Susana Novella

Comparative Genomics of Listeria Species

Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism. We present and compare the genome sequences of L. monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L. innocua (3,011,209 base pairs). We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments. The presence of 270 L. monocytogenes and 149 L. innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events.

A Validated PCR-Based Method To Detect Listeria monocytogenes Using Raw Milk as a Food Model—Towards an International Standard

A PCR assay with an internal amplification control was developed for Listeria monocytogenes. The assay has a 99% detection probability of seven cells per reaction. When tested against 38 L. monocytogenes strains and 52 nontarget strains, the PCR assay was 100% inclusive (positive signal from target) and 100% exclusive (no positive signal from nontarget). The assay was then evaluated in a collaborative trial involving 12 European laboratories, where it was tested against an additional 14 target and 14 nontarget strains. In that trial, the inclusivity was 100% and the exclusivity was 99.4%, and both the accordance (repeatability) and the concordance (reproducibility) were 99.4%. The assay was incorporated within a method for the detection of L. monocytogenes in raw milk, which involves 24 h of enrichment in half-Fraser broth followed by 16 h of enrichment in a medium that can be added directly into the PCR. The performance characteristics of the PCR-based method were evaluated in a collaborative trial involving 13 European laboratories. In that trial, a specificity value (percentage of correct identification of blank samples) of 81.8% was obtained; the accordance was 87.9%, and the concordance was 68.1%. The sensitivity (correct identification of milk samples inoculated with 20 to 200 L. monocytogenes cells per 25 ml) was 89.4%, the accordance was 81.2%, and the concordance was 80.7%. This method provides a basis for the application of routine PCR-based analysis to dairy products and other foodstuffs and should be appropriate for international standardization.

The smcL gene of Listeria ivanovii encodes a sphingomyelinase C that mediates bacterial escape from the phagocytic vacuole

The ruminant pathogen Listeria ivanovii differs from Listeria monocytogenes in that it causes strong, bizonal haemolysis and a characteristic shovel‐shaped co‐operative haemolytic (‘CAMP‐like’) reaction with Rhodococcus equi. We cloned the gene responsible for the differential haemolytic properties of L. ivanovii, smcL. It encodes a sphingomyelinase C (SMase) highly similar (> 50% identity) to the SMases from Staphylococcus aureus (β‐toxin), Bacillus cereus and Leptospira interrogans. smcL was transcribed monocistronically and was expressed independently of PrfA. Low‐stringency Southern blots demonstrated that, within the genus Listeria, smcL was present only in L. ivanovii. We constructed an smcL knock‐out mutant. Its phenotype on blood agar was identical to that of L. monocytogenes (i.e. weak haemolysis and no shovel‐shaped CAMP‐like reaction with R. equi ). This mutant was less virulent for mice, and its intracellular proliferation was impaired in the bovine epithelial‐like cell line MDBK. The role of SmcL in intracellular survival was investigated using an L. monocytogenes mutant lacking the membrane‐damaging determinants hly, plcA and plcB, being thus unable to grow intracellularly. Complementation of this mutant with smcL on a plasmid was sufficient to promote bacterial intracellular proliferation in MDBK cells. Transmission electron microscopy showed that SmcL mediates the disruption of the phagocytic vacuole and the release of bacteria into the cytosol. Therefore, L. ivanovii possesses a third phospholipase with membrane‐damaging activity that, together with PlcA and PlcB, may act in concert with the pore‐forming toxin Hly to mediate efficient escape from the vacuolar compartment. The 5′ end of smcL is contiguous with the internalin locus i‐inlFE, which is also specific to L. ivanovii and is required for full virulence in mice. Thus, smcL forms part of a novel virulence gene cluster in Listeria that is species specific.

Estradiol selectively stimulates endothelial prostacyclin production through estrogen receptor-α

Estradiol (E(2)) acts on the endothelium to promote vasodilatation through the release of several compounds, including prostanoids, which are products of arachidonic acid metabolism. Among these, prostacyclin (PGI2) and thromboxane A2 (TXA2) exert opposite effects on vascular tone. The role of different estrogen receptors (ERs) in the PGI2/TXA2 balance, however, has not been fully elucidated. Our study sought to uncover whether E(2) enhances basal production of PGI2 or TXA2 in cultured human umbilical vein endothelial cells (HUVECs), to analyze the enzymatic mechanisms involved, and to evaluate the different roles of both types of ERs (ERalpha and ERbeta). HUVECs were exposed to E(2), selective ERalpha (1,3,5-tris(4-hydroxyphenyl)-4-propyl-1h-pyrazole, PPT) or ERbeta (diarylpropionitrile, DPN) agonists and antagonists (unspecific: ICI 182 780; specific for ERalpha: methyl-piperidino-pyrazole, MPP). PGI2 and TXA2 production was measured by ELISA. Expression of phospholipases, cyclooxygenases (COX-1 and COX-2), PGI2 synthase (PGIS), and thromboxane synthase (TXAS) was analyzed by western blot and quantitative RT-PCR. E(2) (1-100 nM) dose dependently increased PGI2 production (up to 50%), without affecting TXA2 production. COX-1 and PGIS protein and gene expressions were increased, whereas COX-2, phospholipases, and TXAS expression remained unaltered. All these effects were mediated through ERalpha, since they were produced not only in the presence of E(2), but also in that of PPT, while they were abolished in the presence of MPP. In conclusion, E(2), acting through ERalpha, up-regulates COX-1 and PGIS expression, thus directing prostanoid balance toward increased PGI2 production.

Vascular aging in women: is estrogen the fountain of youth?

Aging is associated with structural and functional changes in the vasculature, including endothelial dysfunction, arterial stiffening and remodeling, impaired angiogenesis, and defective vascular repair, and with increased prevalence of atherosclerosis. Cardiovascular risk is similar for older men and women, but lower in women during their fertile years. This age- and sex-related difference points to estrogen as a protective factor because menopause is marked by the loss of endogenous estrogen production. Experimental and some clinical studies have attributed most of the protective effects of estrogen to its modulatory action on vascular endothelium. Estrogen promotes endothelial-derived NO production through increased expression and activity of endothelial nitric oxide synthase, and modulates prostacyclin and thromboxane A2 release. The thromboxane A2 pathway is key to regulating vascular tone in females. Despite all the experimental evidence, some clinical trials have reported no cardiovascular benefit from estrogen replacement therapy in older postmenopausal women. The “Timing Hypothesis,” which states that estrogen-mediated vascular benefits occur only before the detrimental effects of aging are established in the vasculature, offers a possible explanation for these discrepancies. Nevertheless, a gap remains in current knowledge of cardiovascular aging mechanisms in women. This review comprises clinical and experimental data on the effects of aging, estrogens, and hormone replacement therapy on vascular function of females. We aim to clarify how menopause and aging contribute jointly to vascular aging and how estrogen modulates vascular response at different ages.

Estradiol stimulates vasodilatory and metabolic pathways in cultured human endothelial cells.

Vascular effects of estradiol are being investigated because there are controversies among clinical and experimental studies. DNA microarrays were used to investigate global gene expression patterns in cultured human umbilical vein endothelial cells (HUVEC) exposed to 1 nmol/L estradiol for 24 hours. When compared to control, 187 genes were identified as differentially expressed with 1.9-fold change threshold. Supervised principal component analysis and hierarchical cluster analysis revealed the differences between control and estradiol-treated samples. Physiological concentrations of estradiol are sufficient to elicit significant changes in HUVEC gene expression. Notch signaling, actin cytoskeleton signaling, pentose phosphate pathway, axonal guidance signaling and integrin signaling were the top-five canonical pathways significantly regulated by estrogen. A total of 26 regulatory networks were identified as estrogen responsive. Microarray data were confirmed by quantitative RT-PCR in cardiovascular meaning genes; cyclooxigenase (COX)1, dimethylarginine dimethylaminohydrolase (DDAH)2, phospholipase A2 group IV (PLA2G4) B, and 7-dehydrocholesterol reductase were up-regulated by estradiol in a dose-dependent and estrogen receptor-dependent way, whereas COX2, DDAH1 and PLA2G4A remained unaltered. Moreover, estradiol-induced COX1 gene expression resulted in increased COX1 protein content and enhanced prostacyclin production. DDAH2 protein content was also increased, which in turn decreased asymmetric dimethylarginine concentration and increased NO release. All stimulated effects of estradiol on gene and protein expression were estrogen receptor-dependent, since were abolished in the presence of the estrogen receptor antagonist ICI 182780. This study identifies new vascular mechanisms of action by which estradiol may contribute to a wide range of biological processes.

Effects of Estrogen on Vascular Inflammation: A Matter of Timing

Objective—Our study aims to determine the role of time of menopause on vascular inflammation biomarkers and how it affects their modulation by estrogen and raloxifene in postmenopausal women. Methods and Results—Uterine arteries from 68 postmenopausal women were divided into 3 segments and cultured for 24 hours in tissue culture media containing 17&bgr;-estradiol (100 nmol/L), raloxifene (100 nmol/L), or vehicle. Assessment of arterial concentration of 13 inflammatory biomarkers was performed by multiplex immunobead-based assay. Aging per se has a positive correlation with the generation of several proinflammatory markers. Although short-term estradiol exposure correlates with lower expression of tumor necrosis factor-&agr;, vascular endothelial growth factor, and interleukin-1&bgr; in all age groups, for most biomarkers aging was associated with a switch from a beneficial anti-inflammatory action by estrogen, at earlier stages of menopause, to a proinflammatory profile after 5 years past its onset. Raloxifene has no significant effect on the expression of all proinflammatory markers. Western blot analysis of estrogen receptor expression (estrogen receptor-&agr; and estrogen receptor-&bgr;) showed that estrogen receptor-&bgr; increases with aging, and this increase has a positive correlation with the generation of several proinflammatory markers. Conclusion—Aging alters estrogen-mediated effects on the modulation of inflammatory biomarkers in women. How aging affects estrogen responses on vascular inflammation is not clear, but our data show a positive association between increased estrogen receptor-&bgr; expression with aging and proinflammatory effects by estrogen.

Estradiol induces endothelial cell migration and proliferation through estrogen receptor-enhanced RhoA/ROCK pathway.

Migration and proliferation of endothelial cells are involved in re-endothelialization and angiogenesis, two important cardiovascular processes that are increased in response to estrogens. RhoA, a small GTPase which controls multiple cellular processes, is involved in the control of cell migration and proliferation. Our aim was to study the role of RhoA on estradiol-induced migration and proliferation and its dependence on estrogen receptors activity. Human umbilical vein endothelial cells were stimulated with estradiol, in the presence or absence of ICI 182780 (estrogen receptors antagonist) and Y-27632 (Rho kinase inhibitor). Estradiol increased Rho GEF-1 gene expression and RhoA (gene and protein expression and activity) in an estrogen receptor-dependent manner. Cell migration, stress fiber formation and cell proliferation were increased in response to estradiol and were also dependent on the estrogen receptors and RhoA activation. Estradiol decreased p27 levels, and significantly raised the expression of cyclins and CDK. These effects were counteracted by the use of either ICI 182780 or Y-27632. In conclusion, estradiol enhances the RhoA/ROCK pathway and increases cell cycle-related protein expression by acting through estrogen receptors. This results in an enhanced migration and proliferation of endothelial cells.

Aging Negatively Affects Estrogens-Mediated Effects on Nitric Oxide Bioavailability by Shifting ERα/ERβ Balance in Female Mice

Aims Aging is among the major causes for the lack of cardiovascular protection by estrogen (E2) during postmenopause. Our study aims to determine the mechanisms whereby aging changes E2 effects on nitric oxide (NO) production in a mouse model of accelerated senescence (SAM). Methods and Results Although we found no differences on NO production in females SAM prone (SAMP, aged) compared to SAM resistant (SAMR, young), by either DAF-2 fluorescence or plasmatic nitrite/nitrate (NO2/NO3), in both cases, E2 treatment increased NO production in SAMR but had no effect in SAMP. Those results are in agreement with changes of eNOS protein and gene expression. E2 up-regulated eNOS expression in SAMR but not in SAMP. E2 is also known to increase NO by decreasing its catabolism by superoxide anion (O2 -). Interestingly, E2 treatment decreased O2 − production in young females, while increased O2 − in aged ones. Furthermore, we observed that aging changed expression ratio of estrogen receptors (ERβ/ERα) and levels of DNA methylation. Increased ratio ERβ/ERα in aged females is associated to a lack of estrogen modulation of NO production and with a reversal in its antioxidant effect to a pro-oxidant profile. Conclusions Together, our data suggest that aging has detrimental effects on E2-mediated benefits on NO bioavailability, partially by affecting the ability of E2 to induce up regulation of eNOS and decrease of O2 −. These modifications may be associated to aging-mediated modifications on global DNA methylation status, but not to a specific methylation at 5′flanking region of ERα gene.

Susceptibility of Listeria monocytogenes to antimicrobial peptides

We assessed the susceptibility of several pathogenic and non-pathogenic Listeria species to antimicrobial peptides of animal and plant origin. Human defensins and thionins were highly inhibitory, whereas protamine, snakin and magainin showed an intermediate effect. A temperature dependence in the activity of potato defensin was observed for Listeria monocytogenes and Listeria ivanovii. PrfA* L. monocytogenes mutants, that overexpress constitutively PrfA-dependent genes, were sensitive to the peptide independently of the temperature whereas isogenic PrfA(-) derivatives were constitutively resistant. These data indicate that the thermoregulated transcription factor PrfA controls the expression of bacterial products that influence the susceptibility or resistance to some antimicrobial peptides.