Virulence genes inThe gram-positive, facultative intracellular pathogen Listeria monocytogenes is the causal agent of listeriosis, a severe foodborne, opportunistic infection of humans and animals characterized by meningoencephalitis, abortion, and septicemia (11). Known listerial virulence factors are chromosomally determined, and most of the corresponding genes form a cluster encompassing the hly locus (34). hly is monocistronically transcribed and encodes listeriolysin O (LLO), a cholesterol-binding, SH-activated toxin which confers a hemolytic phenotype to Listeria colonies (9, 29). Downstream from hly is the so-called lecithinase operon, which comprises the genes mpl, actA, and plcB, in that order. Their products are, respectively, Mpl, a metalloprotease which cleaves the plcB product to its active form; ActA, a surface protein which mediates actin-based bacterial motility within infected host cells; and PlcB, a wide-range phospholipase C (PLC), or lecithinase. These proteins are produced from a single mRNA driven by the mpl promoter or from shorter transcripts, one of which starts at a promoter in front of actA and covers actA and plcB (30,46). Upstream, and transcribed divergently from hly, is the plcA-prfA operon, which encodes a phosphatidylinositol-specific PLC, and PrfA, a transcriptional activator. The PrfA protein exerts tight control over all of the above-described transcriptional units, and its presence is absolutely necessary for virulence expression (8,26,30). Elsewhere on the chromosome is the inlA-inlB operon, coding for the surface proteins InlA (or internalin) and InlB, which are involved in invasion and cell tropism (15). This operon is partially regulated by PrfA (3, 28, 41; see references 24, 34, and 39 for reviews on molecular determinants and cell biology of Listeria pathogenesis).PrfA is the only virulence regulator identified so far in L. monocytogenes. It has overall sequence similarity with the cyclic AMP (cAMP) receptor protein (CRP) and other members of the CRP family of transcriptional regulators (22,25). The structural similarities between the listerial regulator and CRP appear to be functionally significant, since, as shown recently (40), single substitutions in the C-terminal DNA-binding helixturn-helix (HTH) motif of PrfA, affecting residues predicted to be involved in DNA contact, alter DNA binding and virulence gene activation. Like CRP-regulated elements, all PrfA-controlled transcriptional units are preceded by conserved DNA sequences with dyad symmetry. These palindromic structures, called PrfA boxes, are 14 bp long and are centered near position Ϫ41.

No abstract

Virulence genes of the facultative intracellular pathogen Listeria monocytogenes are coordinately regulated by the activator protein PrfA, encoded by prfA, a member of the cyclic AMP receptor protein family of bacterial transcription factors. We found that prfA* mutants that constitutively overexpress the virulence regulon due to a Gly145Ser substitution in PrfA (M.-T. Ripio, G. Domínguez-Bernal, M. Lara, M. Suárez, and J.-A. Vázquez-Boland, J. Bacteriol. 179:1533-1540, 1997) rapidly utilized glucose-1-phosphate (G-1-P) as a carbon source for growth, in contrast to wild-type strains, which characteristically do not. Wild-type strains acquired the capacity for readily metabolizing G-1-P upon exposure to environmental conditions that activate the expression of prfA and PrfA-dependent virulence genes (i.e., culture at 37°C in charcoal-treated medium). In these strains, G-1-P utilization followed an expressional pattern identical to that of virulence genes controlled by PrfA, with repression at 20°C. Tn917 insertions in L. monocytogenes mutants selected for G-1-P utilization deficiency mapped to the plcA-prfA operon, a ⌬prfA strain was totally unable to utilize G-1-P, and trans complementation with prfA constructs restored the ability to efficiently metabolize and grow on G-1-P to these mutants. Thus, G-1-P utilization by L. monocytogenes is under the tight positive control of the central virulence regulator, PrfA, and is coexpressed with PrfA-dependent pathogenicity determinants. It was recently reported that readily utilized carbohydrates, such as glucose or cellobiose, repress virulence genes in L. monocytogenes. We confirmed this but, interestingly, found that G-1-P does not inhibit expression of the PrfA regulon, indicating that this sugar follows a catabolic pathway that bypasses the repressor mechanism triggered by other readily metabolized carbon sources. PrfA dependence and coexpression with virulence genes suggest that utilization of exogenous G-1-P may be relevant to Listeria pathogenesis. G-1-P is the precursor metabolite and primary degradation product of glycogen and is therefore available within the mammalian cell. Based on our results, we hypothesize that G-1-P could play an important role as a growth substrate for intracellular Listeria.

Aberrant activation of the phosphoinositide-3-kinase (PI3K)/ PTEN/Akt pathway, leading to increased proliferation and decreased apoptosis, has been implicated in several human pathologies including cancer. Our previous data have shown that Akt-mediated signaling is an essential mediator in the mouse skin carcinogenesis system during both the tumor promotion and progression stages. In addition, overexpression of Akt is also able to transform keratinocytes through transcriptional and posttranscriptional processes. Here, we report the consequences of the increased expression of Akt1 (wtAkt) or constitutively active Akt1 (myrAkt) in the basal layer of stratified epithelia using the bovine keratin K5 promoter. These mice display alterations in epidermal proliferation and differentiation. In addition, transgenic mice with the highest levels of Akt expression developed spontaneous epithelial tumors in multiple organs with age.

Squamous cell carcinomas (SCC) represent the most aggressive type of nonmelanoma skin cancer. Although little is known about the causal alterations of SCCs, in organtransplanted patients the E7 and E6 oncogenes of human papillomavirus, targeting the p53-and pRb-dependent pathways, have been widely involved. Here, we report the functional consequences of the simultaneous elimination of Trp53 and retinoblastoma (Rb) genes in epidermis using Cre-loxP system. Loss of p53, but not pRb, produces spontaneous tumor development, indicating that p53 is the predominant tumor suppressor acting in mouse epidermis. Although the simultaneous inactivation of pRb and p53 does not aggravate the phenotype observed in Rb-deficient epidermis in terms of proliferation and/or differentiation, spontaneous SCC development is severely accelerated in doubly deficient mice. The tumors are aggressive and undifferentiated and display a hair follicle origin. Detailed analysis indicates that the acceleration is mediated by premature activation of the epidermal growth factor receptor/Akt pathway, resulting in increased proliferation in normal and dysplastic hair follicles and augmented tumor angiogenesis. The molecular characteristics of this model provide valuable tools to understand epidermal tumor formation and may ultimately contribute to the development of therapies for the treatment of aggressive squamous cancer. [Cancer Res 2008;68(3):683-92]

No abstract

The mouse skin carcinogenesis represents one of the best models for the understanding of malignant transformation, including the multistage nature of tumor development. The retinoblastoma gene product (pRb) plays a critical role in cell cycle regulation, differentiation, and inhibition of oncogenic transformation. In epidermis, Rb À/À deletion leads to proliferation and differentiation defects. Numerous evidences showed the involvement of the retinoblastoma pathway in this model. However, the actual role of pRb is still unknown. papillomas than the Rb +/+ counterparts. Moreover, the small size of the pRb-deficient tumors is associated with an increase in the apoptotic index. Despite this, pRb-deficient tumors display an increased conversion rate to squamous cell carcinomas. Biochemical analyses revealed that these characteristics correlate with the differential expression and activity of different pathways, including E2F/p19 arf /p53, PTEN/Akt, c-jun NH 2 -terminal kinase/p38, and nuclear factor-KB. Collectively, our findings show unexpected and hitherto nondescribed roles of pRb during the process of epidermal carcinogenesis. (Cancer Res 2005; 65(21): 9678-86)

The development of siRNA-based gene silencing therapies has significant potential for effectively treating debilitating genetic, hyper-proliferative or malignant skin conditions caused by aberrant gene expression. To be efficacious and widely accepted by physicians and patients, therapeutic siRNAs must access the viable skin layers in a stable and functional form, preferably without painful administration. In this study we explore the use of minimally-invasive steel microneedle devices to effectively deliver siRNA into skin. A simple, yet precise microneedle coating method permitted reproducible loading of siRNA onto individual microneedles. Following recovery from the microneedle surface, lamin A/C siRNA retained full activity, as demonstrated by significant reduction in lamin A/C mRNA levels and reduced lamin A/C protein in HaCaT keratinocyte cells. However, lamin A/C siRNA pre-complexed with a commercial lipid-based transfection reagent (siRNA lipoplex) was less functional following microneedle coating. As Accell-modified “self-delivery” siRNA targeted against CD44 also retained functionality after microneedle coating, this form of siRNA was used in subsequent in vivo studies, where gene silencing was determined in a transgenic reporter mouse skin model. Self-delivery siRNA targeting the reporter (luciferase/GFP) gene was coated onto microneedles and delivered to mouse footpad. Quantification of reporter mRNA and intravital imaging of reporter expression in the outer skin layers confirmed functional in vivo gene silencing following microneedle delivery of siRNA. The use of coated metal microneedles represents a new, simple, minimally-invasive, patient-friendly and potentially self-administrable method for the delivery of therapeutic nucleic acids to the skin.