Type IVB pili are required for invasion but not for adhesion of Salmonella enterica serovar Typhi into BHK epithelial cells in a cystic fibrosis transmembrane conductance regulator-independent manner

The cystic fibrosis transmembrane conductance regulator (CFTR) has been proposed as an epithelial cell receptor for the entry of Salmonella Typhi but not Salmonella Typhimurium. The bacterial ligand recognized by CM is thought to reside either in the S. Typhi lipopolysaccharide core region or in the type IV pili. Here, we assessed the ability of virulent strains of S. Typhi and S. Typhimurium to adhere to and invade BHK epithelial cells expressing either the wild-type CFTR protein or the Delta F508 CFTR mutant. Both S. Typhi and S. Typhimurium invaded the epithelial cells in a CFTR-independent fashion. Furthermore and also in a CFTR-independent manner, a S. Typhi pilS mutant adhered normally to BHK cells but displayed a 50% reduction in invasion as compared to wild-type bacteria. Immunofluorescence microscopy revealed that bacteria and CFTR do not colocalize at the epithelial cell surface. Together, our results strongly argue against the established dogma that CFTR is a receptor for entry of Salmonella to epithelial cells. (C) 2011 Elsevier Ltd. All rights reserved.

An integrated transcriptomics and proteomics analysis of the secretome of the helminth pathogen Fasciola hepatica: Proteins associated with invasion and infection of the mammalian host

To infect their mammalian hosts, Fasciola hepatica larvae must penetrate and traverse the intestinal wall of the duodenum, move through the peritoneum, and penetrate the liver. After migrating through and feeding on the liver, causing extensive tissue damage, the parasites move to their final niche in the bile ducts where they mature and produce eggs. Here we integrated a transcriptomics and proteomics approach to profile Fasciola secretory proteins that are involved in host-pathogen interactions and to correlate changes in their expression with the migration of the parasite. Prediction of F. hepatica secretory proteins from 14,031 expressed sequence tags (ESTs) available from the Wellcome Trust Sanger Centre using the semiautomated EST2Secretome pipeline showed that the major components of adult parasite secretions are proteolytic enzymes including cathepsin L, cathepsin B, and asparaginyl endopeptidase cysteine proteases as well as novel trypsin-like serine proteases and carboxypeptidases. Proteomics analysis of proteins secreted by infective larvae, immature flukes, and adult F. hepatica showed that these proteases are developmentally regulated and correlate with the passage of the parasite through host tissues and its encounters with different host macromolecules. Proteases such as FhCL3 and cathepsin B have specific functions in larvae activation and intestinal wall penetration, whereas FhCL1, FhCL2, and FhCL5 are required for liver penetration and tissue and blood feeding. Besides proteases, the parasites secrete an array of antioxidants that are also highly regulated according to their migration through host tissues. However, whereas the proteases of F. hepatica are secreted into the parasite gut via a classical endoplasmic reticulum/Golgi pathway, we speculate that the antioxidants, which all lack a signal sequence, are released via a non-classical trans-tegumental pathway.

Host cell kinases, α5 and β1 integrins, and Rac1 signalling on the microtubule cytoskeleton are important for non-typable Haemophilus influenzae invasion of respiratory epithelial cells

Non-typable Haemophilus influenzae (NTHi) is a common commensal of the human nasopharynx, but causes opportunistic infection when the respiratory tract is compromised by infection or disease. The ability of NTHi to invade epithelial cells has been described, but the underlying molecular mechanisms are poorly characterized. We previously determined that NTHi promotes phosphorylation of the serine-threonine kinase Akt in A549 human lung epithelial cells, and that Akt phosphorylation and NTHi cell invasion are prevented by inhibition of phosphoinositide 3-kinase (PI3K). Because PI3K-Akt signalling is associated with several host cell networks, the purpose of the current study was to identify eukaryotic molecules important for NTHi epithelial invasion. We found that inhibition of Akt activity reduced NTHi internalization; differently, bacterial entry was increased by phospholipase C?1 inhibition but was not affected by protein kinase inhibition. We also found that a5 and ß1 integrins, and the tyrosine kinases focal adhesion kinase and Src, are important for NTHi A549 cell invasion. NTHi internalization was shown to be favoured by activation of Rac1 guanosine triphosphatase (GTPase), together with the guanine nucleotide exchange factor Vav2 and the effector Pak1. Also, Pak1 might be associated with inactivation of the microtubule destabilizing agent Op18/stathmin, to facilitate microtubule polymerization and NTHi entry. Conversely, inhibition of RhoA GTPase and its effector ROCK increased the number of internalized bacteria. Src and Rac1 were found to be important for NTHi-triggered Akt phosphorylation. An increase in host cyclic AMP reduced bacterial entry, which was linked to protein kinase A. These findings suggest that NTHi finely manipulates host signalling molecules to invade respiratory epithelial cells.

Immunophenotypic shift in a case of mycosis fungoides with vitreous invasion

The case of an 82-year-old man who developed intraocular extension from mycosis fungoides, a cutaneous T-cell lymphoma, is presented. The patient died soon after intra-ocular involvement occurred. Immunohistochemistry of a skin biopsy, taken early in the course of the disease, disclosed a predominance of T cells with a helper/inducer phenotype (CD4). However, an intraocular infiltrate obtained 7 years later contained mostly T cells with a suppressor/cytotoxic phenotype (CD8). The occurrence of ocular invasion, the change in immunophenotype, and the predominant proliferation of CD8 lymphocytes may have been related to the poor outcome in this patient.

Gelsolin induces colorectal tumor cell invasion via modulation of the urokinase-type plasminogen activator cascade

Gelsolin is a cytoskeletal protein which participates in actin filament dynamics and promotes cell motility and plasticity. Although initially regarded as a tumor suppressor, gelsolin expression in certain tumors correlates with poor prognosis and therapy-resistance. In vitro, gelsolin has anti-apoptotic and pro-migratory functions and is critical for invasion of some types of tumor cells. We found that gelsolin was highly expressed at tumor borders infiltrating into adjacent liver tissues, as examined by immunohistochemistry. Although gelsolin contributes to lamellipodia formation in migrating cells, the mechanisms by which it induces tumor invasion are unclear. Gelsolin's influence on the invasive activity of colorectal cancer cells was investigated using overexpression and small interfering RNA knockdown. We show that gelsolin is required for invasion of colorectal cancer cells through matrigel. Microarray analysis and quantitative PCR indicate that gelsolin overexpression induces the upregulation of invasion-promoting genes in colorectal cancer cells, including the matrix-degrading urokinase-type plasminogen activator (uPA). Conversely, gelsolin knockdown reduces uPA levels, as well as uPA secretion. The enhanced invasiveness of gelsolin-overexpressing cells was attenuated by treatment with function-blocking antibodies to either uPA or its receptor uPAR, indicating that uPA/uPAR activity is crucial for gelsolin-dependent invasion. In summary, our data reveals novel functions of gelsolin in colorectal tumor cell invasion through its modulation of the uPA/uPAR cascade, with potentially important roles in colorectal tumor dissemination to metastatic sites.

Combined genomic and phenotype screening reveals secretory factor SPINK1 as an invasion and survival factor associated with patient prognosis in breast cancer

Secretory factors that drive cancer progression are attractive immunotherapeutic targets. We used a whole-genome data-mining approach on multiple cohorts of breast tumours annotated for clinical outcomes to discover such factors. We identified Serine protease inhibitor Kazal-type 1 (SPINK1) to be associated with poor survival in estrogen receptor-positive (ER+) cases. Immunohistochemistry showed that SPINK1 was absent in normal breast, present in early and advanced tumours, and its expression correlated with poor survival in ER+ tumours. In ER- cases, the prognostic effect did not reach statistical significance. Forced expression and/or exposure to recombinant SPINK1 induced invasiveness without affecting cell proliferation. However, down-regulation of SPINK1 resulted in cell death. Further, SPINK1 overexpressing cells were resistant to drug-induced apoptosis due to reduced caspase-3 levels and high expression of Bcl2 and phospho-Bcl2 proteins. Intriguingly, these anti-apoptotic effects of SPINK1 were abrogated by mutations of its protease inhibition domain. Thus, SPINK1 affects multiple aggressive properties in breast cancer: survival, invasiveness and chemoresistance. Because SPINK1 effects are abrogated by neutralizing antibodies, we suggest that SPINK1 is a viable potential therapeutic target in breast cancer.

AKT in Stromal Fibroblasts Controls Invasion of Epithelial Cells

The tumour microenvironment has an important role in cancer progression and recent reports have proposed that stromal AKT is activated and regulates tumourigenesis and invasion. We have shown, by immuno-fluorescent analysis of oro-pharyngeal cancer biopsies, an increase in AKT activity in tumour associated stromal fibroblasts compared to normal stromal fibroblasts. Using organotypic raft co-cultures, we show that activation of stromal AKT can induce the invasion of keratinocytes expressing the HPV type 16 E6 and E7 proteins, in a Keratinocyte Growth Factor (KGF) dependent manner. By depleting stromal fibroblasts of each of the three AKT isoforms independently, or through using isoform specific inhibitors, we determined that stromal AKT2 is an essential regulator of invasion and show in oro-pharyngeal cancers that AKT2 specific phosphorylation events are also identified in stromal fibroblasts. Depletion of stromal AKT2 inhibits epithelial invasion through activating a protective pathway counteracting KGF mediated invasions. AKT2 depletion in fibroblasts stimulates the cleavage and release of IL1B from stromal fibroblasts resulting in down-regulation of the KGF receptor (fibroblast growth factor receptor 2B (FGFR2B)) expression in the epithelium. We also show that high IL1B is associated with increased overall survival in a cohort of patients with oro-pharyngeal cancers. Our findings demonstrate the importance of stromal derived growth factors and cytokines in regulating the process of tumour cell invasion.

Advancing impact prediction and hypothesis testing in invasion ecology using a comparative functional response approach

Invasion ecology urgently requires predictive methodologies that can forecast the ecological impacts of existing, emerging and potential invasive species. We argue that many ecologically damaging invaders are characterised by their more efficient use of resources. Consequently, comparison of the classical ‘functional response’ (relationship between resource use and availability) between invasive and trophically analogous native species may allow prediction of invader ecological impact. We review the utility of species trait comparisons and the history and context of the use of functional responses in invasion ecology, then present our framework for the use of comparative functional responses. We show that functional response analyses, by describing the resource use of species over a range of resource availabilities, avoids many pitfalls of ‘snapshot’ assessments of resource use. Our framework demonstrates how comparisons of invader and native functional responses, within and between Type II and III functional responses, allow testing of the likely population-level outcomes of invasions for affected species. Furthermore, we describe how recent studies support the predictive capacity of this method; for example, the invasive ‘bloody red shrimp’ Hemimysis anomala shows higher Type II functional responses than native mysids and this corroborates, and could have predicted, actual invader impacts in the field. The comparative functional response method can also be used to examine differences in the impact of two or more invaders, two or more populations of the same invader, and the abiotic (e.g. temperature) and biotic (e.g. parasitism) context-dependencies of invader impacts. Our framework may also address the previous lack of rigour in testing major hypotheses in invasion ecology, such as the ‘enemy release’ and ‘biotic resistance’ hypotheses, as our approach explicitly considers demographic consequences for impacted resources, such as native and invasive prey species. We also identify potential challenges in the application of comparative functional responses in invasion ecology. These include incorporation of numerical responses, multiple predator effects and trait-mediated indirect interactions, replacement versus non-replacement study designs and the inclusion of functional responses in risk assessment frameworks. In future, the generation of sufficient case studies for a meta-analysis could test the overall hypothesis that comparative functional responses can indeed predict invasive species impacts.

AXL Is a Key Regulator of Inherent and Chemotherapy-Induced Invasion and Predicts a Poor Clinical Outcome in Early-Stage Colon Cancer

Purpose: Despite the use of 5-fluorouracil (5-FU)–based adjuvant treatments, a large proportion of patients with high-risk stage II/III colorectal cancer will relapse. Thus, novel therapeutic strategies are needed for early-stage colorectal cancer. Residual micrometastatic disease from the primary tumor is a major cause of patient relapse.

Experimental Design: To model colorectal cancer tumor cell invasion/metastasis, we have generated invasive (KRASMT/KRASWT/+chr3/p53-null) colorectal cancer cell subpopulations. Receptor tyrosine kinase (RTK) screens were used to identify novel proteins that underpin the migratory/invasive phenotype. Migration/invasion was assessed using the XCELLigence system. Tumors from patients with early-stage colorectal cancer (N = 336) were examined for AXL expression.

Results: Invasive colorectal cancer cell subpopulations showed a transition from an epithelial-to-mesenchymal like phenotype with significant increases in migration, invasion, colony-forming ability, and an attenuation of EGF receptor (EGFR)/HER2 autocrine signaling. RTK arrays showed significant increases in AXL levels in all invasive sublines. Importantly, 5-FU treatment resulted in significantly increased migration and invasion, and targeting AXL using pharmacologic inhibition or RNA interference (RNAi) approaches suppressed basal and 5-FU–induced migration and invasion. Significantly, high AXL mRNA and protein expression were found to be associated with poor overall survival in early-stage colorectal cancer tissues.

Conclusions: We have identified AXL as a poor prognostic marker and important mediator of cell migration/invasiveness in colorectal cancer. These findings provide support for the further investigation of AXL as a novel prognostic biomarker and therapeutic target in colorectal cancer, in particular in the adjuvant disease in which EGFR/VEGF–targeted therapies have failed.

Polemic and apocalyptic in the Cromwellian invasion of Scotland

This article describes the culture of print and polemic associated with the Cromwellian invasion of Scotland and the resistance of the Church of Scotland. It argues that print solidified Parliamentary ideas during the invasion but that it did not continue to do so after English dominance had been established. These texts illustrate the divisions between the mentalité of the Cromwellian rank-and-file and that of the rather small and probably unrepresentative body of opinion formers who sought to control and represent it.

Stressor intensity determines antagonistic interactions between species invasion and multiple stressor effects on ecosystem functioning

Biological invasions, nutrient enrichment and ocean warming are known to threaten biodiversity and ecosystem functioning. The independent effects of these ecological stressors are well studied, however, we lack understanding of their cumulative effects, which may be additive, antagonistic or synergistic. For example, the impacts of biological invasions are often determined by environmental context, which suggests that the effects of invasive species may vary with other stressors such as pollution or climate change. This study examined the effects of an invasive seaweed (Sargassum muticum) on the structure and functioning of a benthic marine assemblage and tested explicitly whether these effects varied with nutrient enrichment and ocean warming. Overall, the presence of Sargassum muticum increased assemblage productivity rates and warming altered algal assemblage structure, which was characterised by a decrease in kelp and an increase in ephemeral green algae. The effects of Sargassum muticum on total algal biomass accumulation, however, varied with nutrient enrichment and warming producing antagonistic cumulative effects on total algal biomass accumulation. These findings show that the nature of stressor interactions may vary with stressor intensity and among response variables, which leads to less predictable consequences for the structure and functioning of communities.

Combining global climate and regional landscape models to improve prediction of invasion risk

Aim
It is widely acknowledged that species distributions result from a variety of biotic and abiotic factors operating at different spatial scales. Here, we aimed to (1) determine the extent to which global climate niche models (CNMs) can be improved by the addition of fine-scale regional data; (2) examine climatic and environmental factors influencing the range of 15 invasive aquatic plant species; and (3) provide a case study for the use of such models in invasion management on an island.

Location
Global, with a case study of species invasions in Ireland.

Methods
Climate niche models of global extent (including climate only) and regional environmental niche models (with additional factors such as human influence, land use and soil characteristics) were generated using maxent for 15 invasive aquatic plants. The performance of these models within the invaded range of the study species in Ireland was assessed, and potential hotspots of invasion suitability were determined. Models were projected forward up to 2080 based on two climate scenarios.

Results
While climate variables are important in defining the global range of species, factors related to land use and nutrient level were of greater importance in regional projections. Global climatic models were significantly improved at the island scale by the addition of fine-scale environmental variables (area under the curve values increased by 0.18 and true skill statistic values by 0.36), and projected ranges decreased from an average of 86% to 36% of the island.

Main conclusions
Refining CNMs with regional data on land use, human influence and landscape may have a substantial impact on predictive capacity, providing greater value for prioritization of conservation management at subregional or local scales.

Invading and expanding: range dynamics and ecological consequences of the greater white-toothed shrew (Crocidura russula) invasion in Ireland

Establishing how invasive species impact upon pre-existing species is a fundamental question in ecology and conservation biology. The greater white-toothed shrew (Crocidura russula) is an invasive species in Ireland that was first recorded in 2007 and which, according to initial data, may be limiting the abundance/distribution of the pygmy shrew (Sorex minutus), previously Ireland’s only shrew species. Because of these concerns, we undertook an intensive live-trapping survey (and used other data from live-trapping, sightings and bird of prey pellets/nest inspections collected between 2006 and 2013) to model the distribution and expansion of C. russula in Ireland and its impacts on Ireland’s small mammal community. The main distribution range of C. russula was found to be approximately 7,600 km2 in 2013, with established outlier populations suggesting that the species is dispersing with human assistance within the island. The species is expanding rapidly for a small mammal, with a radial expansion rate of 5.5 km/yr overall (2008–2013), and independent estimates from live-trapping in 2012–2013 showing rates of 2.4–14.1 km/yr, 0.5–7.1 km/yr and 0–5.6 km/yr depending on the landscape features present. S. minutus is negatively associated with C. russula. S. minutus is completely absent at sites where C. russula is established and is only present at sites at the edge of and beyond the invasion range of C. russula. The speed of this invasion and the homogenous nature of the Irish landscape may mean that S. minutus has not had sufficient time to adapt to the sudden appearance of C. russula. This may mean the continued decline/disappearance of S. minutus as C. russula spreads throughout the island.