Ecological Modeling from Time-Series Inference: Insight into Dynamics and Stability of Intestinal Microbiota

The intestinal microbiota is a microbial ecosystem of crucial importance to human health. Understanding how the microbiota confers resistance against enteric pathogens and how antibiotics disrupt that resistance is key to the prevention and cure of intestinal infections. We present a novel method to infer microbial community ecology directly from time-resolved metagenomics. This method extends generalized Lotka–Volterra dynamics to account for external perturbations. Data from recent experiments on antibiotic-mediated Clostridium difficile infection is analyzed to quantify microbial interactions, commensal-pathogen interactions, and the effect of the antibiotic on the community. Stability analysis reveals that the microbiota is intrinsically stable, explaining how antibiotic perturbations and C. difficile inoculation can produce catastrophic shifts that persist even after removal of the perturbations. Importantly, the analysis suggests a subnetwork of bacterial groups implicated in protection against C. difficile. Due to its generality, our method can be applied to any high-resolution ecological time-series data to infer community structure and response to external stimuli.     

Ab Initio Modeling and Experimental Assessment of Janus Kinase 2 (JAK2) Kinase-Pseudokinase Complex Structure

The Janus Kinase 2 (JAK2) plays essential roles in transmitting signals from multiple cytokine receptors, and constitutive activation of JAK2 results in hematopoietic disorders and oncogenesis. JAK2 kinase activity is negatively regulated by its pseudokinase domain (JH2), where the gain-of-function mutation V617F that causes myeloproliferative neoplasms resides. In the absence of a crystal structure of full-length JAK2, how JH2 inhibits the kinase domain (JH1), and how V617F hyperactivates JAK2 remain elusive. We modeled the JAK2 JH1–JH2 complex structure using a novel informatics-guided protein-protein docking strategy. A detailed JAK2 JH2-mediated auto-inhibition mechanism is proposed, where JH2 traps the activation loop of JH1 in an inactive conformation and blocks the movement of kinase αC helix through critical hydrophobic contacts and extensive electrostatic interactions. These stabilizing interactions are less favorable in JAK2-V617F. Notably, several predicted binding interfacial residues in JH2 were confirmed to hyperactivate JAK2 kinase activity in site-directed mutagenesis and BaF3/EpoR cell transformation studies. Although there may exist other JH2-mediated mechanisms to control JH1, our JH1–JH2 structural model represents a verifiable working hypothesis for further experimental studies to elucidate the role of JH2 in regulating JAK2 in both normal and pathological settings.     

Binding of HIV-1 gp120 to DC-SIGN Promotes ASK-1-Dependent Activation-Induced Apoptosis of Human Dendritic Cells

During disease progression to AIDS, HIV-1 infected individuals become increasingly immunosuppressed and susceptible to opportunistic infections. It has also been demonstrated that multiple subsets of dendritic cells (DC), including DC-SIGN(+) cells, become significantly depleted in the blood and lymphoid tissues of AIDS patients, which may contribute to the failure in initiating effective host immune responses. The mechanism for DC depletion, however, is unclear. It is also known that vast quantities of viral envelope protein gp120 are shed from maturing HIV-1 virions and form circulating immune complexes in the serum of HIV-1-infected individuals, but the pathological role of gp120 in HIV-1 pathogenesis remains elusive. Here we describe a previously unrecognized mechanism of DC death in chronic HIV-1 infection, in which ligation of DC-SIGN by gp120 sensitizes DC to undergo accelerated apoptosis in response to a variety of activation stimuli. The cultured monocyte-derived DC and also freshly-isolated DC-SIGN(+) blood DC that were exposed to either cross-linked recombinant gp120 or immune-complex gp120 in HIV(+) serum underwent considerable apoptosis after CD40 ligation or exposure to bacterial lipopolysaccharide (LPS) or pro-inflammatory cytokines such as TNFα and IL-1β. Furthermore, circulating DC-SIGN(+) DC that were isolated directly from HIV-1(+) individuals had actually been pre-sensitized by serum gp120 for activation-induced exorbitant apoptosis. In all cases the DC apoptosis was substantially inhibited by DC-SIGN blockade. Finally, we showed that accelerated DC apoptosis was a direct consequence of excessive activation of the pro-apoptotic molecule ASK-1 and transfection of siRNA against ASK-1 significantly prevented the activation-induced excessive DC death. Our study discloses a previously unknown mechanism of immune modulation by envelope protein gp120, provides new insights into HIV immunopathogenesis, and suggests potential therapeutic approaches to prevent DC depletion in chronic HIV infection.     

Zinc Piracy as a Mechanism of Neisseria meningitidis for Evasion of Nutritional Immunity

The outer membrane of Gram-negative bacteria functions as a permeability barrier that protects these bacteria against harmful compounds in the environment. Most nutrients pass the outer membrane by passive diffusion via pore-forming proteins known as porins. However, diffusion can only satisfy the growth requirements if the extracellular concentration of the nutrients is high. In the vertebrate host, the sequestration of essential nutrient metals is an important defense mechanism that limits the growth of invading pathogens, a process known as “nutritional immunity.” The acquisition of scarce nutrients from the environment is mediated by receptors in the outer membrane in an energy-requiring process. Most characterized receptors are involved in the acquisition of iron. In this study, we characterized a hitherto unknown receptor from Neisseria meningitidis, a causative agent of sepsis and meningitis. Expression of this receptor, designated CbpA, is induced when the bacteria are grown under zinc limitation. We demonstrate that CbpA functions as a receptor for calprotectin, a protein that is massively produced by neutrophils and other cells and that has been shown to limit bacterial growth by chelating Zn²⁺ and Mn²⁺ ions. Expression of CbpA enables N. meningitidis to survive and propagate in the presence of calprotectin and to use calprotectin as a zinc source. Besides CbpA, also the TonB protein, which couples energy of the proton gradient across the inner membrane to receptor-mediated transport across the outer membrane, is required for the process. CbpA was found to be expressed in all N. meningitidis strains examined, consistent with a vital role for the protein when the bacteria reside in the host. Together, our results demonstrate that N. meningitidis is able to subvert an important defense mechanism of the human host and to utilize calprotectin to promote its growth.     

Atomic Model of Rabbit Hemorrhagic Disease Virus by Cryo-Electron Microscopy and Crystallography

Rabbit hemorrhagic disease, first described in China in 1984, causes hemorrhagic necrosis of the liver. Its etiological agent, rabbit hemorrhagic disease virus (RHDV), belongs to the Lagovirus genus in the family Caliciviridae. The detailed molecular structure of any lagovirus capsid has yet to be determined. Here, we report a cryo-electron microscopic (cryoEM) reconstruction of wild-type RHDV at 6.5 Å resolution and the crystal structures of the shell (S) and protruding (P) domains of its major capsid protein, VP60, each at 2.0 Å resolution. From these data we built a complete atomic model of the RHDV capsid. VP60 has a conserved S domain and a specific P2 sub-domain that differs from those found in other caliciviruses. As seen in the shell portion of the RHDV cryoEM map, which was resolved to ∼5.5 Å, the N-terminal arm domain of VP60 folds back onto its cognate S domain. Sequence alignments of VP60 from six groups of RHDV isolates revealed seven regions of high variation that could be mapped onto the surface of the P2 sub-domain and suggested three putative pockets might be responsible for binding to histo-blood group antigens. A flexible loop in one of these regions was shown to interact with rabbit tissue cells and contains an important epitope for anti-RHDV antibody production. Our study provides a reliable, pseudo-atomic model of a Lagovirus and suggests a new candidate for an efficient vaccine that can be used to protect rabbits from RHDV infection.     

Human Spermatogenic Failure Purges Deleterious Mutation Load from the Autosomes and Both Sex Chromosomes,including the Gene DMRT1

Gonadal failure, along with early pregnancy loss and perinatal death, may be an important filter that limits the propagation of harmful mutations in the human population. We hypothesized that men with spermatogenic impairment, a disease with unknown genetic architecture and a common cause of male infertility, are enriched for rare deleterious mutations compared to men with normal spermatogenesis. After assaying genomewide SNPs and CNVs in 323 Caucasian men with idiopathic spermatogenic impairment and more than 1,100 controls, we estimate that each rare autosomal deletion detected in our study multiplicatively changes a man''s risk of disease by 10% (OR 1.10 [1.04–1.16], p<2×10⁻³), rare X-linked CNVs by 29%, (OR 1.29 [1.11–1.50], p<1×10⁻³), and rare Y-linked duplications by 88% (OR 1.88 [1.13–3.13], p<0.03). By contrasting the properties of our case-specific CNVs with those of CNV callsets from cases of autism, schizophrenia, bipolar disorder, and intellectual disability, we propose that the CNV burden in spermatogenic impairment is distinct from the burden of large, dominant mutations described for neurodevelopmental disorders. We identified two patients with deletions of DMRT1, a gene on chromosome 9p24.3 orthologous to the putative sex determination locus of the avian ZW chromosome system. In an independent sample of Han Chinese men, we identified 3 more DMRT1 deletions in 979 cases of idiopathic azoospermia and none in 1,734 controls, and found none in an additional 4,519 controls from public databases. The combined results indicate that DMRT1 loss-of-function mutations are a risk factor and potential genetic cause of human spermatogenic failure (frequency of 0.38% in 1306 cases and 0% in 7,754 controls, p = 6.2×10⁻⁵). Our study identifies other recurrent CNVs as potential causes of idiopathic azoospermia and generates hypotheses for directing future studies on the genetic basis of male infertility and IVF outcomes.     

Mitochondrial respiratory chain complex activity and bioenergetic alterations in human platelets derived from pre-symptomatic and symptomatic Huntington's disease carriers

Mitochondrial dysfunction has been implicated in Huntington's disease (HD) pathogenesis. We analyzed the activity of mitochondrial complexes (Cx) I–IV, protein levels of selected Cx subunits and adenine nucleotides in platelet mitochondria from pre-symptomatic versus symptomatic HD human carriers and age-matched control individuals. Mitochondrial platelets exhibited reduced activity of citrate synthase in pre-symptomatic and Cx-I in pre-symptomatic and symptomatic HD carriers. Positive correlation between Cx activity and protein subunits was observed for Cx-I in symptomatic HD patient's mitochondria. Moreover, AMP increased in mitochondria from pre-symptomatic HD carriers. Results highlight mitochondrial changes occurring before the onset of HD clinical symptoms.     

A novel familial case of diffuse leukodystrophy related to NDUFV1 compound heterozygous mutations

NDUFV1 mutations have been related to encephalopathic phenotypes due to mitochondrial energy metabolism disturbances. In this study, we report two siblings affected by a diffuse leukodystrophy, who carry the NDUFV1 c.1156C>T (p.Arg386Cys) missense mutation and a novel 42-bp deletion. Bioinformatic and molecular analysis indicated that this deletion lead to the synthesis of mRNA molecules carrying a premature stop codon, which might be degraded by the nonsense-mediated decay system. Our results add information on the molecular basis and the phenotypic features of mitochondrial disease caused by NDUFV1 mutations.