Microbiology and immunology: An ideal partnership for a tango at the gut surface—A tribute to Philippe Sansonetti

Over the past 20 years, the highly dynamic interactions that take place between hosts and the gut microbiota have emerged as a major determinant in health and disease. The complexity of the gut microbiota represents, however, a considerable challenge, and reductionist approaches are indispensable to define the contribution of individual bacteria to host responses and to dissect molecular mechanisms. In this tribute to Philippe Sansonetti, I would like to show how rewarding collaborations with microbiologists have guided our team of immunologists in the study of host–microbiota interactions and, thanks to the use of controlled colonisation experiments in gnotobiotic mice, toward the demonstration that segmented filamentous bacteria (SFB) are indispensable to drive the post‐natal maturation of the gut immune barrier in mice. The work led with Philippe Sansonetti to set up in vitro culture conditions has been one important milestone that laid the ground for in‐depth characterization of the molecular attributes of this unusual symbiont. Recent suggestions that SFB may be present in the human microbiota encourage further cross‐fertilising interactions between microbiologists and immunologists to define whether results from mice can be translated to humans and, if so, how SFB may be used to promote human intestinal defences against enteropathogens. Nurturing the competences to pursue this inspiring project is one legacy of Philippe Sansonetti.     

Characterization of an Additional Splice Acceptor Site Introduced into CYP4B1 in Hominoidae during Evolution

CYP4B1 belongs to the cytochrome P450 family 4, one of the oldest P450 families whose members have been highly conserved throughout evolution. The CYP4 monooxygenases typically oxidize fatty acids to both inactive and active lipid mediators, although the endogenous ligand(s) is largely unknown. During evolution, at the transition of great apes to humanoids, the CYP4B1 protein acquired a serine instead of a proline at the canonical position 427 in the meander region. Although this alteration impairs P450 function related to the processing of naturally occurring lung toxins, a study in transgenic mice suggested that an additional serine insertion at position 207 in human CYP4B1 can rescue the enzyme stability and activity. Here, we report that the genomic insertion of a CAG triplet at the intron 5–exon 6 boundary in human CYP4B1 introduced an additional splice acceptor site in frame. During evolution, this change occurred presumably at the stage of Hominoidae and leads to two major isoforms of the CYP4B1 enzymes of humans and great apes, either with or without a serine 207 insertion (insSer207). We further demonstrated that the CYP4B1 enzyme with insSer207 is the dominant isoform (76%) in humans. Importantly, this amino acid insertion did not affect the 4-ipomeanol metabolizing activities or stabilities of the native rabbit or human CYP4B1 enzymes, when introduced as transgenes in human primary cells and cell lines. In our 3D modeling, this functional neutrality of insSer207 is compatible with its predicted location on the exterior surface of CYP4B1 in a flexible side chain. Therefore, the Ser207 insertion does not rescue the P450 functional activity of human CYP4B1 that has been lost during evolution.     

Genome‐wide mapping of copy number variations in patients with both anorectal malformations and central nervous system abnormalities

Background: Anorectal malformations (ARM) have a prevalence of around 1 in 2500 live births. In around 50% of patients, the malformation is isolated, while in the remainder it arises within the context of complex genetic abnormalities or a defined genetic syndrome. Recent studies have implicated rare copy number variations (CNVs) in both isolated and nonisolated ARM, and identified plausible candidate genes. Methods: In the present study, array‐based molecular karyotyping was performed to identify causative CNVs in 32 sporadic ARM patients with comorbid abnormalities of the central nervous system (CNS). This phenotype was selected to enrich for rare CNVs, since previous research has implicated rare CNVs in both CNS abnormalities and ARM. Results: In five patients, a probable disease‐causing CNV was identified (del6q14.3q16.3, del14q32.2, del17q12q21.2, and two patients with del22q11.21). In three of these patients, the CNVs were de novo. For the remaining two patients, no parental DNA was available. Deletions at 22q11.21 and 6q14.3 have been associated with both CNS abnormalities and ARM. In contrast, deletions at 14q32.2 have only been described in patients with CNS abnormalities, and the del17q12q21.2 is a novel CNV. Expression studies in mice suggest that NEUROD2 and RARA, which reside within the newly identified del17q12q21.2 region, are candidate genes for the formation of microcephaly and ARM. Conclusion: The present data suggest that CNVs are a frequent cause of the ARM with CNS abnormalities phenotype, and that array‐analysis is indicated in such patients. Birth Defects Research (Part A) 103:235–242, 2015. © 2014 Wiley Periodicals, Inc.     

Essential Amino Acids in the Gluten-Free Diet and Serum in Relation to Depression in Patients with Celiac Disease

IntroductionCeliac disease (CD) is associated with an increased risk of major depressive disorder, possibly due to deficiencies in micronutrients in the gluten-free diet. We aimed to investigate whether essential amino acids (i.e., the precursors of serotonin, dopamine and other neurotransmitters) are depleted in the diet and serum of CD patients with major depressive disorder.MethodsIn a cross-sectional study we assessed dietary intake of amino acids and serum levels of amino acids, in 77 CD patients on a gluten-free diet and in 33 healthy controls. Major depressive disorder was assessed with structured interviews (using the Mini International Neuropsychiatric Interview Plus). Dietary intake was assessed using a 203-item food frequency questionnaire.ResultsParticipants had a mean age of 55 years and 74% were women. The intake of vegetable protein was significantly lower in CD patients than in healthy controls (mean difference of 7.8 g/d; 95% CI: 4.7–10.8), as were serum concentrations of tyrosine, phenylalanine and tryptophan (all p < 0.005). However, within the CD patient group, the presence of major depressive disorder (n = 42) was not associated with intake or serum levels of essential amino acids.ConclusionsPatients with CD on a long-term gluten-free diet, with good adherence, consume significantly less vegetable protein than controls, and their serum levels of several essential amino acids were also lower. Despite its potential adverse effect, intake and serum levels of essential amino acids were not related to major depression.     

Beta-TrCP1-mediated degradation of PERIOD2 is essential for circadian dynamics

Regulated degradation of circadian clock proteins is a crucial step for rhythm generation per se but also for establishing a normal circadian period. Here, the authors show that the F-box protein beta-transducin repeat containing protein 1 (beta-TrCP1) as part of the E3 ubiquitin ligase complex is an essential component of the mammalian circadian oscillator. Down-regulation of endogenous beta-TrCP1 as well as expression of a dominant-negative form both result in lengthening of the circadian period in oscillating fibroblasts. These phenotypes are due to an impaired degradation of PERIOD (PER) proteins, since expression of beta-TrCP interaction-deficient PER2 variants--but not wild-type PER2--results in a dramatic stabilization of PER2 protein as well as in the disruption of circadian rhythmicity. Mathematical modeling conceptualizes the authors' findings and suggests that loss of sustained rhythmicity in cells with eliminated beta-TrCP-mediated PER2 degradation is due to excessive nuclear repression, a prediction they verified experimentally.     

Distinct roles of chromatin-associated proteins MDC1 and 53BP1 in mammalian double-strand break repair

Phosphorylated histone H2AX ("gamma-H2AX") recruits MDC1, 53BP1, and BRCA1 to chromatin near a double-strand break (DSB) and facilitates efficient repair of the break. It is unclear to what extent gamma-H2AX-associated proteins act in concert and to what extent their functions within gamma-H2AX chromatin are distinct. We addressed this question by comparing the mechanisms of action of MDC1 and 53BP1 in DSB repair (DSBR). We find that MDC1 functions primarily in homologous recombination/sister chromatid recombination, in a manner strictly dependent upon its ability to interact with gamma-H2AX but, unexpectedly, not requiring recruitment of 53BP1 or BRCA1 to gamma-H2AX chromatin. In contrast, 53BP1 functions in XRCC4-dependent nonhomologous end-joining, likely mediated by its interaction with dimethylated lysine 20 of histone H4 but, surprisingly, independent of H2AX. These results suggest a specialized adaptation of the "histone code" in which distinct histone tail-protein interactions promote engagement of distinct DSBR pathways.     

Identification and characterization of onchoastacin, an astacin-like metalloproteinase from the filaria Onchocerca volvulus

The tissue-invasive nematode Onchocerca volvulus causes skin and eye pathology in human onchocerciasis. While the adult females reside sessile in subcutaneous nodules, the microfilariae are abundantly released from the nodules, males and juvenile worms migrate through the host tissue. Matrix-degrading metallo- and serine proteinases have been detected in excretory-secretory worm products that may be essential for migration of the mobile stages. In this study, a 1713bp long cDNA encoding for a putative proteinase of O. volvulus has been isolated. The predicted protein sequence includes a signal peptide indicating secretion to the extracellular space, a propeptide, an astacin-like protease domain, an EGF-like and a CUB-domain, thereby identifying the protein as a member of the astacin family of zinc endopeptidases. Onchoastacin, Ov-AST-1, is most closely related to a subfamily comprising nematode astacins including Caenorhabditis and Ancylostoma. Ov-AST-1 was expressed as a recombinant protein in baculovirus-infected insect cells and exhibited enzymatic activity. The exposure of onchoastacin to the host immune system is indicated by demonstration of IgG reacting with the recombinant Ov-AST-1 and with two peptides of the protein. Since a homologous metalloproteinase is part of a promising hookworm vaccine, Ov-AST-1 may be a candidate for intervention strategies in filarial infections.