Are CDI Systems Multicolored, Facultative, Helping Greenbeards?

Competitive and cooperative interactions between organisms, including bacteria, can significantly impact the composition of a community and the fitness of its members, as well as the fitness of their hosts when communities are living on or within other organisms. Understanding the underlying mechanisms is critical to the development of strategies to control microbiological communities that impact animal and plant health and also for understanding the evolution of social behaviors, which has been challenging for evolutionary biologists. Contact-dependent growth inhibition (CDI) is a phenomenon defined by the delivery of a protein toxin to the cytoplasm of neighboring bacteria upon cell-cell contact, resulting in growth inhibition or death unless a specific immunity protein is present. CDI was first described based on observations of interbacterial killing and has been assumed to function primarily as a means of eliminating competitor cells. However, recent molecular evidence indicates that multiple levels of specificity restrict CDI toxin delivery and activity to the same bacterial strain, and that CDI system proteins can mediate cooperative behaviors among 'self' cells, a phenomenon called contact-dependent signaling (CDS). Here we review these recent findings and discuss potential biological and evolutionary implications of CDI system-mediated interbacterial competition and cooperation.

Cathelicidin augments epithelial receptivity and pathogenesis in experimental Escherichia coli cystitis.

BACKGROUND: Cathelicidin is a proposed defender against infection of the urinary tract via its antimicrobial properties, but its activity has not been delineated in a dedicated cystitis model. METHODS: Female C57Bl/6 mice, wild type or deficient in cathelin-related antimicrobial peptide (CRAMP; an ortholog of the sole human cathelicidin, LL-37), were infected transurethrally with the cystitis-derived uropathogenic Escherichia coli (UPEC) strain UTI89. Infection course was evaluated by bladder titers, intracellular bacterial community quantification, and histological analysis. Immune responses and resolution were characterized through cytokine profiling, microscopy, and quantitation of epithelial recovery from exfoliation. RESULTS: CRAMP-deficient mice exhibited significantly lower bladder bacterial loads and fewer intracellular bacterial communities during acute cystitis. Although differences in bacterial titers were evident as early as 1 hour after infection, CRAMP-deficient mice showed no baseline alterations in immune activation, uroepithelial structure, apical expression of uroplakins (which serve as bacterial receptors), or intracellular bacterial growth rate. CRAMP-deficient hosts demonstrated less intense cytokine responses, diminished neutrophil infiltration, and accelerated uroepithelial recovery. CONCLUSIONS: Mice lacking the antimicrobial peptide cathelicidin experienced less severe infection than wild-type mice in a well-established model of cystitis. Although CRAMP exhibits in vitro antibacterial activity against UPEC, it may enhance UPEC infection in the bladder by promoting epithelial receptivity and local inflammation.

Reading, Writing, and Presenting Original Scientific Research: A Nine-Week Course in Scientific Communication for High School Students.

High school students are not often given opportunities to communicate scientific findings to their peers, the general public, and/or people in the scientific community, and therefore they do not develop scientific communication skills. We present a nine-week course that can be used to teach high school students, who may have no previous experience, how to read and write primary scientific articles and how to discuss scientific findings with a broad audience. Various forms of this course have been taught for the past 10 years as part of an intensive summer research program for rising high school seniors that is coordinated by the Young Scientist Program at Washington University in St. Louis. The format presented here includes assessments for efficacy through both rubric-based methods and student self-assessment surveys.

Bacterial lysis liberates the neutrophil migration suppressor YbcL from the periplasm of uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) modulates aspects of the innate immune response during urinary tract infection to facilitate bacterial invasion of the bladder epithelium, a requirement for the propagation of infection. For example, UPEC-encoded YbcL suppresses the traversal of bladder epithelia by neutrophils in both an in vitro model and an in vivo murine cystitis model. The suppressive activity of YbcL requires liberation from the bacterial periplasm, though the mechanism of release is undefined. Here we present findings on the site of action of YbcL and demonstrate a novel mode of secretion for a UPEC exoprotein. Suppression of neutrophil migration by purified YbcL(UTI), encoded by cystitis isolate UTI89, required the presence of a uroepithelial layer; YbcL(UTI) did not inhibit neutrophil chemotaxis directly. YbcL(UTI) was released to a greater extent during UPEC infection of uroepithelial cells than during that of neutrophils. Release of YbcL(UTI) was maximal when UPEC and bladder epithelial cells were in close proximity. Established modes of secretion, including outer membrane vesicles, the type II secretion system, and the type IV pilus, were dispensable for YbcL(UTI) release from UPEC. Instead, YbcL(UTI) was liberated during bacterial death, which was augmented upon exposure to bladder epithelial cells, as confirmed by detection of bacterial cytoplasmic proteins and DNA in the supernatant and enumeration of bacteria with compromised membranes. As YbcL(UTI) acts on the uroepithelium to attenuate neutrophil migration, this mode of release may represent a type of altruistic cooperation within a UPEC population during colonization of the urinary tract.

RNA interference in marine and freshwater sponges: actin knockdown in Tethya wilhelma and Ephydatia muelleri by ingested dsRNA expressing bacteria.

BACKGROUND: The marine sponge Tethya wilhelma and the freshwater sponge Ephydatia muelleri are emerging model organisms to study evolution, gene regulation, development, and physiology in non-bilaterian animal systems. Thus far, functional methods (i.e., loss or gain of function) for these organisms have not been available. RESULTS: We show that soaking developing freshwater sponges in double-stranded RNA and/or feeding marine and freshwater sponges bacteria expressing double-stranded RNA can lead to RNA interference and reduction of targeted transcript levels. These methods, first utilized in C. elegans, have been adapted for the development and feeding style of easily cultured marine and freshwater poriferans. We demonstrate phenotypic changes result from 'knocking down' expression of the actin gene. CONCLUSION: This technique provides an easy, efficient loss-of-function manipulation for developmental and gene regulatory studies in these important non-bilaterian animals.

Leucine leucine-37 uses formyl peptide receptor-like 1 to activate signal transduction pathways, stimulate oncogenic gene expression, and enhance the invasiveness of ovarian cancer cells.

Emerging evidence suggests that the antimicrobial peptide, leucine leucine-37 (LL-37), could play a role in the progression of solid tumors. LL-37 is expressed as the COOH terminus of human cationic antimicrobial protein-18 (hCAP-18) in ovarian, breast, and lung cancers. Previous studies have shown that the addition of LL-37 to various cancer cell lines in vitro stimulates proliferation, migration, and invasion. Similarly, overexpression of hCAP-18/LL-37 in vivo accelerates tumor growth. However, the receptor or receptors through which these processes are mediated have not been thoroughly examined. In the present study, expression of formyl peptide receptor-like 1 (FPRL1) was confirmed on ovarian cancer cells. Proliferation assays indicated that LL-37 does not signal through a G protein-coupled receptor, such as FPRL1, to promote cancer cell growth. By contrast, FPRL1 was required for LL-37-induced invasion through Matrigel. The peptide stimulated mitogen-activated protein kinase and Janus-activated kinase/signal transducers and activators of transcription signaling cascades and led to the significant activation of several transcription factors, through both FPRL1-dependent and FPRL1-independent pathways. Likewise, expression of some LL-37-stimulated genes was attenuated by the inhibition of FPRL1. Increased expression of CXCL10, EGF, and PDGF-BB as well as other soluble factors was confirmed from conditioned medium of LL-37-treated cells. Taken together, these data suggest that LL-37 potentiates a more aggressive behavior from ovarian cancer cells through its interaction with FPRL1.

The pro-inflammatory peptide LL-37 promotes ovarian tumor progression through recruitment of multipotent mesenchymal stromal cells.

Bone marrow-derived mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs) have been shown to engraft into the stroma of several tumor types, where they contribute to tumor progression and metastasis. However, the chemotactic signals mediating MSC migration to tumors remain poorly understood. Previous studies have shown that LL-37 (leucine, leucine-37), the C-terminal peptide of human cationic antimicrobial protein 18, stimulates the migration of various cell types and is overexpressed in ovarian, breast, and lung cancers. Although there is evidence to support a pro-tumorigenic role for LL-37, the function of the peptide in tumors remains unclear. Here, we demonstrate that neutralization of LL-37 in vivo significantly reduces the engraftment of MSCs into ovarian tumor xenografts, resulting in inhibition of tumor growth as well as disruption of the fibrovascular network. Migration and invasion experiments conducted in vitro indicated that the LL-37-mediated migration of MSCs to tumors likely occurs through formyl peptide receptor like-1. To assess the response of MSCs to the LL-37-rich tumor microenvironment, conditioned medium from LL-37-treated MSCs was assessed and found to contain increased levels of several cytokines and pro-angiogenic factors compared with controls, including IL-1 receptor antagonist, IL-6, IL-10, CCL5, VEGF, and matrix metalloproteinase-2. Similarly, Matrigel mixed with LL-37, MSCs, or the combination of the two resulted in a significant number of vascular channels in nude mice. These data indicate that LL-37 facilitates ovarian tumor progression through recruitment of progenitor cell populations to serve as pro-angiogenic factor-expressing tumor stromal cells.

Toll-like receptors on human mesenchymal stem cells drive their migration and immunomodulating responses.

Adult human bone marrow-derived mesenchymal stem cells (hMSCs) are under study as therapeutic delivery agents that assist in the repair of damaged tissues. To achieve the desired clinical outcomes for this strategy requires a better understanding of the mechanisms that drive the recruitment, migration, and engraftment of hMSCs to the targeted tissues. It is known that hMSCs are recruited to sites of stress or inflammation to fulfill their repair function. It is recognized that toll-like receptors (TLRs) mediate stress responses of other bone marrow-derived cells. This study explored the role of TLRs in mediating stress responses of hMSCs. Accordingly, the presence of TLRs in hMSCs was initially established by reverse transcription-polymerase chain reaction assays. Flow cytometry and fluorescence immunocytochemical analyses confirmed these findings. The stimulation of hMSCs with TLR agonists led to the activation of downstream signaling pathways, including nuclear factor kappaB, AKT, and MAPK. Consequently, activation of these pathways triggered the induction and secretion of cytokines, chemokines, and related TLR gene products as established from cDNA array, immunoassay, and cytokine antibody array analyses. Interestingly, the unique patterns of affected genes, cytokines, and chemokines measured identify these receptors as critical players in the clinically established immunomodulation observed for hMSCs. Lastly, hMSC migration was promoted by TLR ligand exposure as demonstrated by transwell migration assays. Conversely, disruption of TLRs by neutralizing TLR antibodies compromised hMSC migration. This study defines a novel TLR-driven stress and immune modulating response for hMSCs that is critical to consider in the design of stem cell-based therapies.

Ovarian cancers overexpress the antimicrobial protein hCAP-18 and its derivative LL-37 increases ovarian cancer cell proliferation and invasion.

The role of the pro-inflammatory peptide, LL-37, and its pro-form, human cationic antimicrobial protein 18 (hCAP-18), in cancer development and progression is poorly understood. In damaged and inflamed tissue, LL-37 functions as a chemoattractant, mitogen and pro-angiogenic factor suggesting that the peptide may potentiate tumor progression. The aim of this study was to characterize the distribution of hCAP-18/LL-37 in normal and cancerous ovarian tissue and to examine the effects of LL-37 on ovarian cancer cells. Expression of hCAP-18/LL-37 was localized to immune and granulosa cells of normal ovarian tissue. By contrast, ovarian tumors displayed significantly higher levels of hCAP-18/LL-37 where expression was observed in tumor and stromal cells. Protein expression was statistically compared to the degree of immune cell infiltration and microvessel density in epithelial-derived ovarian tumors and a significant correlation was observed for both. It was demonstrated that ovarian tumor tissue lysates and ovarian cancer cell lines express hCAP-18/LL-37. Treatment of ovarian cancer cell lines with recombinant LL-37 stimulated proliferation, chemotaxis, invasion and matrix metalloproteinase expression. These data demonstrate for the first time that hCAP-18/LL-37 is significantly overexpressed in ovarian tumors and suggest LL-37 may contribute to ovarian tumorigenesis through direct stimulation of tumor cells, initiation of angiogenesis and recruitment of immune cells. These data provide further evidence of the existing relationship between pro-inflammatory molecules and ovarian cancer progression.