Engineering of a Bacterial Biosensor for the Detection of Chlorate in Food.

Chlorate can contaminate food due to the use of chlorinated water for processing or equipment disinfection. Chronic exposure to chlorate in food and drinking water is a potential health concern. The current methods for detecting chlorate in liquids and foods are expensive and not easily accessible to all laboratories, highlighting an urgent need for a simple and cost-effective method. The discovery of the adaptation mechanism of Escherichia coli to chlorate stress, which involves the production of the periplasmic Methionine Sulfoxide Reductase (MsrP), prompted us to use an E. coli strain with an msrP-lacZ fusion as a biosensor for detecting chlorate. Our study aimed to optimize the bacterial biosensor's sensitivity and efficiency to detect chlorate in various food samples using synthetic biology and adapted growth conditions. Our results demonstrate successful biosensor enhancement and provide proof of concept for detecting chlorate in food samples.

Gfi1 Loss Protects against Two Models of Induced Diabetes.

Background: Although several approaches have revealed much about individual factors that regulate pancreatic development, we have yet to fully understand their complicated interplay during pancreas morphogenesis. Gfi1 is transcription factor specifically expressed in pancreatic acinar cells, whose role in pancreas cells fate identity and specification is still elusive. Methods: In order to gain further insight into the function of this factor in the pancreas, we generated animals deficient for Gfi1 specifically in the pancreas. Gfi1 conditional knockout animals were phenotypically characterized by immunohistochemistry, RT-qPCR, and RNA scope. To assess the role of Gfi1 in the pathogenesis of diabetes, we challenged Gfi1-deficient mice with two models of induced hyperglycemia: long-term high-fat/high-sugar feeding and streptozotocin injections. Results: Interestingly, mutant mice did not show any obvious deleterious phenotype. However, in depth analyses demonstrated a significant decrease in pancreatic amylase expression, leading to a diminution in intestinal carbohydrates processing and thus glucose absorption. In fact, Gfi1-deficient mice were found resistant to diet-induced hyperglycemia, appearing normoglycemic even after long-term high-fat/high-sugar diet. Another feature observed in mutant acinar cells was the misexpression of ghrelin, a hormone previously suggested to exhibit anti-apoptotic effects on ß-cells in vitro. Impressively, Gfi1 mutant mice were found to be resistant to the cytotoxic and diabetogenic effects of high-dose streptozotocin administrations, displaying a negligible loss of ß-cells and an imperturbable normoglycemia. Conclusions: Together, these results demonstrate that Gfi1 could turn to be extremely valuable for the development of new therapies and could thus open new research avenues in the context of diabetes research.

Neisseria meningitidis Type IV Pili Trigger Ca2+-Dependent Lysosomal Trafficking of the Acid Sphingomyelinase To Enhance Surface Ceramide Levels.

Acid sphingomyelinase (ASM) is a lipid hydrolase that converts sphingomyelin to ceramide and that can be activated by various cellular stress mechanisms, including bacterial pathogens. Vesicle transportation or trafficking of ASM from the lysosomal compartment to the cell membrane is a prerequisite for its activation in response to bacterial infections; however, the effectors and mechanisms of ASM translocation and activation are poorly defined. Our recent work documented the key importance of ASM for Neisseria meningitidis uptake into human brain microvascular endothelial cells (HBMEC). We clearly identified OpcA to be one bacterial effector promoting ASM translocation and activity, though it became clear that additional bacterial components were involved, as up to 80% of ASM activity and ceramide generation was retained in cells infected with an opcA-deficient mutant. We hypothesized that N. meningitidis might use pilus components to promote the translocation of ASM into HBMEC. Indeed, we found that both live, piliated N. meningitidis and pilus-enriched fractions trigger transient ASM surface display, followed by the formation of ceramide-rich platforms (CRPs). By using indirect immunocytochemistry and direct stochastic optical reconstruction microscopy, we show that the overall number of CRPs with a size of ∼80 nm in the plasma membrane is significantly increased after exposure to pilus-enriched fractions. Infection with live bacteria as well as exposure to pilus-enriched fractions transiently increased cytosolic Ca2+ levels in HBMEC, and this was found to be important for ASM surface display mediated by lysosomal exocytosis, as depletion of cytosolic Ca2+ resulted in a significant decrease in ASM surface levels, ASM activity, and CRP formation.

Antibacterial activity of ceramide and ceramide analogs against pathogenic Neisseria.

Certain fatty acids and sphingoid bases found at mucosal surfaces are known to have antibacterial activity and are thought to play a more direct role in innate immunity against bacterial infections. Herein, we analysed the antibacterial activity of sphingolipids, including the sphingoid base sphingosine as well as short-chain C6 and long-chain C16-ceramides and azido-functionalized ceramide analogs against pathogenic Neisseriae. Determination of the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) demonstrated that short-chain ceramides and a ω-azido-functionalized C6-ceramide were active against Neisseria meningitidis and N. gonorrhoeae, whereas they were inactive against Escherichia coli and Staphylococcus aureus. Kinetic assays showed that killing of N. meningitidis occurred within 2 h with ω-azido-C6-ceramide at 1 X the MIC. Of note, at a bactericidal concentration, ω-azido-C6-ceramide had no significant toxic effect on host cells. Moreover, lipid uptake and localization was studied by flow cytometry and confocal laser scanning microscopy (CLSM) and revealed a rapid uptake by bacteria within 5 min. CLSM and super-resolution fluorescence imaging by direct stochastic optical reconstruction microscopy demonstrated homogeneous distribution of ceramide analogs in the bacterial membrane. Taken together, these data demonstrate the potent bactericidal activity of sphingosine and synthetic short-chain ceramide analogs against pathogenic Neisseriae.

Comparison of the inflammatory response of brain microvascular and peripheral endothelial cells following infection with Neisseria meningitidis.

The interaction of Neisseria meningitidis with both peripheral and brain endothelial cells is a critical event in the development of invasive meningococcal disease. In this study, we used in vitro models based on human brain microvascular endothelial cells (HBMEC), and peripheral endothelial EA.hy926 cells, to investigate their roles in the inflammatory response towards meningococcal infection. Both cell lines were infected with two pathogenic N. meningitidis isolates and secretion of the cytokine interleukin-6 (IL-6), the CXC chemokine IL-8 and the monocyte chemoattractant protein-1 (MCP-1) were estimated by ELISA. Neisseria meningitidis was able to stimulate the production of IL-6 and IL-8 by HBMEC and EA.hy926 cells in a time- and concentration-dependent manner. Interestingly, HBMEC released significant higher amounts of IL-6 and IL-8. Moreover, we observed that heat-killed bacteria stimulated high levels of IL-8. In addition, capsule expression had an inhibitory effect on IL-8 release. We extended our study and included serogroup C strains belonging to sequence type 11 clonal complex (cc) from a recent outbreak in France, as well as isolates belonging to the hypervirulent clonal complexes cc8, cc18, cc32 and cc269 and analyzed their ability to induce the secretion of IL-8 from both cell lines. Although individual variations were observed among different isolates, no clear correlations were observed between strain origin, clinical presentation and IL-8 levels.

Characterization of Plasma Membrane Ceramides by Super-Resolution Microscopy.

The sphingolipid ceramide regulates cellular processes such as differentiation, proliferation, growth arrest, and apoptosis. Ceramide-rich membrane areas promote structural changes within the plasma membrane that segregate membrane receptors and affect membrane curvature and vesicle formation, fusion, and trafficking. Ceramides were labeled by immunocytochemistry to visualize their distribution on the plasma membrane of different cells with virtually molecular resolution by direct stochastic optical reconstruction microscopy (dSTORM). Super-resolution images show that independent of labeling conditions and cell type 50-60 % of all membrane ceramides are located in ceramide-rich platforms (CRPs) with a size of about 75 nm that are composed of at least about 20 ceramides. Treatment of cells with Bacillus cereus sphingomyelinase (bSMase) increases the overall ceramide concentration in the plasma membrane, the quantity of CRPs, and their size. Simultaneously, the ceramide concentration in CRPs increases approximately twofold.

Disease and Carrier Isolates of Neisseria meningitidis Cause G1 Cell Cycle Arrest in Human Epithelial Cells.

Microbial pathogens have developed several mechanisms to modulate and interfere with host cell cycle progression. In this study, we analyzed the effect of the human pathogen Neisseria meningitidis on the cell cycle of epithelial cells. Two pathogenic isolates, as well as two carrier isolates, were tested for their ability to adhere to and invade into the epithelial cell lines Detroit 562 and NP69 and to modulate the cell cycle. We found that all isolates adhered equally well to both Detroit 562 and NP69 cells, whereas the carrier isolates were significantly less invasive. Using propidium iodide staining and 5-ethynyl-2'-deoxyuridine pulse-labeling, we provide evidence that meningococcal infection arrested cells in the G1 phase of the cell cycle at 24 h postinfection. In parallel, a significant decrease of cells in the S phase was observed. Interestingly, G1-phase arrest was only induced after infection with live bacteria but not with heat-killed bacteria. By Western blotting we demonstrate that bacterial infection resulted in a decreased protein level of the cell cycle regulator cyclin D1, whereas cyclin E expression levels were increased. Furthermore, N. meningitidis infection induced an accumulation of the cyclin-dependent kinase inhibitor (CKI) p21(WAF1/CIP1) that was accompanied by a redistribution of this CKI to the cell nucleus, as shown by immunofluorescence analysis. Moreover, the p27(CIP1) CKI was redistributed and showed punctate foci in infected cells. In summary, we present data that N. meningitidis can interfere with the processes of host cell cycle regulation.

A putative regulatory genetic locus modulates virulence in the pathogen Leptospira interrogans.

Limited research has been conducted on the role of transcriptional regulators in relation to virulence in Leptospira interrogans, the etiological agent of leptospirosis. Here, we identify an L. interrogans locus that encodes a sensor protein, an anti-sigma factor antagonist, and two genes encoding proteins of unknown function. Transposon insertion into the gene encoding the sensor protein led to dampened transcription of the other 3 genes in this locus. This lb139 insertion mutant (the lb139(-) mutant) displayed attenuated virulence in the hamster model of infection and reduced motility in vitro. Whole-transcriptome analyses using RNA sequencing revealed the downregulation of 115 genes and the upregulation of 28 genes, with an overrepresentation of gene products functioning in motility and signal transduction and numerous gene products with unknown functions, predicted to be localized to the extracellular space. Another significant finding encompassed suppressed expression of the majority of the genes previously demonstrated to be upregulated at physiological osmolarity, including the sphingomyelinase C precursor Sph2 and LigB. We provide insight into a possible requirement for transcriptional regulation as it relates to leptospiral virulence and suggest various biological processes that are affected due to the loss of native expression of this genetic locus.

Risk factors and predictors of severe leptospirosis in New Caledonia.

BACKGROUND: Leptospirosis is a major public health concern in New Caledonia (NC) and in other tropical countries. Severe manifestations of the disease are estimated to occur in 5-15% of all human infections worldwide and factors associated with these forms are poorly understood. Our objectives were to identify risk factors and predictors of severe forms of leptospirosis in adults. METHODS AND FINDINGS: We conducted a retrospective case-control study of inpatients with laboratory-confirmed leptospirosis who were admitted to two public hospitals in NC in 2008-2011. Cases were patients with fatal or severe leptospirosis, as determined by clinical criteria. This approach was meant to be pragmatic and to reflect the routine medical management of patients. Controls were defined as patients hospitalized for milder leptospirosis. Risk and prognostic factors were identified by multivariate logistic regression. Among the 176 patients enrolled in the study, 71 had criteria of severity including 10 deaths (Case Fatality Rate = 14.1%). Three risk factors were independently associated with severe leptospirosis: current cigarette smoking (OR = 2.94 [CI 1.45-5.96]); delays >2 days between the onset of symptoms and the initiation of antibiotherapy (OR = 2.78 [CI 1.31-5.91]); and Leptospira interrogans serogroup Icterohaemorrhagiae as the infecting strain (OR = 2.79 [CI 1.26-6.18]). The following post-admission laboratory results correlated with poor prognoses: platelet count ≤50,000/µL (OR = 6.36 [CI 1.79-22.62]), serum creatinine >200 mM (OR = 5.86 [CI 1.61-21.27]), serum lactate >2.5 mM (OR = 5.14 [CI 1.57-16.87]), serum amylase >250 UI/L (OR = 4.66 [CI 1.39-15.69]) and leptospiremia >1000 leptospires/mL (OR = 4.31 [CI 1.17-15.92]). CONCLUSIONS: To assess the risk of developing severe leptospirosis, our study illustrates the benefit for clinicians to have: i) the identification of the infective strain, ii) a critical threshold of qPCR-determined leptospiremia and iii) early laboratory results. In New Caledonia, preventative measures should focus on early presumptive antibacterial therapy and on rodent (reservoir of Icterohaemorrhagiae serogroup) control.

Differential in vivo gene expression of major Leptospira proteins in resistant or susceptible animal models.

Transcripts of Leptospira 16S rRNA, FlaB, LigB, LipL21, LipL32, LipL36, LipL41, and OmpL37 were quantified in the blood of susceptible (hamsters) and resistant (mice) animal models of leptospirosis. We first validated adequate reference genes and then evaluated expression patterns in vivo compared to in vitro cultures. LipL32 expression was downregulated in vivo and differentially regulated in resistant and susceptible animals. FlaB expression was also repressed in mice but not in hamsters. In contrast, LigB and OmpL37 were upregulated in vivo. Thus, we demonstrated that a virulent strain of Leptospira differentially adapts its gene expression in the blood of infected animals.

Rodent abundance dynamics and leptospirosis carriage in an area of hyper-endemicity in New Caledonia.

BACKGROUND: Widespread but particularly incident in the tropics, leptospirosis is transmitted to humans directly or indirectly by virtually any Mammal species. However, rodents are recognized as the most important reservoir. In endemic regions, seasonal outbreaks are observed during hot rainy periods. In such regions, hot spots can be evidenced, where leptospirosis is "hyper-endemic", its incidence reaching 500 annual cases per 100,000. A better knowledge of how rodent populations and their Leptospira prevalence respond to seasonal and meteorological fluctuations might help implement relevant control measures. METHODOLOGY/PRINCIPAL FINDINGS: In two tribes in New Caledonia with hyper-endemic leptospirosis, rodent abundance and Leptospira prevalence was studied twice a year, in hot and cool seasons for two consecutive years. Highly contrasted meteorological situations, particularly rainfall intensities, were noted between the two hot seasons studied. Our results show that during a hot and rainy period, both the rodent populations and their Leptospira carriage were higher. This pattern was more salient in commensal rodents than in the sylvatic rats. CONCLUSIONS/SIGNIFICANCE: The dynamics of rodents and their Leptospira carriage changed during the survey, probably under the influence of meteorology. Rodents were both more numerous and more frequently carrying (therefore disseminating) leptospires during a hot rainy period, also corresponding to a flooding period with higher risks of human exposure to waters and watered soils. The outbreaks of leptospirosis in hyper-endemic areas could arise from meteorological conditions leading to both an increased risk of exposure of humans and an increased volume of the rodent reservoir. Rodent control measures would therefore be most effective during cool and dry seasons, when rodent populations and leptospirosis incidence are low.