Interclonal Variations in the Molecular Karyotype of Trypanosoma cruzi: Chromosome Rearrangements in a Single Cell-Derived Clone of the G Strain

Trypanosoma cruzi comprises a pool of populations which are genetically diverse in terms of DNA content, growth and infectivity. Inter- and intra-strain karyotype heterogeneities have been reported, suggesting that chromosomal rearrangements occurred during the evolution of this parasite. Clone D11 is a single-cell-derived clone of the T. cruzi G strain selected by the minimal dilution method and by infecting Vero cells with metacyclic trypomastigotes. Here we report that the karyotype of clone D11 differs from that of the G strain in both number and size of chromosomal bands. Large chromosomal rearrangement was observed in the chromosomes carrying the tubulin loci. However, most of the chromosome length polymorphisms were of small amplitude, and the absence of one band in clone D11 in relation to its reference position in the G strain could be correlated to the presence of a novel band migrating above or below this position. Despite the presence of chromosomal polymorphism, large syntenic groups were conserved between the isolates. The appearance of new chromosomal bands in clone D11 could be explained by chromosome fusion followed by a chromosome break or interchromosomal exchange of large DNA segments. Our results also suggest that telomeric regions are involved in this process. The variant represented by clone D11 could have been induced by the stress of the cloning procedure or could, as has been suggested for Leishmania infantum, have emerged from a multiclonal, mosaic parasite population submitted to frequent DNA amplification/deletion events, leading to a 'mosaic' structure with different individuals having differently sized versions of the same chromosomes. If this is the case, the variant represented by clone D11 would be better adapted to survive the stress induced by cloning, which includes intracellular development in the mammalian cell. Karyotype polymorphism could be part of the T. cruzi arsenal for responding to environmental pressure. © 2013 Lima et al.

Dissection of the role of pili and type 2 and 3 secretion systems in adherence and biofilm formation of an atypical enteropathogenic escherichia coli strain

Atypical enteropathogenic Escherichia coli (aEPEC) strains are diarrheal pathogens that lack bundle-forming pilus production but possess the virulence-associated locus of enterocyte effacement. aEPEC strain 1551-2 produces localized adherence (LA) on HeLa cells; however, its isogenic intimin (eae) mutant produces a diffuse-adherence (DA) pattern. In this study, we aimed to identify the DA-associated adhesin of the 1551-2 eae mutant. Electron microscopy of 1551-2 identified rigid rod-like pili composed of an 18-kDa protein, which was identified as the major pilin subunit of type 1 pilus (T1P) by mass spectrometry analysis. Deletion of fimA in 1551-2 affected biofilm formation but had no effect on adherence properties. Analysis of secreted proteins in supernatants of this strain identified a 150-kDa protein corresponding to SslE, a type 2 secreted protein that was recently reported to be involved in biofilm formation of rabbit and human EPEC strains. However, neither adherence nor biofilm formation was affected in a 1551-2 sslE mutant. We then investigated the role of the EspA filament associated with the type 3 secretion system (T3SS) in DA by generating a double eae espA mutant. This strain was no longer adherent, strongly suggesting that the T3SS translocon is the DA adhesin. In agreement with these results, specific anti-EspA antibodies blocked adherence of the 1551-2 eae mutant. Our data support a role for intimin in LA, for the T3SS translocon in DA, and for T1P in biofilm formation, all of which may act in concert to facilitate host intestinal colonization by aEPEC strains. ©2013, American Society for Microbiology.

Caracterização do fenótipo comportamental e de linguagem na síndrome de Williams-Beuren

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Cocaine effects on mouse incentive-learning and human addiction are linked to alpha 2 subunit-containing GABA(A) receptors

Because GABA(A) receptors containing alpha 2 subunits are highly represented in areas of the brain, such as nucleus accumbens (NAcc), frontal cortex, and amygdala, regions intimately involved in signaling motivation and reward, we hypothesized that manipulations of this receptor subtype would influence processing of rewards. Voltage-clamp recordings from NAcc medium spiny neurons of mice with alpha 2 gene deletion showed reduced synaptic GABA(A) receptor-mediated responses. Behaviorally, the deletion abolished cocaine`s ability to potentiate behaviors conditioned to rewards (conditioned reinforcement), and to support behavioral sensitization. In mice with a point mutation in the benzodiazepine binding pocket of alpha 2-GABA(A) receptors (alpha 2H101R), GABAergic neurotransmission in medium spiny neurons was identical to that of WT (i.e., the mutation was silent), but importantly, receptor function was now facilitated by the atypical benzodiazepine Ro 15-4513 (ethyl 8-amido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo [1,5-a] [1,4] benzodiazepine-3-carboxylate). In alpha 2H101R, but not WT mice, Ro 15-4513 administered directly into the NAcc-stimulated locomotor activity, and when given systemically and repeatedly, induced behavioral sensitization. These data indicate that activation of alpha 2-GABA(A) receptors (most likely in NAcc) is both necessary and sufficient for behavioral sensitization. Consistent with a role of these receptors in addiction, we found specific markers and haplotypes of the GABRA2 gene to be associated with human cocaine addiction.

RNA Interference of Endochitinases in the Sugarcane Endophyte Trichoderma virens 223 Reduces Its Fitness as a Biocontrol Agent of Pineapple Disease

The sugarcane root endophyte Trichoderma virens 223 holds enormous potential as a sustainable alternative to chemical pesticides in the control of sugarcane diseases. Its efficacy as a biocontrol agent is thought to be associated with its production of chitinase enzymes, including N-acetyl-beta-D-glucosaminidases, chitobiosidases and endochitinases. We used targeted gene deletion and RNA-dependent gene silencing strategies to disrupt N-acetyl-beta-D-glucosaminidase and endochitinase activities of the fungus, and to determine their roles in the biocontrol of soil-borne plant pathogens. The loss of N-acetyl-beta-D-glucosaminidase activities was dispensable for biocontrol of the plurivorous damping-off pathogens Rhizoctonia solani and Sclerotinia sclerotiorum, and of the sugarcane pathogen Ceratocystis paradoxa, the causal agent of pineapple disease. Similarly, suppression of endochitinase activities had no effect on R. solani and S. sclerotiorum disease control, but had a pronounced effect on the ability of T. virens 223 to control pineapple disease. Our work demonstrates a critical requirement for T. virens 223 endochitinase activity in the biocontrol of C. paradoxa sugarcane disease, but not for general antagonism of other soil pathogens. This may reflect its lifestyle as a sugarcane root endophyte.

The Effects of Oxybutynin on Urinary Symptoms in Children with Williams-Beuren Syndrome

Purpose: Williams-Beuren syndrome is a genomic disorder caused by a hemizygous contiguous gene deletion on chromosome 7q11.23. Lower urinary tract symptoms are common in children with Williams-Beuren syndrome. However, there are few data on the management of voiding symptoms in this population. We report our experience using oxybutynin to treat urinary symptoms in children with Williams-Beuren syndrome. Materials and Methods: We prospectively analyzed 42 patients with Williams-Beuren syndrome and significant lower urinary tract symptoms due to detrusor overactivity diagnosed on urodynamics in a 12-week, open-label study. Urological assessment included symptomatic evaluation, the impact of lower urinary tract symptoms on quality of life, frequency-volume chart, urodynamics and urinary tract sonography. After 12 weeks of treatment with 0.6 mg/kg oxybutynin per day given in 3 daily doses, patients were assessed for treatment efficacy and side effects. Results: A total of 17 girls and 19 boys completed medical therapy and were assessed at 12 weeks. Mean +/- SD patient age was 9.2 +/- 4.3 years (range 3 to 18). The most common urinary complaint was urgency, which occurred in 31 patients (86.1%), followed by urge incontinence, which was seen in 29 (80.5%). Compared to baseline, urinary symptoms were substantially improved. The negative impact of storage symptoms on quality of life was significantly decreased from a mean +/- SD of 3.3 +/- 1.7 to 0.5 +/- 0.9 (p <0.001). Mean +/- SD maximum urinary flow improved from 14.2 +/- 15.0 to 20.5 +/- 6.4 ml per second (p <0.001). Conclusions: A total of 12 weeks of therapy with 0.6 mg/kg oxybutynin daily resulted in improvement of lower urinary tract symptoms, quality of life and maximum flow rate in most patients with Williams-Beuren syndrome.

Inflammasome-dependent IFN-γ drives pathogenesis in Streptococcus pneumoniae meningitis

The pathology associated with Streptococcus pneumoniae meningitis results largely from activation of immune-associated pathways. We systematically investigated the production of IFN subtypes, as well as their influence on pathology, in a mouse model of S. pneumoniae meningitis. Despite the occurrence of a mixed IFN type I/II gene signature, no evidence for production or involvement of type I IFNs in disease progression was found. In contrast, type II IFN (IFN-γ) was strongly induced, and IFN-γ(-/-) mice were significantly protected from severe disease. Using intracellular cytokine staining and targeted cell-depletion approaches, NK cells were found to be the dominant source of IFN-γ. Furthermore, production of IFN-γ was found to be dependent upon ASC and IL-18, indicating that an ASC-dependent inflammasome pathway was responsible for mediating IFN-γ induction. The influence of IFN-γ gene deletion on a range of processes known to be involved in bacterial meningitis pathogenesis was examined. Although neutrophil numbers in the brain were similar in infected wild-type and IFN-γ(-/-) mice, both monocyte recruitment and CCL2 production were less in infected IFN-γ(-/-) mice compared with infected wild-type controls. Additionally, gene expression of NO synthase was strongly diminished in infected IFN-γ(-/-) mice compared with infected controls. Finally, bacterial clearance was enhanced in IFN-γ(-/-) mice, although the underlying mechanism remains unclear. Together, these data suggest that inflammasome-dependent IFN-γ contributes via multiple pathways to pathology during S. pneumoniae meningitis.

Assessing The Role Of Multi-protein Complexes In Determining Phenotype

Understanding regulatory mechanisms in complex biological systems is an important challenge, in particular to understand disease mechanisms, and to discover new therapies and drugs. In this paper, we consider the important question of cellular regulation of phenotype. Using single gene deletion data, we address the problem of linking a phenotype to underlying functional roles in the organism and provide a sound computational and statistical paradigm that can be extended to address more complex experimental settings such as multiple deletions. We apply the proposed approaches to publicly available data sets to demonstrate strong evidence for the involvement of multi-protein complexes in the phenotypes studied.

Characterization of the ebp(fm) pilus-encoding operon of Enterococcus faecium and its role in biofilm formation and virulence in a murine model of urinary tract infection.

We recently identified 15 genes encoding putative surface proteins with features of MSCRAMMs and/or pili in the Enterococcus faecium TX0016 (DO) genome, including four predicted pilus-encoding gene clusters; we also demonstrated that one of these, ebpABC(fm), is transcribed as an operon, that its putative major pilus subunit, EbpC(fm) (also called pilB), is polymerized into high molecular weight complexes, and that it is enriched among clinical E. faecium isolates. Here, we created a deletion of the ebpABC(fm) operon in an endocarditis-derived E. faecium strain (TX82) and showed, by a combination of whole-cell ELISA, flow cytometry, immunoblot and immunogold electron microscopy, that this deletion abolished EbpC(fm) expression and eliminated EbpC(fm)-containing pili from the cell surface. However, transcription of the downstream sortase, bps(fm), was not affected. Importantly, the ebpABC(fm) deletion resulted in significantly reduced biofilm formation (p < 0.0001) and initial adherence (p < 0.0001) versus the wild-type; both were restored by complementing ebpABC(fm) in trans, which also restored cell surface expression of EbpC(fm) and pilus production. Furthermore, the deletion mutant was significantly attenuated in two independent mixed infection mouse urinary tract experiments, i.e., outnumbered by the wild-type in kidneys (p = 0.0003 and < 0.0001, respectively) and urinary bladders (p = 0.0003 and = 0.002). In conclusion, we have shown that the ebpABC(fm) locus encodes pili on the E. faecium TX82 cell surface and provide the first evidence that pili of this emerging pathogen are important for its ability to form biofilm and to cause infection in an ascending UTI model.

Contribution of the collagen adhesin Acm to pathogenesis of Enterococcus faecium in experimental endocarditis.

Enterococcus faecium is a multidrug-resistant opportunist causing difficult-to-treat nosocomial infections, including endocarditis, but there are no reports experimentally demonstrating E. faecium virulence determinants. Our previous studies showed that some clinical E. faecium isolates produce a cell wall-anchored collagen adhesin, Acm, and that an isogenic acm deletion mutant of the endocarditis-derived strain TX0082 lost collagen adherence. In this study, we show with a rat endocarditis model that TX0082 Deltaacm::cat is highly attenuated versus wild-type TX0082, both in established (72 h) vegetations (P < 0.0001) and for valve colonization 1 and 3 hours after infection (P or=50-fold reduction relative to an Acm producer) were found in three of these five nonadherent isolates, including the sequenced strain TX0016, by quantitative reverse transcription-PCR, indicating that acm transcription is downregulated in vitro in these isolates. However, examination of TX0016 cells obtained directly from infected rat vegetations by flow cytometry showed that Acm was present on 40% of cells grown during infection. Finally, we demonstrated a significant reduction in E. faecium collagen adherence by affinity-purified anti-Acm antibodies from E. faecium endocarditis patient sera, suggesting that Acm may be a potential immunotarget for strategies to control this emerging pathogen.

Endocarditis and biofilm-associated pili of Enterococcus faecalis.

Increasing multidrug resistance in Enterococcus faecalis, a nosocomial opportunist and common cause of bacterial endocarditis, emphasizes the need for alternative therapeutic approaches such as immunotherapy or immunoprophylaxis. In an earlier study, we demonstrated the presence of antibodies in E. faecalis endocarditis patient sera to recombinant forms of 9 E. faecalis cell wall-anchored proteins; of these, we have now characterized an in vivo-expressed locus of 3 genes and an associated sortase gene (encoding sortase C; SrtC). Here, using mutation analyses and complementation, we demonstrated that both the ebp (encoding endocarditis and biofilm-associated pili) operon and srtC are important for biofilm production of E. faecalis strain OG1RF. In addition, immunogold electron microscopy using antisera against EbpA-EbpC proteins as well as patient serum demonstrated that E. faecalis produces pleomorphic surface pili. Assembly of pili and their cell wall attachment appeared to occur via a mechanism of cross-linking of the Ebp proteins by the designated SrtC. Importantly, a nonpiliated, allelic replacement mutant was significantly attenuated in an endocarditis model. These biologically important surface pili, which are antigenic in humans during endocarditis and encoded by a ubiquitous E. faecalis operon, may be a useful immunotarget for studies aimed at prevention and/or treatment of this pathogen.

EbpR is important for biofilm formation by activating expression of the endocarditis and biofilm-associated pilus operon (ebpABC) of Enterococcus faecalis OG1RF.

We identify ef1090 (renamed ebpR) and show its importance for the transcriptional regulation of expression of the Enterococcus faecalis pilus operon, ebpABC. An ebpR deletion (DeltaebpR) mutant was found to have reduced ebpABC expression with loss of pilus production and a defect in primary adherence with, as a consequence, reduced biofilm formation.

Relative contributions of Enterococcus faecalis OG1RF sortase-encoding genes, srtA and bps (srtC), to biofilm formation and a murine model of urinary tract infection.

Deletion mutants of the two sortase genes of Enterococcus faecalis OG1RF were constructed. srtC (renamed here bps for biofilm and pilus-associated sortase) was previously shown to be necessary for the production of Ebp pili and important for biofilm formation and endocarditis. Here, we report that a srtA deletion mutant showed a small (5%) yet significant (P = 0.037) reduction in biofilm relative to OG1RF, while a DeltasrtA Deltabps double mutant showed a much greater reduction (74% versus OG1RF and 44% versus the Deltabps mutant). In a murine urinary tract infection (UTI), the 50% infective doses of both the DeltasrtA Deltabps and Deltabps mutants were approximately 2 log10 greater than that of OG1RF or the DeltasrtA mutant. Similarly, approximately 2 log10 fewer bacteria were recovered from the kidneys after infection with the Deltabps mutant (P = 0.017) and the DeltasrtA Deltabps double mutant (P = 0.022) compared to wild-type strain OG1RF. In a competition UTI, the Deltabps mutant was slightly, but not significantly, less attenuated than the DeltasrtA Deltabps double mutant. Fluorescence-activated cell sorter analysis with Ebp-specific antibodies confirmed that a minority of OG1RF cells express Ebp pili on their surface in vitro and that Bps has a major role in Ebp pilus biogenesis but also indicated a function for SrtA in surface localization of the pilus subunit protein EbpA. In conclusion, deletion of bps had a major effect on virulence in murine UTIs, as well as biofilm; deletion of srtA from OG1RF had little effect on these phenotypes, but its deletion from a bps mutant had a pronounced effect on biofilm, suggesting that Bps and/or the proteins it anchors may compensate for the loss of some SrtA function(s).

Relative contributions of Enterococcus faecalis OG1RF sortase-encoding genes, srtA and bps (srtC), to biofilm formation and a murine model of urinary tract infection.

Deletion mutants of the two sortase genes of Enterococcus faecalis OG1RF were constructed. srtC (renamed here bps for biofilm and pilus-associated sortase) was previously shown to be necessary for the production of Ebp pili and important for biofilm formation and endocarditis. Here, we report that a srtA deletion mutant showed a small (5%) yet significant (P = 0.037) reduction in biofilm relative to OG1RF, while a DeltasrtA Deltabps double mutant showed a much greater reduction (74% versus OG1RF and 44% versus the Deltabps mutant). In a murine urinary tract infection (UTI), the 50% infective doses of both the DeltasrtA Deltabps and Deltabps mutants were approximately 2 log10 greater than that of OG1RF or the DeltasrtA mutant. Similarly, approximately 2 log10 fewer bacteria were recovered from the kidneys after infection with the Deltabps mutant (P = 0.017) and the DeltasrtA Deltabps double mutant (P = 0.022) compared to wild-type strain OG1RF. In a competition UTI, the Deltabps mutant was slightly, but not significantly, less attenuated than the DeltasrtA Deltabps double mutant. Fluorescence-activated cell sorter analysis with Ebp-specific antibodies confirmed that a minority of OG1RF cells express Ebp pili on their surface in vitro and that Bps has a major role in Ebp pilus biogenesis but also indicated a function for SrtA in surface localization of the pilus subunit protein EbpA. In conclusion, deletion of bps had a major effect on virulence in murine UTIs, as well as biofilm; deletion of srtA from OG1RF had little effect on these phenotypes, but its deletion from a bps mutant had a pronounced effect on biofilm, suggesting that Bps and/or the proteins it anchors may compensate for the loss of some SrtA function(s).

The Role of the Androgen Receptor Cofactor p44/WDR77 in Astrocyte Activation

Astrogliosis is induced by neuronal damage and is also a pathological feature of the major aging-related neurodegenerative disorders. The mechanisms that control the cascade of astrogliosis have not been well established. In a previous study, we identified a novel androgen receptor (AR)-interacting protein (p44/WDR77) and found that it plays a critical role in the control of proliferation and differentiation of prostate epithelial cells. In the present study, we found that deletion of the p44 gene in the mouse brain caused accelerated aging with dramatic astrogliosis. The p44/WDR77 is expressed in astrocytes and loss of p44/WDR77 expression in astrocytes leads to astrogliosis. Our results reveal a novel role of p44/WDR77 in astrocytes, which may explain the well-documented role of androgens in suppression of astrogliosis. While many of detailed mechanisms of astrocyte activation remain to be elucidated, a number pathways have been implicated in astrocyte activation including p21Cip1 and the NF-kB pathway. Astrocytic activation induced by p44/WDR77 gene deletion was associated with a significant increase of p21Cip1 expression and NF-kB activation characterized by p65 nuclear localization. We found that down-regulation of p21Cip1 expression inhibited astrocyte activation induced by the p44/WDR77 deletion and was accompanied by a decreased p65 nuclear localization. While p21Cip1 role in astrocyte activation and NF-kB activation is not well understood, studies of other cell cycle regulators have implicated cell cycle control systems as modulators of astrocyte activation, thus p21Cip1 could induce secondary effect to induce p65 nuclear localization. However, p65 knockdown completely relieved the inhibition of astrocyte growth induced by the p44/WDR77 deletion, while p21Cip1 knockdown only partially recovered this inhibition. Thus, NF-kB activity performs additional regulatory actions not mediated by p21Cip1. These analyses imply that p4/WDR77 suppresses astrocyte activation through modulating p21Cip1 expression and NF-kB activation.