Interactions between the Nse3 and Nse4 Components of the SMC5-6 Complex Identify Evolutionarily Conserved Interactions between MAGE and EID Families

The SMC5-6 protein complex is involved in the cellular response to DNA damage. It is composed of 6-8 polypeptides, of which Nse1, Nse3 and Nse4 form a tight sub-complex. MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and Nse4 is related to the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors.Using site-directed mutagenesis, protein-protein interaction analyses and molecular modelling, we have identified a conserved hydrophobic surface on the C-terminal domain of Nse3 that interacts with Nse4 and identified residues in its N-terminal domain that are essential for interaction with Nse1. We show that these interactions are conserved in the human orthologs. Furthermore, interaction of MAGEG1, the mammalian ortholog of Nse3, with NSE4b, one of the mammalian orthologs of Nse4, results in transcriptional co-activation of the nuclear receptor, steroidogenic factor 1 (SF1). In an examination of the evolutionary conservation of the Nse3-Nse4 interactions, we find that several MAGE proteins can interact with at least one of the NSE4/EID proteins.We have found that, despite the evolutionary diversification of the MAGE family, the characteristic hydrophobic surface shared by all MAGE proteins from yeast to humans mediates its binding to NSE4/EID proteins. Our work provides new insights into the interactions, evolution and functions of the enigmatic MAGE proteins

B Cells Regulate Neutrophilia during Mycobacterium tuberculosis Infection and BCG Vaccination by Modulating the Interleukin-17 Response

We have previously demonstrated that B cells can shape the immune response to Mycobacterium tuberculosis, including the level of neutrophil infiltration and granulomatous inflammation at the site of infection. The present study examined the mechanisms by which B cells regulate the host neutrophilic response upon exposure to mycobacteria and how neutrophilia may influence vaccine efficacy. To address these questions, a murine aerosol infection tuberculosis (TB) model and an intradermal (ID) ear BCG immunization mouse model, involving both the μMT strain and B cell-depleted C57BL/6 mice, were used. IL (interleukin)-17 neutralization and neutrophil depletion experiments using these systems provide evidence that B cells can regulate neutrophilia by modulating the IL-17 response during M. tuberculosis infection and BCG immunization. Exuberant neutrophilia at the site of immunization in B cell-deficient mice adversely affects dendritic cell (DC) migration to the draining lymph nodes and attenuates the development of the vaccine-induced Th1 response. The results suggest that B cells are required for the development of optimal protective anti-TB immunity upon BCG vaccination by regulating the IL-17/neutrophilic response. Administration of sera derived from M. tuberculosis-infected C57BL/6 wild-type mice reverses the lung neutrophilia phenotype in tuberculous μMT mice. Together, these observations provide insight into the mechanisms by which B cells and humoral immunity modulate vaccine-induced Th1 response and regulate neutrophila during M. tuberculosis infection and BCG immunization. © 2013 Kozakiewicz et al

Smc5/6 coordinates formation and resolution of joint molecules with chromosome morphology to ensure meiotic divisions

During meiosis, Structural Maintenance of Chromosome (SMC) complexes underpin two fundamental features of meiosis: homologous recombination and chromosome segregation. While meiotic functions of the cohesin and condensin complexes have been delineated, the role of the third SMC complex, Smc5/6, remains enigmatic. Here we identify specific, essential meiotic functions for the Smc5/6 complex in homologous recombination and the regulation of cohesin. We show that Smc5/6 is enriched at centromeres and cohesin-association sites where it regulates sister-chromatid cohesion and the timely removal of cohesin from chromosomal arms, respectively. Smc5/6 also localizes to recombination hotspots, where it promotes normal formation and resolution of a subset of joint-molecule intermediates. In this regard, Smc5/6 functions independently of the major crossover pathway defined by the MutLγ complex. Furthermore, we show that Smc5/6 is required for stable chromosomal localization of the XPF-family endonuclease, Mus81-Mms4Eme1. Our data suggest that the Smc5/6 complex is required for specific recombination and chromosomal processes throughout meiosis and that in its absence, attempts at cell division with unresolved joint molecules and residual cohesin lead to severe recombination-induced meiotic catastroph

No evidence for cardiac dysfunction in Kif6 mutant mice.

A KIF6 variant in man has been reported to be associated with adverse cardiovascular outcomes after myocardial infarction. No clear biological or physiological data exist for Kif6. We sought to investigate the impact of a deleterious KIF6 mutation on cardiac function in mice. Kif6 mutant mice were generated and verified. Cardiac function was assessed by serial echocardiography at baseline, after ageing and after exercise. Lipid levels were also measured. No discernable adverse lipid or cardiac phenotype was detected in Kif6 mutant mice. These data suggest that dysfunction of Kif6 is linked to other more complex biological/biochemical parameters or is unlikely to be of material consequence in cardiac function

Hydrogen cross-feeders of the human gastrointestinal tract.

Hydrogen plays a key role in many microbial metabolic pathways in the human gastrointestinal tract (GIT) that have an impact on human nutrition, health and wellbeing. Hydrogen is produced by many members of the GIT microbiota, and may be subsequently utilized by cross-feeding microbes for growth and in the production of larger molecules. Hydrogenotrophic microbes fall into three functional groups: sulfate-reducing bacteria, methanogenic archaea and acetogenic bacteria, which can convert hydrogen into hydrogen sulfide, methane and acetate, respectively. Despite different energy yields per molecule of hydrogen used between the functional groups, all three can coexist in the human GIT. The factors affecting the numerical balance of hydrogenotrophs in the GIT remain unconfirmed. There is increasing evidence linking both hydrogen sulfide and methane to GIT diseases such as irritable bowel syndrome, and strategies for the mitigation of such health problems through targeting of hydrogenotrophs constitute an important field for further investigation.fals

Examination of hydrogen cross-feeders using a colonic microbiota model

BACKGROUND: Hydrogen cross-feeding microbes form a functionally important subset of the human colonic microbiota. The three major hydrogenotrophic functional groups of the colon: sulphate-reducing bacteria (SRB), methanogens and reductive acetogens, have been linked to wide ranging impacts on host physiology, health and wellbeing. RESULTS: An existing mathematical model for microbial community growth and metabolism was combined with models for each of the three hydrogenotrophic functional groups. The model was further developed for application to the colonic environment via inclusion of responsive pH, host metabolite absorption and the inclusion of host mucins. Predictions of the model, using two existing metabolic parameter sets, were compared to experimental faecal culture datasets. Model accuracy varied between experiments and measured variables and was most successful in predicting the growth of high relative abundance functional groups, such as the Bacteroides, and short chain fatty acid (SCFA) production. Two versions of the colonic model were developed: one representing the colon with sequential compartments and one utilising a continuous spatial representation. When applied to the colonic environment, the model predicted pH dynamics within the ranges measured in vivo and SCFA ratios comparable to those in the literature. The continuous version of the model simulated relative abundances of microbial functional groups comparable to measured values, but predictions were sensitive to the metabolic parameter values used for each functional group. Sulphate availability was found to strongly influence hydrogenotroph activity in the continuous version of the model, correlating positively with SRB and sulphide concentration and negatively with methanogen concentration, but had no effect in the compartmentalised model version. CONCLUSIONS: Although the model predictions compared well to only some experimental measurements, the important features of the colon environment included make it a novel and useful contribution to modelling the colonic microbiota.fals

Mathematical modelling supports the existence of a threshold hydrogen concentration and media-dependent yields in the growth of a reductive acetogen.

The bacterial production of acetate via reductive acetogenesis along the Wood-Ljungdahl metabolic pathway is an important source of this molecule in several environments, ranging from industrial bioreactors to the human gastrointestinal tract. Here, we contributed to the study of reductive acetogens by considering mathematical modelling techniques for the prediction of bacterial growth and acetate production. We found that the incorporation of a hydrogen uptake concentration threshold into the models improves their predictions and we calculated this threshold as 86.2 mM (95% confidence interval 6.1-132.6 mM). Monod kinetics and first-order kinetics models, with the inclusion of two candidate threshold terms or reversible Michaelis-Menten kinetics, were compared to experimental data and the optimal formulation for predicting both growth and metabolism was found. The models were then used to compare the efficacy of two growth media for acetogens. We found that the recently described general acetogen medium was superior to the DSMZ medium in terms of unbiased estimation of acetogen growth and investigated the contribution of yeast extract concentration to acetate production and bacterial growth in culture. The models and their predictions will be useful to those studying both industrially and environmentally relevant reductive acetogenesis and allow for straightforward adaptation to similar cases with different organisms.fals

Competition for Hydrogen Prevents Coexistence of Human Gastrointestinal Hydrogenotrophs in Continuous Culture.

Understanding the metabolic dynamics of the human gastrointestinal tract (GIT) microbiota is of growing importance as research continues to link the microbiome to host health status. Microbial strains that metabolize hydrogen have been associated with a variety of both positive and negative host nutritional and health outcomes, but limited data exists for their competition in the GIT. To enable greater insight into the behaviour of these microbes, a mathematical model was developed for the metabolism and growth of the three major hydrogenotrophic groups: sulphate-reducing bacteria (SRB), methanogens and reductive acetogens. In batch culture simulations with abundant sulphate and hydrogen, the SRB outcompeted the methanogen for hydrogen due to having a half-saturation constant 106 times lower than that of the methanogen. The acetogen, with a high model threshold for hydrogen uptake of around 70 mM, was the least competitive. Under high lactate and zero sulphate conditions, hydrogen exchange between the SRB and the methanogen was the dominant interaction. The methanogen grew at 70% the rate of the SRB, with negligible acetogen growth. In continuous culture simulations, both the SRB and the methanogen were washed out at dilution rates above 0.15 h-1 regardless of substrate availability, whereas the acetogen could survive under abundant hydrogen conditions. Specific combinations of conditions were required for survival of more than one hydrogenotroph in continuous culture, and survival of all three was not possible. The stringency of these requirements and the inability of the model to simulate survival of all three hydrogenotrophs in continuous culture demonstrates that factors outside of those modelled are vital to allow hydrogenotroph coexistence in the GIT.fals

A Mathematical Model for the Hydrogenotrophic Metabolism of Sulphate-Reducing Bacteria.

Sulphate-reducing bacteria (SRB) are studied across a range of scientific fields due to their characteristic ability to metabolise sulphate and produce hydrogen sulphide, which can lead to significant consequences for human activities. Importantly, they are members of the human gastrointestinal microbial population, contributing to the metabolism of dietary and host secreted molecules found in this environment. The role of the microbiota in host digestion is well studied, but the full role of SRB in this process has not been established. Moreover, from a human health perspective, SRB have been implicated in a number of functional gastrointestinal disorders such as Irritable Bowel Syndrome and the development of colorectal cancer. To assist with the study of SRB, we present a mathematical model for the growth and metabolism of the well-studied SRB, Desulfovibrio vulgaris in a closed system. Previous attempts to model SRB have resulted in complex or highly specific models that are not easily adapted to the study of SRB in different environments, such as the gastrointestinal tract. We propose a simpler, Monod-based model that allows for easy alteration of both key parameter values and the governing equations to enable model adaptation. To prevent any incorrect assumptions about the nature of SRB metabolic pathways, we structure the model to consider only the concentrations of initial and final metabolites in a pathway, which circumvents the current uncertainty around hydrogen cycling by SRB. We parameterise our model using experiments with varied initial substrate conditions, obtaining parameter values that compare well with experimental estimates in the literature. We then validate our model against four independent experiments involving D. vulgaris with further variations to substrate availability. Further use of the model will be possible in a number of settings, notably as part of larger models studying the metabolic interactions between SRB and other hydrogenotrophic microbes in the human gastrointestinal tract and how this relates to functional disorders.fals