Clostridium difficile ribotypes in humans and animals in Brazil

Clostridium difficile is an emerging enteropathogen responsible for pseudomembranous colitis in humans and diarrhoea in several domestic and wild animal species. Despite its known importance, there are few studies aboutC. difficile polymerase chain reaction (PCR) ribotypes in Brazil and the actual knowledge is restricted to studies on human isolates. The aim of the study was therefore to compare C. difficileribotypes isolated from humans and animals in Brazil. Seventy-six C. difficile strains isolated from humans (n = 25), dogs (n = 23), piglets (n = 12), foals (n = 7), calves (n = 7), one cat, and one manned wolf were distributed into 24 different PCR ribotypes. Among toxigenic strains, PCR ribotypes 014/020 and 106 were the most common, accounting for 14 (18.4%) and eight (10.5%) samples, respectively. Fourteen different PCR ribotypes were detected among human isolates, nine of them have also been identified in at least one animal species. PCR ribotype 027 was not detected, whereas 078 were found only in foals. This data suggests a high diversity of PCR ribotypes in humans and animals in Brazil and support the discussion of C. difficile as a zoonotic pathogen.     

Clostridium difficile Toxin B Causes Epithelial Cell Necrosis through an Autoprocessing-Independent Mechanism

Clostridium difficile is the most common cause of antibiotic-associated nosocomial infection in the United States. C. difficile secretes two homologous toxins, TcdA and TcdB, which are responsible for the symptoms of C. difficile associated disease. The mechanism of toxin action includes an autoprocessing event where a cysteine protease domain (CPD) releases a glucosyltransferase domain (GTD) into the cytosol. The GTD acts to modify and inactivate Rho-family GTPases. The presumed importance of autoprocessing in toxicity, and the apparent specificity of the CPD active site make it, potentially, an attractive target for small molecule drug discovery. In the course of exploring this potential, we have discovered that both wild-type TcdB and TcdB mutants with impaired autoprocessing or glucosyltransferase activities are able to induce rapid, necrotic cell death in HeLa and Caco-2 epithelial cell lines. The concentrations required to induce this phenotype correlate with pathology in a porcine colonic explant model of epithelial damage. We conclude that autoprocessing and GTD release is not required for epithelial cell necrosis and that targeting the autoprocessing activity of TcdB for the development of novel therapeutics will not prevent the colonic tissue damage that occurs in C. difficile – associated disease.     

Caenorhabditis elegans Bacterial Pathogen Resistant bus-4 Mutants Produce Altered Mucins

Caenorabditis elegans bus-4 glycosyltransferase mutants are resistant to infection by Microbacterium nematophilum, Yersinia pestis and Yersinia pseudotuberculosis and have altered susceptibility to two Leucobacter species Verde1 and Verde2. Our objective in this study was to define the glycosylation changes leading to this phenotype to better understand how these changes lead to pathogen resistance. We performed MALDI-TOF MS, tandem MS and GC/MS experiments to reveal fine structural detail for the bus-4 N- and O-glycan pools. We observed dramatic changes in O-glycans and moderate ones in N-glycan pools compared to the parent strain. Ce core-I glycans, the nematode''s mucin glycan equivalent, were doubled in abundance, halved in charge and bore shifts in terminal substitutions. The fucosyl O-glycans, Ce core-II and neutral fucosyl forms, were also increased in abundance as were fucosyl N-glycans. Quantitative expression analysis revealed that two mucins, let-653 and osm-8, were upregulated nearly 40 fold and also revealed was a dramatic increase in GDP-Man 4,6 dehydratease expression. We performed detailed lectin binding studies that showed changes in glycoconjugates in the surface coat, cuticle surface and intestine. The combined changes in cell surface glycoconjugate distribution, increased abundance and altered properties of mucin provide an environment where likely the above pathogens are not exposed to normal glycoconjugate dependent cues leading to barriers to these bacterial infections.     

Chenodeoxycholate Is an Inhibitor of Clostridium difficile Spore Germination

Some cholate derivatives that are normal components of bile can act with glycine to induce the germination of Clostridium difficile spores, but at least one bile component, chenodeoxycholate, does not induce germination. Here we show that chenodeoxycholate inhibits the germination of C. difficile spores in response to cholate and taurocholate.The anaerobic human pathogen Clostridium difficile must be in the spore form to survive for extended periods of time outside the colonic environment (6). Spores are also the form of the organism most likely to be ingested by a host. To cause disease, however, C. difficile spores must germinate in the gastrointestinal tract and reach the anaerobic environment of the colon, where they can grow out as vegetative bacteria (2). The vegetative form produces two toxins that damage the colonic epithelium and lead to C. difficile-associated diseases, such as diarrhea, pseudomembranous colitis, and toxic megacolon (4, 15). Extending the work of Wilson and colleagues (17, 18), we have shown that certain bile salts and glycine act as cogerminants for C. difficile spores (13). Primary bile salts produced by the liver are composed mainly of cholate (CA) and chenodeoxycholate (CDCA) derivatives conjugated with either taurine or glycine (11). Since CA derivatives are found in the relatively aerobic proximal ileum (9), we reasoned that C. difficile might benefit if its germination were inhibited until the spores reached the anaerobic environment of the large intestine.Inhibitors of germination are typically structurally similar to the germinant whose activities they inhibit. For example, l-alanine-mediated germination of Bacillus subtilis spores is inhibited by d-alanine (16) and 6-thioguanosine inhibits inosine-mediated germination in Bacillus anthracis (1, 16). Since CA and CDCA are structurally similar but CA induces the germination of C. difficile spores (13) and CDCA does not, we tested whether CDCA could act as an inhibitor of germination. C. difficile strain CD196 (10) spores were produced and their concentration determined as described previously (13). After the vegetative bacteria were killed by incubation at 60°C for 20 min, spores were incubated in water containing various concentrations and combinations of bile salts for 10 min. Here we took advantage of the finding by Wilson et al. that C. difficile spores germinate very inefficiently on rich medium plates lacking bile salts (18) unless they are preincubated with bile salts (13, 17). After incubation, spores were serially diluted and plated on brain heart infusion agar supplemented with 5 g yeast extract per liter-0.1% l-cysteine (BHIS) (Difco) in the absence of any bile salt (BHIS contains enough glycine to act as a cogerminant). After overnight growth at 37°C, colonies were enumerated. As a positive or negative control, spores were plated on BHIS containing 0.1% taurocholate (TA) [BHIS(TA)] or on BHIS agar alone, respectively. Preincubation of spores with 0.1% TA in water resulted in the recovery of approximately 0.5% of the total number of spores as colonies compared to results for spores plated directly on BHIS(TA). These results are similar to our previous findings that spores germinate and grow out as colonies more efficiently on agar medium containing TA (13). As reported previously, 0.1% CDCA poorly stimulated colony formation by C. difficile spores (13), yielding only 0.006% spore recovery (Fig. ​(Fig.1A).1A). When TA and CDCA were combined, both at 0.1%, colony formation by C. difficile spores was reduced 21-fold to 0.024% compared to the effect of TA alone. This result indicates that CDCA blocks TA-stimulated colony formation and suggests that CDCA may be an inhibitor of C. difficile spore germination. Increasing the ratio of TA to CDCA suppressed the inhibitory effect of CDCA, increasing colony formation by spores (Fig. ​(Fig.1A).1A). Thus, CDCA seems to block colony formation by competing with TA.Open in a separate windowFIG. 1.CDCA inhibits colony formation by C. difficile spores in response to TA and CA. (A) Spores were prepared and preincubated with TA or CDCA or both in water for 10 min before serial dilution and plating on BHIS agar in the absence of TA. Spores plated on BHIS(TA) served as a positive control for 100% colony formation (CFU). Based on comparisons of total spore counts obtained by microscopy and by colony formation on BHIS(TA) plates, the efficiency of colony formation on BHIS(TA) was estimated at 83%. (B) Spores were prepared as described for panel A and exposed to CA or CDCA or both. Values shown are the averages for three independent experiments, and error bars represent one standard deviation from the mean.CA and other cholate derivatives (e.g., TA, glycocholate, and deoxycholate [DCA]) are also germinants for C. difficile spores (13, 17). To test if CDCA prevents colony formation induced by CA, spores were preexposed to 0.1% CA with and without CDCA. Exposure to CA alone resulted in approximately 1% spore recovery, whereas exposure to 0.1% CA and 0.1% CDCA together led to a decrease in colony formation to 0.075% (Fig. ​(Fig.1B).1B). The effect of CDCA on CA-mediated colony formation was relieved by increasing the concentration of CA to 1.0%, raising colony formation to 2.6% (Fig. ​(Fig.1B).1B). These results indicate that CDCA blocks colony formation induced by CA, as well as that induced by TA, and may be an inhibitor of germination by C. difficile spores that acts competitively in both cases.Spore germination per se is classically measured as a decrease in the optical density of a spore suspension occurring concomitantly with a release of Ca²⁺-dipicolinate from the spore core, rehydration of the core, and degradation of the cortex (8, 12). As determined by this assay, TA is the most effective bile salt for inducing rapid germination (13). To test if CDCA is an inhibitor of germination as opposed to an inhibitor of some other step between germination and colony formation, spores were purified as described previously (13). Spores did not germinate in BHIS medium alone or when this medium was supplemented with 0.1% CDCA (Fig. ​(Fig.2).2). When C. difficile spores were suspended in BHIS containing 0.1% TA, the optical density of the suspension rapidly decreased, indicating that the spores were germinating. However, the optical density of the spores suspended in BHIS with 0.1% TA plus 0.1% CDCA did not decrease over time, indicating that CDCA inhibited TA-mediated germination (Fig. ​(Fig.2).2). When the concentration of TA was increased from 0.1% to 1.0% in the presence of 0.1% CDCA, spores were able to germinate (Fig. ​(Fig.2).2). After overnight incubation in BHIS with 0.1% TA plus 0.1% CDCA, 84% of the spores remained phase bright, while only 11% of spores remained phase bright in BHIS with 1.0% TA plus 0.1% CDCA, indicating that CDCA blocks germination at a very early step. Thus, CDCA is an inhibitor of germination by C. difficile spores that functions by competing with TA and possibly with CA.Open in a separate windowFIG. 2.CDCA inhibits germination of Clostridium difficile spores. Spores were prepared as described previously (13). C. difficile spores were suspended in BHIS alone (•), BHIS plus 0.1% CDCA (▾), BHIS plus 0.1% TA (⧫), BHIS plus 0.1% TA-0.1% CDCA (▪), or BHIS plus 1.0% TA-0.1% CDCA (▴). The ratio of the OD₆₀₀ at the various time points to the OD₆₀₀ at time zero is plotted versus time. Data points are the averages of three independent experiments, and error bars represent one standard deviation from the mean.We previously suggested a role for bile salts in determining the ability of C. difficile to colonize and cause disease (13). In this model, germination of C. difficile spores depends on interaction with glycine and certain bile salts. We show here that the primary bile salt CDCA inhibits germination of C. difficile spores. As mentioned above, germination inhibitors are commonly structurally related to the germinant they inhibit. The structures of CA derivatives and CDCA derivatives are very similar; they differ only insofar as CDCA lacks the 12α hydroxyl group (11).CDCA and CA derivatives are present in approximately equal concentrations in the cecum (5). Under such conditions, CDCA would compete with CA derivatives for binding to putative germinant receptors on C. difficile spores. Mekhjian and colleagues measured the colonic absorption rates of CDCA, CA, and DCA that were introduced into the cecum and collected at the distal colon (7). They found that CDCA was absorbed by the colon at 10 times the rate for CA (7). Thus, when spores reach the distal large intestine, they encounter a decreased ratio of CDCA to CA. Such a change in ratio might allow CA derivatives to act as effective germinants. Thus, C. difficile spores would not be expected to germinate until they reach the colon, which also provides the anaerobic environment required for C. difficile growth.The colonic microflora, which is known to protect the host against C. difficile infection, plays a significant role in the metabolism of bile salts (3, 11). Many different species express on their cell surfaces bile salt hydrolases that serve to remove the conjugated tauryl or glycyl groups from primary bile salts (11). After deconjugation, CA and CDCA are further metabolized by a small percentage of the bacterial species in the cecum to the secondary bile salts deoxycholate and lithocholate, respectively (11, 14). Deoxycholate is an inhibitor of C. difficile growth (13, 17). CDCA inhibits both germination and growth (13). The use of CDCA either as prophylaxis or as a therapy for C. difficile-associated disease might be helpful for patients who are undergoing antibiotic regimens or who are colonized by this bacterium. For example, when an antibiotic that is known to be associated with an increased risk of inciting C. difficile-associated disease is administered, the coadministration of CDCA might protect that individual from colonization by C. difficile through inhibiting spore germination. Alternatively, administering CDCA to individuals who are already being given vancomycin or metronidazole for C. difficile-associated disease may have the benefit of preventing spore germination and further vegetative growth (13) after antibiotic therapy is stopped. This strategy may reduce the already significant risk of a relapse.     

Clostridium difficile Modulates Host Innate Immunity via Toxin-Independent and Dependent Mechanism(s)

Clostridium difficile infection (CDI) is the leading cause of hospital and community-acquired antibiotic-associated diarrhoea and currently represents a significant health burden. Although the role and contribution of C. difficile toxins to disease pathogenesis is being increasingly understood, at present other facets of C. difficile-host interactions, in particular, bacterial-driven effects on host immunity remain less studied. Using an ex-vivo model of infection, we report that the human gastrointestinal mucosa elicits a rapid and significant cytokine response to C. difficile. Marked increase in IFN-γ with modest increase in IL-22 and IL-17A was noted. Significant increase in IL-8 suggested potential for neutrophil influx while presence of IL-12, IL-23, IL-1β and IL-6 was indicative of a cytokine milieu that may modulate subsequent T cell immunity. Majority of C. difficile-driven effects on murine bone-marrow-derived dendritic cell (BMDC) activation were toxin-independent; the toxins were however responsible for BMDC inflammasome activation. In contrast, human monocyte-derived DCs (mDCs) released IL-1β even in the absence of toxins suggesting host-specific mediation. Infected DC-T cell crosstalk revealed the ability of {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291 and 630 WT strains to elicit a differential DC IL-12 family cytokine milieu which culminated in significantly greater Th1 immunity in response to {"type":"entrez-nucleotide","attrs":{"text":"R20291","term_id":"774925","term_text":"R20291"}}R20291. Interestingly, both strains induced a similar Th17 response. Elicitation of mucosal IFN-γ/IL-17A and Th1/Th17 immunity to C. difficile indicates a central role for this dual cytokine axis in establishing antimicrobial immunity to CDI.     

Clostridium difficile Toxin CDT Induces Formation of Microtubule-Based Protrusions and Increases Adherence of Bacteria

Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis by production of the Rho GTPase-glucosylating toxins A and B. Recently emerging hypervirulent Clostridium difficile strains additionally produce the binary ADP-ribosyltransferase toxin CDT (Clostridium difficile transferase), which ADP-ribosylates actin and inhibits actin polymerization. Thus far, the role of CDT as a virulence factor is not understood. Here we report by using time-lapse- and immunofluorescence microscopy that CDT and other binary actin-ADP-ribosylating toxins, including Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin, induce redistribution of microtubules and formation of long (up to >150 µm) microtubule-based protrusions at the surface of intestinal epithelial cells. The toxins increase the length of decoration of microtubule plus-ends by EB1/3, CLIP-170 and CLIP-115 proteins and cause redistribution of the capture proteins CLASP2 and ACF7 from microtubules at the cell cortex into the cell interior. The CDT-induced microtubule protrusions form a dense meshwork at the cell surface, which wrap and embed bacterial cells, thereby largely increasing the adherence of Clostridia. The study describes a novel type of microtubule structure caused by less efficient microtubule capture and offers a new perspective for the pathogenetic role of CDT and other binary actin-ADP-ribosylating toxins in host–pathogen interactions.     

Butanol Production from Crystalline Cellulose by Cocultured Clostridium thermocellum and Clostridium saccharoperbutylacetonicum N1-4

We investigated butanol production from crystalline cellulose by cocultured cellulolytic Clostridium thermocellum and the butanol-producing strain, Clostridium saccharoperbutylacetonicum (strain N1-4). Butanol was produced from Avicel cellulose after it was incubated with C. thermocellum for at least 24 h at 60°C before the addition of strain N1-4. Butanol produced by strain N1-4 on 4% Avicel cellulose peaked (7.9 g/liter) after 9 days of incubation at 30°C, and acetone was undetectable in this coculture system. Less butanol was produced by cocultured Clostridium acetobutylicum and Clostridium beijerinckii than by strain N1-4, indicating that strain N1-4 was the optimal strain for producing butanol from crystalline cellulose in this coculture system.     

Wolbachia-Associated Bacterial Protection in the Mosquito Aedes aegypti

Background

Wolbachia infections confer protection for their insect hosts against a range of pathogens including bacteria, viruses, nematodes and the malaria parasite. A single mechanism that might explain this broad-based pathogen protection is immune priming, in which the presence of the symbiont upregulates the basal immune response, preparing the insect to defend against subsequent pathogen infection. A study that compared natural Wolbachia infections in Drosophila melanogaster with the mosquito vector Aedes aegypti artificially transinfected with the same strains has suggested that innate immune priming may only occur in recent host-Wolbachia associations. This same study also revealed that while immune priming may play a role in viral protection it cannot explain the entirety of the effect.

Methodology/Findings

Here we assess whether the level of innate immune priming induced by different Wolbachia strains in A. aegypti is correlated with the degree of protection conferred against bacterial pathogens. We show that Wolbachia strains wMel and wMelPop, currently being tested for field release for dengue biocontrol, differ in their protective abilities. The wMelPop strain provides stronger, more broad-based protection than wMel, and this is likely explained by both the higher induction of immune gene expression and the strain-specific activation of particular genes. We also show that Wolbachia densities themselves decline during pathogen infection, likely as a result of the immune induction.

Conclusions/Significance

This work shows a correlation between innate immune priming and bacterial protection phenotypes. The ability of the Toll pathway, melanisation and antimicrobial peptides to enhance viral protection or to provide the basis of malaria protection should be further explored in the context of this two-strain comparison. This work raises the questions of whether Wolbachia may improve the ability of wild mosquitoes to survive pathogen infection or alter the natural composition of gut flora, and thus have broader consequences for host fitness.     

Clostridium difficile 027/BI/NAP1 Encodes a Hypertoxic and Antigenically Variable Form of TcdB

The Clostridium difficile exotoxin, TcdB, which is a major virulence factor, varies between strains of this pathogen. Herein, we show that TcdB from the epidemic BI/NAP1/027 strain of C. difficile is more lethal, causes more extensive brain hemorrhage, and is antigenically variable from TcdB produced by previously studied strains of this pathogen (TcdB₀₀₃). In mouse intoxication assays, TcdB from a ribotype 027 strain (TcdB₀₂₇) was at least four fold more lethal than TcdB₀₀₃. TcdB₀₂₇ caused a previously undescribed brain hemorrhage in mice and this correlated with a heightened sensitivity of brain microvascular endothelial cells to the toxin. TcdB₀₀₃ and TcdB₀₂₇ also differed in their antigenic profiles and did not share cross-neutralizing epitopes in a major immunogenic region of the protein. Solid phase humoral mapping of epitopes in the carboxy-terminal domains (CTD) of TcdB₀₂₇ and TcdB₀₀₃ identified 11 reactive epitopes that varied between the two forms of TcdB, and 13 epitopes that were shared or overlapping. Despite the epitope differences and absence of neutralizing epitopes in the CTD of TcdB₀₂₇, a toxoid form of this toxin primed a strong protective response. These findings indicate TcdB₀₂₇ is a more potent toxin than TcdB₀₀₃ as measured by lethality assays and pathology, moreover the sequence differences between the two forms of TcdB alter antigenic epitopes and reduce cross-neutralization by antibodies targeting the CTD.     

In Vivo Yeast Cell Morphogenesis Is Regulated by a p21-Activated Kinase in the Human Pathogen Penicillium marneffei

Pathogens have developed diverse strategies to infect their hosts and evade the host defense systems. Many pathogens reside within host phagocytic cells, thus evading much of the host immune system. For dimorphic fungal pathogens which grow in a multicellular hyphal form, a central attribute which facilitates growth inside host cells without rapid killing is the capacity to switch from the hyphal growth form to a unicellular yeast form. Blocking this transition abolishes or severely reduces pathogenicity. Host body temperature (37°C) is the most common inducer of the hyphal to yeast transition in vitro for many dimorphic fungi, and it is often assumed that this is the inducer in vivo. This work describes the identification and analysis of a new pathway involved in sensing the environment inside a host cell by a dimorphic fungal pathogen, Penicillium marneffei. The pakB gene, encoding a p21-activated kinase, defines this pathway and operates independently of known effectors in P. marneffei. Expression of pakB is upregulated in P. marneffei yeast cells isolated from macrophages but absent from in vitro cultured yeast cells produced at 37°C. Deletion of pakB leads to a failure to produce yeast cells inside macrophages but no effect in vitro at 37°C. Loss of pakB also leads to the inappropriate production of yeast cells at 25°C in vitro, and the mechanism underlying this requires the activity of the central regulator of asexual development. The data shows that this new pathway is central to eliciting the appropriate morphogenetic response by the pathogen to the host environment independently of the common temperature signal, thus clearly separating the temperature- and intracellular-dependent signaling systems.     

Clostridium difficile Cell Attachment Is Modified by Environmental Factors

Adherence of Clostridium difficile to Vero cells under anaerobic conditions was increased by a high sodium concentration, calcium-rich medium, an acidic pH, and iron starvation. The level of adhesion of nontoxigenic strains was comparable to that of toxigenic strains. Depending on the bacterial culture conditions, Vero cells could bind to one, two, or three bacterial surface proteins with molecular masses of 70, 50, and 40 kDa.     

De novo Genome Assembly of the Fungal Plant Pathogen Pyrenophora semeniperda

Pyrenophora semeniperda (anamorph Drechslera campulata) is a necrotrophic fungal seed pathogen that has a wide host range within the Poaceae. One of its hosts is cheatgrass (Bromus tectorum), a species exotic to the United States that has invaded natural ecosystems of the Intermountain West. As a natural pathogen of cheatgrass, P. semeniperda has potential as a biocontrol agent due to its effectiveness at killing seeds within the seed bank; however, few genetic resources exist for the fungus. Here, the genome of P. semeniperda isolate assembled from sequence reads of 454 pyrosequencing is presented. The total assembly is 32.5 Mb and includes 11,453 gene models encoding putative proteins larger than 24 amino acids. The models represent a variety of putative genes that are involved in pathogenic pathways typically found in necrotrophic fungi. In addition, extensive rearrangements, including inter- and intrachromosomal rearrangements, were found when the P. semeniperda genome was compared to P. tritici-repentis, a related fungal species.     

The Spore Differentiation Pathway in the Enteric Pathogen Clostridium difficile

Endosporulation is an ancient bacterial developmental program that culminates with the differentiation of a highly resistant endospore. In the model organism Bacillus subtilis, gene expression in the forespore and in the mother cell, the two cells that participate in endospore development, is governed by cell type-specific RNA polymerase sigma subunits. σ^F in the forespore, and σ^E in the mother cell control early stages of development and are replaced, at later stages, by σ^G and σ^K, respectively. Starting with σ^F, the activation of the sigma factors is sequential, requires the preceding factor, and involves cell-cell signaling pathways that operate at key morphological stages. Here, we have studied the function and regulation of the sporulation sigma factors in the intestinal pathogen Clostridium difficile, an obligate anaerobe in which the endospores are central to the infectious cycle. The morphological characterization of mutants for the sporulation sigma factors, in parallel with use of a fluorescence reporter for single cell analysis of gene expression, unraveled important deviations from the B. subtilis paradigm. While the main periods of activity of the sigma factors are conserved, we show that the activity of σ^E is partially independent of σ^F, that σ^G activity is not dependent on σ^E, and that the activity of σ^K does not require σ^G. We also show that σ^K is not strictly required for heat resistant spore formation. In all, our results indicate reduced temporal segregation between the activities of the early and late sigma factors, and reduced requirement for the σ^F-to-σ^E, σ^E-to-σ^G, and σ^G-to-σ^K cell-cell signaling pathways. Nevertheless, our results support the view that the top level of the endosporulation network is conserved in evolution, with the sigma factors acting as the key regulators of the pathway, established some 2.5 billion years ago upon its emergence at the base of the Firmicutes Phylum.     

CRY2 Is Associated with Depression

Background

Abnormalities in the circadian clockwork often characterize patients with major depressive and bipolar disorders. Circadian clock genes are targets of interest in these patients. CRY2 is a circadian gene that participates in regulation of the evening oscillator. This is of interest in mood disorders where a lack of switch from evening to morning oscillators has been postulated.

Principal Findings

We observed a marked diurnal variation in human CRY2 mRNA levels from peripheral blood mononuclear cells and a significant up-regulation (P = 0.020) following one-night total sleep deprivation, a known antidepressant. In depressed bipolar patients, levels of CRY2 mRNA were decreased (P = 0.029) and a complete lack of increase was observed following sleep deprivation. To investigate a possible genetic contribution, we undertook SNP genotyping of the CRY2 gene in two independent population-based samples from Sweden (118 cases and 1011 controls) and Finland (86 cases and 1096 controls). The CRY2 gene was significantly associated with winter depression in both samples (haplotype analysis in Swedish and Finnish samples: OR = 1.8, P = 0.0059 and OR = 1.8, P = 0.00044, respectively).

Conclusions

We propose that a CRY2 locus is associated with vulnerability for depression, and that mechanisms of action involve dysregulation of CRY2 expression.     

Multiparameter Viability Assay for Stress Profiling Applied to the Food Pathogen Listeria monocytogenes F2365

A novel generic approach for stress profiling was applied to Listeria monocytogenes strain F2365. This food-borne pathogen was exposed to gradients of five different stresses of increasing intensity, typically ranging from moderate to lethal conditions. The stress factors included heat, acidic pH, a detergent disinfectant, an oxidant, and hyperosmotic conditions. In addition to CFU counts and lag time, five different molecular viability parameters were measured by fluorescence-based assays, including membrane integrity, membrane potential, esterase activity, redox activity, and intracellular pH stability. The last was measured by our recently invented real-time viability assay. Exposure to all stresses resulted in clear dose-response relationships for all viability parameters with the exception of hyperosmotic conditions. A statistical analysis showed strong correlations for (i) the growth parameters plate counts and lag times, (ii) the enzyme-associated functions redox and esterase activity, and (iii) the membrane-associated pH stability and membrane integrity. Results indicated a pronounced difference in the susceptibilities of the measured parameters depending on the stress factor applied. However, at relatively high stress intensities, all of the viability parameters became affected independent of the stress factor. Applications of the approach presented here include studies on the mechanism of action of unknown compounds with biocidal activity and a comparative analysis of the severities of the impact of stress conditions of interest. It appears that a meaningful evaluation of the impact of mild stress conditions can be obtained only through measurement of multiple viability parameters.     

Glomus fasciculatum,a Weak Pathogen of Heterodera glycines

The occurrence ofchlamydospores of Glomus fasciculatum (Gf) within cysts of the soybean cyst nematode, Heterodera glycines, and the effects of vesicular-arbuscular mycorrhizae on nematode population dynamics and soybean (Glycine max) plant growth were investigated. Chlamydospores occupied 1-24% of cysts recovered from field soil samples. Hyphae of Missouri isolate Gfl penetrated the female nematode cuticle shortly after she ruptured the root epidermis. Convoluted hyphae filled infected eggs, and sporogenesis occurred within infected eggs. G. microcarpum, G. mosseae, and two isolates of Gf were inoculated with H. glycines on plants of ''Essex'' soybeans. Each of the two Gf isolates infected about 1% of the nematode eggs in experimental pot cuhures. The Gfl isolate decreased the number of first-generation adult females 26%, compared with the nonmycorrhizal control. The total numbers of first-generation plus second-generation adult females were similar for both Gf isolates and 29-41% greater than the nonmycorrhizal control. Soybean plants with Gf and H. glycines produced more biomass than did nonmycorrhizal plants with nematodes, but only Gfl delayed leaf senescence.     

MiR-101 Is Involved in Human Breast Carcinogenesis by Targeting Stathmin1

Background

MicroRNA-101 (miR-101) expression is negatively associated with tumor growth and blood vessel formation in several solid epithelial cancers. However, the role of miR-101 in human breast cancer remains elusive.

Results

MiR-101 was significantly decreased in different subtypes of human breast cancer tissues compared with that in adjacent normal breast tissues (P<0.01). Up-regulation of miR-101 inhibited cell proliferation, migration and invasion, and promoted cell apoptosis in ER alpha-positive and ER alpha-negative breast cancer cells and normal breast cells. Down-regulation of miR-101 displayed opposite effects on cell growth and metastasis. Further investigation revealed a significant inverse correlation between the expression of miR-101 and Stathmin1 (Stmn1), and miR-101 could bind to the 3′-untranslated region (UTR) of Stmn1 to inhibit Stmn1 translation. The inhibition of cell growth and metastasis induced by up-regulation of miR-101 was partially restored by overexpresson of Stmn1. Knockdown of Stmn1 attenuates the down-regulation of miR-101-mediated enhancement of cell growth and metastasis. More importantly, in vivo analysis found that Stmn1 mRNA and protein level in different subtypes of human breast cancer tissues, contrary to the down-regulation of miR-101, were significantly elevated.

Conclusions

This study demonstrates that down-regulation of miR-101 in different subtypes of human breast cancer tissues is linked to the increase of cellular proliferation and invasiveness via targeting Stmn1, which highlights novel regulatory mechanism in breast cancer and may provide valuable clues for the future clinical diagnosis of breast cancer.     

In Vitro Identification of Novel Plasminogen-Binding Receptors of the Pathogen Leptospira interrogans

Background

Leptospirosis is a multisystem disease caused by pathogenic strains of the genus Leptospira. We have reported that Leptospira are able to bind plasminogen (PLG), to generate active plasmin in the presence of activator, and to degrade purified extracellular matrix fibronectin.

Methodology/Principal Findings

We have now cloned, expressed and purified 14 leptospiral recombinant proteins. The proteins were confirmed to be surface exposed by immunofluorescence microscopy and were evaluated for their ability to bind plasminogen (PLG). We identified eight as PLG-binding proteins, including the major outer membrane protein LipL32, the previously published rLIC12730, rLIC10494, Lp29, Lp49, LipL40 and MPL36, and one novel leptospiral protein, rLIC12238. Bound PLG could be converted to plasmin by the addition of urokinase-type PLG activator (uPA), showing specific proteolytic activity, as assessed by its reaction with the chromogenic plasmin substrate, D-Val-Leu-Lys 4-nitroanilide dihydrochloride. The addition of the lysine analog 6-aminocaproic acid (ACA) inhibited the protein-PLG interaction, thus strongly suggesting the involvement of lysine residues in plasminogen binding. The binding of leptospiral surface proteins to PLG was specific, dose-dependent and saturable. PLG and collagen type IV competed with LipL32 protein for the same binding site, whereas separate binding sites were observed for plasma fibronectin.

Conclusions/Significance

PLG-binding/activation through the proteins/receptors on the surface of Leptospira could help the bacteria to specifically overcome tissue barriers, facilitating its spread throughout the host.     

Prdm6 Is Essential for Cardiovascular Development In Vivo

Members of the PRDM protein family have been shown to play important roles during embryonic development. Previous in vitro and in situ analyses indicated a function of Prdm6 in cells of the vascular system. To reveal physiological functions of Prdm6, we generated conditional Prdm6-deficient mice. Complete deletion of Prdm6 results in embryonic lethality due to cardiovascular defects associated with aberrations in vascular patterning. However, smooth muscle cells could be regularly differentiated from Prdm6-deficient embryonic stem cells and vascular smooth muscle cells were present and proliferated normally in Prdm6-deficient embryos. Conditional deletion of Prdm6 in the smooth muscle cell lineage using a SM22-Cre driver line resulted in perinatal lethality due to hemorrhage in the lungs. We thus identified Prdm6 as a factor that is essential for the physiological control of cardiovascular development.     

Nondigestible Oligosaccharides Enhance Bacterial Colonization Resistance against Clostridium difficile In Vitro

Clostridium difficile is the principal etiologic agent of pseudomembranous colitis and is a major cause of nosocomial antibiotic-associated diarrhea. A limited degree of success in controlling C. difficile infection has been achieved by using probiotics; however, prebiotics can also be used to change bacterial community structure and metabolism in the large gut, although the effects of these carbohydrates on suppression of clostridial pathogens have not been well characterized. The aims of this study were to investigate the bifidogenicity of three nondigestible oligosaccharide (NDO) preparations in normal and antibiotic-treated fecal microbiotas in vitro and their abilities to increase barrier resistance against colonization by C. difficile by using cultural and molecular techniques. Fecal cultures from three healthy volunteers were challenged with a toxigenic strain of C. difficile, and molecular probes were used to monitor growth of the pathogen, together with growth of bifidobacterial and bacteroides populations, over a time course. Evidence of colonization resistance was assessed by determining viable bacterial counts, short-chain fatty acid formation, and cytotoxic activity. Chemostat studies were then performed to determine whether there was a direct correlation between bifidobacteria and C. difficile suppression. NDO were shown to stimulate bifidobacterial growth, and there were concomitant reductions in C. difficile populations. However, in the presence of clindamycin, activity against bifidobacteria was augmented in the presence of NDO, resulting in a further loss of colonization resistance. In the absence of clindamycin, NDO enhanced colonization resistance against C. difficile, although this could not be attributed to bifidobacterium-induced inhibitory phenomena.