Escherichia coli ClbS is a colibactin resistance protein

The genomic pks island codes for the biosynthetic machinery that produces colibactin, a peptide‐polyketide metabolite. Colibactin is a genotoxin that contributes to the virulence of extra‐intestinal pathogenic Escherichia coli and promotes colorectal cancer. In this work, we examined whether the pks‐encoded clbS gene of unknown function could participate in the self‐protection of E. coli‐producing colibactin. A clbS mutant was not impaired in the ability to inflict DNA damage in HeLa cells, but the bacteria activated the SOS response and ceased to replicate. This autotoxicity phenotype was markedly enhanced in a clbS uvrB double mutant inactivated for DNA repair by nucleotide excision but was suppressed in a clbS clbA double mutant unable to produce colibactin. In addition, ectopic expression of clbS protected infected HeLa cells from colibactin. Thus, ClbS is a resistance protein blocking the genotoxicity of colibactin both in the procaryotic and the eucaryotic cells.     

Study of rabies virus by Differential Scanning Calorimetry

Differential Scanning Calorimetry (DSC) has been used in the past to study the thermal unfolding of many different viruses. Here we present the first DSC analysis of rabies virus. We show that non-inactivated, purified rabies virus unfolds cooperatively in two events centered at approximately 62 and 73 °C. Beta-propiolactone (BPL) treatment does not alter significantly viral unfolding behavior, indicating that viral inactivation does not alter protein structure significantly. The first unfolding event was absent in bromelain treated samples, causing an elimination of the G-protein ectodomain, suggesting that this event corresponds to G-protein unfolding. This hypothesis was confirmed by the observation that this first event was shifted to higher temperatures in the presence of three monoclonal, G-protein specific antibodies. We show that dithiothreitol treatment of the virus abolishes the first unfolding event, indicating that the reduction of G-protein disulfide bonds causes dramatic alterations to protein structure. Inactivated virus samples heated up to 70 °C also showed abolished recognition of conformational G-protein specific antibodies by Surface Plasmon Resonance analysis. The sharpness of unfolding transitions and the low standard deviations of the Tm values as derived from multiple analysis offers the possibility of using this analytical tool for efficient monitoring of the vaccine production process and lot to lot consistency.     

T cell apoptosis and induction of foxp3+ regulatory T cells underlie the therapeutic efficacy of CD4 blockade in experimental autoimmune encephalomyelitis

The pathogenesis of multiple sclerosis requires the participation of effector neuroantigen-specific T cells. Thus, T cell targeting has been proposed as a promising therapeutic strategy. However, the mechanism underlying effective disease prevention following T cell targeting remains incompletely known. We found, using several TCR-transgenic strains, that CD4 blockade is effective in preventing experimental autoimmune encephalopathy and in treating mice after the disease onset. The mechanism does not rely on direct T cell depletion, but the anti-CD4 mAb prevents the proliferation of naive neuroantigen-specific T cells, as well as acquisition of effector Th1 and Th17 phenotypes. Simultaneously, the mAb favors peripheral conversion of Foxp3(+) regulatory T cells. Pre-existing effector cells, or neuroantigen-specific cells that undergo cell division despite the presence of anti-CD4, are committed to apoptosis. Therefore, protection from experimental autoimmune encephalopathy relies on a combination of dominant mechanisms grounded on regulatory T cell induction and recessive mechanisms based on apoptosis of neuropathogenic cells. We anticipate that the same mechanisms may be implicated in other T cell-mediated autoimmune diseases that can be treated or prevented with Abs targeting T cell molecules, such as CD4 or CD3.     

BLyS-mediated modulation of naive B cell subsets impacts HIV Env-induced antibody responses

Neutralizing Abs provide the protective effect of the majority of existing human vaccines. For a prophylactic vaccine against HIV-1, broadly neutralizing Abs targeting conserved epitopes of the viral envelope glycoproteins (Env) are likely required, because the pool of circulating HIV-1 variants is extremely diverse. The failure to efficiently induce broadly neutralizing Abs by vaccination may be due to the use of suboptimal immunogens or immunization regimens, or it may indicate that B cells specific for broadly neutralizing Env determinants are selected against during peripheral checkpoints, either before or after Ag encounter. To investigate whether perturbation of B cell subsets prior to immunization with recombinant Env protein affects the vaccine-induced Ab response in mice, we used B lymphocyte stimulator (BLyS), a cytokine that regulates survival and selection of peripheral B cells. We show that the transient BLyS treatment used in this study substantially affected naive B cell populations; in particular, it resulted in more B cells surviving counter-selection at the transitional stages. We also observed more mature naive B cells, especially marginal zone B cells, in BLyS-treated mice. Intriguingly, provision of excess BLyS prior to immunization led to a consistent improvement in the frequency and potency of HIV-1 Env vaccine-induced neutralizing Ab responses, without increasing the number of Env-specific Ab-secreting cells or the Ab-binding titers measured after boosting. The results presented in this article suggest that an increased understanding of BLyS-regulated processes may help the design of vaccine regimens aimed at eliciting improved neutralizing Ab responses against HIV-1.     

Trpc1 ion channel modulates phosphatidylinositol 3-kinase/Akt pathway during myoblast differentiation and muscle regeneration

We previously showed in vitro that calcium entry through Trpc1 ion channels regulates myoblast migration and differentiation. In the present work, we used primary cell cultures and isolated muscles from Trpc1(-/-) and Trpc1(+/+) murine model to investigate the role of Trpc1 in myoblast differentiation and in muscle regeneration. In these models, we studied regeneration consecutive to cardiotoxin-induced muscle injury and observed a significant hypotrophy and a delayed regeneration in Trpc1(-/-) muscles consisting in smaller fiber size and increased proportion of centrally nucleated fibers. This was accompanied by a decreased expression of myogenic factors such as MyoD, Myf5, and myogenin and of one of their targets, the developmental MHC (MHCd). Consequently, muscle tension was systematically lower in muscles from Trpc1(-/-) mice. Importantly, the PI3K/Akt/mTOR/p70S6K pathway, which plays a crucial role in muscle growth and regeneration, was down-regulated in regenerating Trpc1(-/-) muscles. Indeed, phosphorylation of both Akt and p70S6K proteins was decreased as well as the activation of PI3K, the main upstream regulator of the Akt. This effect was independent of insulin-like growth factor expression. Akt phosphorylation also was reduced in Trpc1(-/-) primary myoblasts and in control myoblasts differentiated in the absence of extracellular Ca(2+) or pretreated with EGTA-AM or wortmannin, suggesting that the entry of Ca(2+) through Trpc1 channels enhanced the activity of PI3K. Our results emphasize the involvement of Trpc1 channels in skeletal muscle development in vitro and in vivo, and identify a Ca(2+)-dependent activation of the PI3K/Akt/mTOR/p70S6K pathway during myoblast differentiation and muscle regeneration.     

C. elegans ATAD-3 Is Essential for Mitochondrial Activity and Development

Background

Mammalian ATAD3 is a mitochondrial protein, which is thought to play an important role in nucleoid organization. However, its exact function is still unresolved.

Results

Here, we characterize the Caenorhabditis elegans (C. elegans) ATAD3 homologue (ATAD-3) and investigate its importance for mitochondrial function and development. We show that ATAD-3 is highly conserved among different species and RNA mediated interference against atad-3 causes severe defects, characterized by early larval arrest, gonadal dysfunction and embryonic lethality. Investigation of mitochondrial physiology revealed a disturbance in organellar structure while biogenesis and function, as indicated by complex I and citrate synthase activities, appeared to be unaltered according to the developmental stage. Nevertheless, we observed very low complex I and citrate synthase activities in L1 larvae populations in comparison to higher larval and adult stages. Our findings indicate that atad-3(RNAi) animals arrest at developmental stages with low mitochondrial activity. In addition, a reduced intestinal fat storage and low lysosomal content after depletion of ATAD-3 suggests a central role of this protein for metabolic activity.

Conclusions

In summary, our data clearly indicate that ATAD-3 is essential for C. elegans development in vivo. Moreover, our results suggest that the protein is important for the upregulation of mitochondrial activity during the transition to higher larval stages.     

Farnesol promotes epithelial cell defense against Candida albicans through Toll-like receptor 2 expression,interleukin-6 and human β-defensin 2 production

Farnesol, a quorum-sensing molecule, regulates virulence and morphogenesis in Candida albicans and is involved in various human pathologies including oral candidiasis. Oral epithelial cells are involved in innate immunity against Candida infections via Toll-like receptors (TLRs) and inflammatory mediators. We investigated the effects of farnesol on host cells and its possible synergistic interaction with gingival epithelial cells against C. albicans infection by studying the expression of TLR2, 4 and 6. The production of IL-6, IL-8, and human β-defensins 1 and 2 was also examined using engineered human oral mucosa tissue put in contact with various concentrations of farnesol with and without C. albicans. Our findings indicate that 24 h after contact with C. albicans, epithelial cells expressed more TLR2 than did non-infected cells. The addition of exogenous farnesol upregulated the TLR2 expression by the gingival epithelial cells in the presence or absence of C. albicans. In contrast, TLR4 was down regulated when farnesol was added to the tissue with or without C. albicans. Finally, farnesol alone was shown to have no effect on TLR6, yet in the presence of both C. albicans and farnesol, TLR6 expression was down regulated. Farnesol modulated TLR2 expression by the epithelial cells following tissue contact with C. albicans. This effect was paralleled by IL-6 but not IL-8 secretion. Farnesol’s effect on innate immunity was strengthened by its capacity to increase human β-defensin 2 production, and by the efficacy of β-defensin against C. albicans growth. Overall results showed that exogenous farnesol promoted epithelial cell defense against C. albicans infection through the involvement of TLR2, IL-6, and human β-defensin 2.