEGFA, and FLT1 were upregulated in 2 h/4 h AD-cultures. The ACTB, TUBB, EZR, RDX, FN1, VEGFA, FLK1 Casp9, Casp3, PRKCA mRNAs were downregulated in 5d-MCS-samples. ESR1 was upregulated in AD, and PGR1 in both phenotypes after 5d. A pathway analysis revealed that the corresponding gene products are involved in organization and regulation of the cell shape, in cell tip formation and membrane to membrane docking

The Arp2/3 complex is essential for the actin-based motility of Listeria monocytogenes

AbstractActin polymerisation is thought to drive the movement of eukaryotic cells and some intracellular pathogens such as Listeria monocytogenes. The Listeria surface protein ActA synergises with recruited host proteins to induce actin polymerisation, propelling the bacterium through the host cytoplasm [1]. The Arp2/3 complex is one recruited host factor [2,3]; it is also believed to regulate actin dynamics in lamellipodia [4,5]. The Arp2/3 complex promotes actin filament nucleation in vitro, which is further enhanced by ActA [6,7]. The Arp2/3 complex also interacts with members of the Wiskott–Aldrich syndrome protein (WASP) [8] family – Scar1 [9,10] and WASP itself [11]. We interfered with the targeting of the Arp2/3 complex to Listeria by using carboxy-terminal fragments of Scar1 that bind the Arp2/3 complex [11]. These fragments completely blocked actin tail formation and motility of Listeria, both in mouse brain extract and in Ptk2 cells overexpressing Scar1 constructs. In both systems, Listeria could initiate actin cloud formation, but tail formation was blocked. Full motility in vitro was restored by adding purified Arp2/3 complex. We conclude that the Arp2/3 complex is a host-cell factor essential for the actin-based motility of L. monocytogenes, suggesting that it plays a pivotal role in regulating the actin cytoskeleton

Morphological and Molecular Changes in Juvenile Normal Human Fibroblasts Exposed to Simulated Microgravity

The literature suggests morphological alterations and molecular biological changes within the cellular milieu of human cells, exposed to microgravity (mu g), as many cell types assemble to multicellular spheroids (MCS). In this study we investigated juvenile normal human dermal fibroblasts (NHDF) grown in simulated mu g (s-mu g) on a random positioning machine (RPM), aiming to study changes in cell morphology, cytoskeleton, extracellular matrix (ECM), focal adhesion and growth factors. On the RPM, NHDF formed an adherent monolayer and compact MCS. For the two cell populations we found a differential regulation of fibronectin, laminin, collagen-IV, aggrecan, osteopontin, TIMP-1, integrin-beta(1), caveolin-1, E-cadherin, talin-1, vimentin, alpha-SM actin, TGF-beta(1), IL-8, MCP-1, MMP-1, and MMP-14 both on the transcriptional and/or translational level. Immunofluorescence staining revealed only slight structural changes in cytoskeletal components. Flow cytometry showed various membrane-bound proteins with considerable variations. In silico analyses of the regulated proteins revealed an interaction network, contributing to MCS growth via signals mediated by integrin-beta(1), E-cadherin, caveolin-1 and talin-1. In conclusion, s-mu g-conditions induced changes in the cytoskeleton, ECM, focal adhesion and growth behavior of NHDF and we identified for the first time factors involved in fibroblast 3D-assembly. This new knowledge might be of importance in tissue engineering, wound healing and cancer metastasis

Pathways Regulating Spheroid Formation of Human Follicular Thyroid Cancer Cells under Simulated Microgravity Conditions: A Genetic Approach

Microgravity induces three-dimensional (3D) growth in numerous cell types. Despite substantial efforts to clarify the underlying mechanisms for spheroid formation, the precise molecular pathways are still not known. The principal aim of this paper is to compare static 1g-control cells with spheroid forming (MCS) and spheroid non-forming (AD) thyroid cancer cells cultured in the same flask under simulated microgravity conditions. We investigated the morphology and gene expression patterns in human follicular thyroid cancer cells (UCLA RO82-W-1 cell line) after a 24 h-exposure on the Random Positioning Machine (RPM) and focused on 3D growth signaling processes. After 24 h, spheroid formation was observed in RPM-cultures together with alterations in the F-actin cytoskeleton. qPCR indicated more changes in gene expression in MCS than in AD cells. Of the 24 genes analyzed VEGFA, VEGFD, MSN, and MMP3 were upregulated in MCS compared to 1g-controls, whereas ACTB, ACTA2, KRT8, TUBB, EZR, RDX, PRKCA, CAV1, MMP9, PAI1, CTGF, MCP1 were downregulated. A pathway analysis revealed that the upregulated genes code for proteins, which promote 3D growth (angiogenesis) and prevent excessive accumulation of extracellular proteins, while genes coding for structural proteins are downregulated. Pathways regulating the strength/rigidity of cytoskeletal proteins, the amount of extracellular proteins, and 3D growth may be involved in MCS formation

Proteomic analysis of the U1 snRNP of Schizosaccharomyces pombe reveals three essential organism-specific proteins

Characterization of spliceosomal complexes in the fission yeast Schizosaccharomyces pombe revealed particles sedimenting in the range of 30–60S, exclusively containing U1 snRNA. Here, we report the tandem affinity purification (TAP) of U1-specific protein complexes. The components of the complexes were identified using (LC-MS/MS) mass spectrometry. The fission yeast U1 snRNP contains 16 proteins, including the 7 Sm snRNP core proteins. In both fission and budding yeast, the U1 snRNP contains 9 and 10 U1 specific proteins, respectively, whereas the U1 particle found in mammalian cells contains only 3. Among the U1-specific proteins in S. pombe, three are homolog to the mammalian and six to the budding yeast Saccharomyces cerevisiae U1-specific proteins, whereas three, called U1H, U1J and U1L, are proteins specific to S. pombe. Furthermore, we demonstrate that the homolog of U1-70K and the three proteins specific to S. pombe are essential for growth. We will discuss the differences between the U1 snRNPs with respect to the organism-specific proteins found in the two yeasts and the resulting effect it has on pre-mRNA splicing

Nε−Lysine Acetylation of a Bacterial Transcription Factor Inhibits Its DNA-Binding Activity

Evidence suggesting that eukaryotes and archaea use reversible Nε-lysine (Nε-Lys) acetylation to modulate gene expression has been reported, but evidence for bacterial use of Nε-Lys acetylation for this purpose is lacking. Here, we report data in support of the notion that bacteria can control gene expression by modulating the acetylation state of transcription factors (TFs). We screened the E. coli proteome for substrates of the bacterial Gcn5-like protein acetyltransferase (Pat). Pat acetylated four TFs, including the RcsB global regulatory protein, which controls cell division, and capsule and flagellum biosynthesis in many bacteria. Pat acetylated residue Lys180 of RcsB, and the NAD+-dependent Sir2 (sirtuin)-like protein deacetylase (CobB) deacetylated acetylated RcsB (RcsBAc), demonstrating that Nε-Lys acetylation of RcsB is reversible. Analysis of RcsBAc and variant RcsB proteins carrying substitutions at Lys180 provided biochemical and physiological evidence implicating Lys180 as a critical residue for RcsB DNA-binding activity. These findings further the likelihood that reversible Nε-Lys acetylation of transcription factors is a mode of regulation of gene expression used by all cells