Source Of Phenotypic Heterogeneity Research Articles

Lipoarabinomannan modification as a source of phenotypic heterogeneity in host-adapted Mycobacterium abscessus isolates

Mycobacterium abscessus is increasingly recognized as the causative agent of chronic pulmonary infections in humans. One of the genes found to be under strong evolutionary pressure during adaptation of M. abscessus to the human lung is embC which encodes an arabinosyltransferase required for the biosynthesis of the cell envelope lipoglycan, lipoarabinomannan (LAM). To assess the impact of patient-derived embC mutations on the physiology and virulence of M. abscessus, mutations were introduced in the isogenic background of M. abscessus ATCC 19977 and the resulting strains probed for phenotypic changes in a variety of in vitro and host cell-based assays relevant to infection. We show that patient-derived mutational variations in EmbC result in an unexpectedly large number of changes in the physiology of M. abscessus, and its interactions with innate immune cells. Not only did the mutants produce previously unknown forms of LAM with a truncated arabinan domain and 3-linked oligomannoside chains, they also displayed significantly altered cording, sliding motility, and biofilm-forming capacities. The mutants further differed from wild-type M. abscessus in their ability to replicate and induce inflammatory responses in human monocyte-derived macrophages and epithelial cells. The fact that different embC mutations were associated with distinct physiologic and pathogenic outcomes indicates that structural alterations in LAM caused by nonsynonymous nucleotide polymorphisms in embC may be a rapid, one-step, way for M. abscessus to generate broad-spectrum diversity beneficial to survival within the heterogeneous and constantly evolving environment of the infected human airway.

Metabolic heterogeneity and cross-feeding within isogenic yeast populations captured by DILAC

Genetically identical cells are known to differ in many physiological parameters such as growth rate and drug tolerance. Metabolic specialization is believed to be a cause of such phenotypic heterogeneity, but detection of metabolically divergent subpopulations remains technically challenging. We developed a proteomics-based technology, termed differential isotope labelling by amino acids (DILAC), that can detect producer and consumer subpopulations of a particular amino acid within an isogenic cell population by monitoring peptides with multiple occurrences of the amino acid. We reveal that young, morphologically undifferentiated yeast colonies contain subpopulations of lysine producers and consumers that emerge due to nutrient gradients. Deconvoluting their proteomes using DILAC, we find evidence for in situ cross-feeding where rapidly growing cells ferment and provide the more slowly growing, respiring cells with ethanol. Finally, by combining DILAC with fluorescence-activated cell sorting, we show that the metabolic subpopulations diverge phenotypically, as exemplified by a different tolerance to the antifungal drug amphotericin B. Overall, DILAC captures previously unnoticed metabolic heterogeneity and provides experimental evidence for the role of metabolic specialization and cross-feeding interactions as a source of phenotypic heterogeneity in isogenic cell populations.

Standing genetic variation affects phenotypic heterogeneity in an SCN5A-mutation founder population with excess sudden cardiac death

The Worm Study, ascertained from a multigeneration pedigree segregating a single amino acid deletion in SCN5A (c.4850_4852delTCT, p.(Phe1617del), rs749697698), is characterized by substantial phenotypic heterogeneity and overlap of sudden cardiac death, long-QT syndrome, cardiac conduction disease, Brugada syndrome, and isorhythmic atrioventricular dissociation. Linkage analysis for a synthetic trait derived from these phenotypes identified a single peak (logarithm of the odds [LOD] = 4.52) at the SCN5A/SCN10A/SCN11A locus on chromosome3. This study explored the role of additional genetic variation in the chromosome 3 locus as a source of phenotypic heterogeneity in the Worm Study population. Genotypes underlying the linkage peak (n = 70) were characterized using microarrays. Haplotypes were determined using family-aware phasing and a population-specific reference panel. Variants with minor allele frequencies >0.10 were tested for association with cardiac conduction disease and isorhythmic dissociation using LAMP and logistic regression. Only 1 haplotype carried the p.Phe1617del/rs749697698 deletion, suggesting relatively recent development (∼18 generations); this haplotype contained 5 other missense variants spanning SCN5A/SCN10A/SCN11A. Noncarrier haplotypes (n = 74) ranged in frequency from 0.5% to 5%. Although no variants were associated with cardiac conduction disease, a homozygous missense variant in SCN10A was associated with isorhythmic dissociation after correction for multiple comparisons (odds ratio 11.23; 95% confidence interval 2.76-23.39; P = 1.2 × 10-4). This variant (rs12632942) was previously associated with PR interval. Our data suggest that other variants, alongside a pathogenic mutation, are associated with phenotypic heterogeneity. Single-mutation screening may be insufficient to predict electrical heart disease in patients and family members. In the Worm Study population, segregating a pathogenic SCN5A mutation, compound variation in the SCN5A/SCN10A/SCN11A locus determines arrhythmic outcome.

Chlorate Contamination in Commercial Growth Media as a Source of Phenotypic Heterogeneity within Bacterial Populations.

Under anaerobic conditions, chlorate is reduced to chlorite, a cytotoxic compound that triggers oxidative stress within bacterial cultures. We previously found that BD Bacto Casamino Acids were contaminated with chlorate. In this study, we investigated whether chlorate contamination is detectable in other commercial culture media. We provide evidence that in addition to different batches of BD Bacto Casamino Acids, several commercial agar powders are contaminated with chlorate. A direct consequence of this contamination is that, during anaerobic growth, Escherichia coli cells activate the expression of msrP, a gene encoding periplasmic methionine sulfoxide reductase, which repairs oxidized protein-bound methionine. We further demonstrate that during aerobic growth, progressive oxygen depletion triggers msrP expression in a subpopulation of cells due to the presence of chlorate. Hence, we propose that chlorate contamination in commercial growth media is a source of phenotypic heterogeneity within bacterial populations. IMPORTANCE Agar is arguably the most utilized solidifying agent for microbiological media. In this study, we show that agar powders from different suppliers, as well as certain batches of BD Bacto Casamino Acids, contain significant levels of chlorate. We demonstrate that this contamination induces the expression of a methionine sulfoxide reductase, suggesting the presence of intracellular oxidative damage. Our results should alert the microbiology community to a pitfall in the cultivation of microorganisms under laboratory conditions.

Gabra2 is a genetic modifier of Dravet syndrome in mice

Pathogenic variants in epilepsy genes result in a spectrum of clinical severity. One source of phenotypic heterogeneity is modifier genes that affect expressivity of a primary pathogenic variant. Mouse epilepsy models also display varying degrees of clinical severity on different genetic backgrounds. Mice with heterozygous deletion of Scn1a (Scn1a+/−) model Dravet syndrome, a severe epilepsy most often caused by SCN1A haploinsufficiency. Scn1a+/− mice recapitulate features of Dravet syndrome, including spontaneous seizures, sudden death, and cognitive/behavioral deficits. Scn1a+/− mice maintained on the 129S6/SvEvTac (129) strain have normal lifespan and no spontaneous seizures. In contrast, admixture with C57BL/6J (B6) results in epilepsy and premature lethality. We previously mapped Dravet Survival Modifier loci (Dsm1-Dsm5) responsible for strain-dependent differences in survival. Gabra2, encoding the GABAA α2 subunit, was nominated as a candidate modifier at Dsm1. Direct measurement of GABAA receptors found lower abundance of α2-containing receptors in hippocampal synapses of B6 mice relative to 129. We also identified a B6-specific single nucleotide deletion within Gabra2 that lowers mRNA and protein by nearly 50%. Repair of this deletion reestablished normal levels of Gabra2 expression. In this study, we used B6 mice with a repaired Gabra2 allele to evaluate Gabra2 as a genetic modifier of severity in Scn1a+/− mice. Gabra2 repair restored transcript and protein expression, increased abundance of α2-containing GABAA receptors in hippocampal synapses, and rescued epilepsy phenotypes of Scn1a+/− mice. These findings validate Gabra2 as a genetic modifier of Dravet syndrome, and support enhancing function of α2-containing GABAA receptors as treatment strategy for Dravet syndrome.

Contribution of DNA adenine methylation to gene expression heterogeneity in Salmonella enterica.

Expression of Salmonella enterica loci harboring undermethylated GATC sites at promoters or regulatory regions was monitored by single cell analysis. Cell-to-cell differences in expression were detected in ten such loci (carA, dgoR, holA, nanA, ssaN, STM1290, STM3276, STM5308, gtr and opvAB), with concomitant formation of ON and OFF subpopulations. The ON and OFF subpopulation sizes varied depending on the growth conditions, suggesting that the population structure can be modulated by environmental control. All the loci under study except STM5308 displayed altered patterns of expression in strains lacking or overproducing Dam methylase, thereby confirming control by Dam methylation. Bioinformatic analysis identified potential binding sites for transcription factors OxyR, CRP and Fur, and analysis of expression in mutant backgrounds confirmed transcriptional control by one or more of such factors. Surveys of gene expression in pairwise combinations of Dam methylation-dependent loci revealed independent switching, thus predicting the formation of a high number of cell variants. This study expands the list of S. enterica loci under transcriptional control by Dam methylation, and underscores the relevance of the DNA adenine methylome as a source of phenotypic heterogeneity.

Dissecting diagnostic heterogeneity in depression by integrating neuroimaging and genetics.

Depression is a heterogeneous and etiologically complex psychiatric syndrome, not a unitary disease entity, encompassing a broad spectrum of psychopathology arising from distinct pathophysiological mechanisms. Motivated by a need to advance our understanding of these mechanisms and develop new treatment strategies, there is a renewed interest in investigating the neurobiological basis of heterogeneity in depression and rethinking our approach to diagnosis for research purposes. Large-scale genome-wide association studies have now identified multiple genetic risk variants implicating excitatory neurotransmission and synapse function and underscoring a highly polygenic inheritance pattern that may be another important contributor to heterogeneity in depression. Here, we review various sources of phenotypic heterogeneity and approaches to defining and studying depression subtypes, including symptom-based subtypes and biology-based approaches to decomposing the depression syndrome. We review "dimensional," "categorical," and "hybrid" approaches to parsing phenotypic heterogeneity in depression and defining subtypes using functional neuroimaging. Next, we review recent progress in neuroimaging genetics (correlating neuroimaging patterns of brain function with genetic data) and its potential utility for generating testable hypotheses concerning molecular and circuit-level mechanisms. We discuss how genetic variants and transcriptomic profiles may confer risk for depression by modulating brain structure and function. We conclude by highlighting several promising areas for future research into the neurobiological underpinnings of heterogeneity, including efforts to understand sexually dimorphic mechanisms, the longitudinal dynamics of depressive episodes, and strategies for developing personalized treatments and facilitating clinical decision-making.

Combinatorial complexity and compositional drift in protein interaction networks.

The assembly of molecular machines and transient signaling complexes does not typically occur under circumstances in which the appropriate proteins are isolated from all others present in the cell. Rather, assembly must proceed in the context of large-scale protein-protein interaction (PPI) networks that are characterized both by conflict and combinatorial complexity. Conflict refers to the fact that protein interfaces can often bind many different partners in a mutually exclusive way, while combinatorial complexity refers to the explosion in the number of distinct complexes that can be formed by a network of binding possibilities. Using computational models, we explore the consequences of these characteristics for the global dynamics of a PPI network based on highly curated yeast two-hybrid data. The limited molecular context represented in this data-type translates formally into an assumption of independent binding sites for each protein. The challenge of avoiding the explicit enumeration of the astronomically many possibilities for complex formation is met by a rule-based approach to kinetic modeling. Despite imposing global biophysical constraints, we find that initially identical simulations rapidly diverge in the space of molecular possibilities, eventually sampling disjoint sets of large complexes. We refer to this phenomenon as “compositional drift”. Since interaction data in PPI networks lack detailed information about geometric and biological constraints, our study does not represent a quantitative description of cellular dynamics. Rather, our work brings to light a fundamental problem (the control of compositional drift) that must be solved by mechanisms of assembly in the context of large networks. In cases where drift is not (or cannot be) completely controlled by the cell, this phenomenon could constitute a novel source of phenotypic heterogeneity in cell populations.

Organization of H locus conserved repeats in Leishmania (Viannia) braziliensis correlates with lack of gene amplification and drug resistance

Resistance to antimonials is a major problem when treating visceral leishmaniasis in India and has already been described for New World parasites. Clinical response to meglumine antimoniate in patients infected with parasites of the Viannia sub-genus can be widely variable, suggesting the presence of mechanisms of drug resistance. In this work, we have compared L. major and L. braziliensis mutants selected in different drugs. The cross-resistance profiles of some cell lines resembled those of mutants bearing H locus amplicons. However, amplified episomal molecules were exclusively detected in L. major mutants. The analysis of the L. braziliensis H region revealed a strong conservation of gene synteny. The typical intergenic repeats that are believed to mediate the amplification of the H locus in species of the Leishmania sub-genus are partially conserved in the Viannia species. The conservation of these non-coding elements in equivalent positions in both species is indicative of their relevance within this locus. The absence of amplicons in L. braziliensis suggests that this species may not favour extra-chromosomal gene amplification as a source of phenotypic heterogeneity and fitness maintenance in changing environments.