A Transient Luminal Chitinous Matrix Is Required to Model Epithelial Tube Diameter in the Drosophila Trachea

SummaryEpithelial tubes are found in many vital organs and require uniform and correct tube diameters for optimal function. Tube size depends on apical membrane growth and subapical cytoskeletal reorganization, but the cues that coordinate these events to ensure functional tube shape remain elusive. We find that epithelial tubes in the Drosophila trachea require luminal chitin polysaccharides to attain the correct diameter. Tracheal chitin forms a broad transient filament within the tubes during the restricted period of expansion. Loss of chitin causes tubular constrictions and cysts associated with irregular subapical cytoskeletal organization, without affecting epithelial integrity and polarity. Analysis of previously identified tube expansion mutants in genes encoding septate junction proteins further suggests that septate junction components may function in tubulogenesis through their role in luminal matrix assembly. We propose that the transient luminal protein/polysaccharide matrix is sensed by the epithelial cells and coordinates cytoskeletal organization to ensure uniform lumen diameter

Resolving taxonomic confusion : establishing the genus Phytobacter on the list of clinically relevant Enterobacteriaceae

Although many clinically significant strains belonging to the family Enterobacteriaceae fall into a restricted number of genera and species, there is still a substantial number of isolates that elude this classification and for which proper identification remains challenging. With the current improvements in the field of genomics, it is not only possible to generate high-quality data to accurately identify individual nosocomial isolates at the species level and understand their pathogenic potential but also to analyse retrospectively the genome sequence databases to identify past recurrences of a specific organism, particularly those originally published under an incorrect or outdated taxonomy. We propose a general use of this approach to classify further clinically relevant taxa, i.e., Phytobacter spp., that have so far gone unrecognised due to unsatisfactory identification procedures in clinical diagnostics. Here, we present a genomics and literature-based approach to establish the importance of the genus Phytobacter as a clinically relevant member of the Enterobacteriaceae family

Trafficking through COPII Stabilises Cell Polarity and Drives Secretion during Drosophila Epidermal Differentiation

BACKGROUND: The differentiation of an extracellular matrix (ECM) at the apical side of epithelial cells implies massive polarised secretion and membrane trafficking. An epithelial cell is hence engaged in coordinating secretion and cell polarity for a correct and efficient ECM formation. PRINCIPAL FINDINGS: We are studying the molecular mechanisms that Drosophila tracheal and epidermal cells deploy to form their specific apical ECM during differentiation. In this work we demonstrate that the two genetically identified factors haunted and ghost are essential for polarity maintenance, membrane topology as well as for secretion of the tracheal luminal matrix and the cuticle. We show that they code for the Drosophila COPII vesicle-coating components Sec23 and Sec24, respectively, that organise vesicle transport from the ER to the Golgi apparatus. CONCLUSION: Taken together, epithelial differentiation during Drosophila embryogenesis is a concerted action of ECM formation, plasma membrane remodelling and maintenance of cell polarity that all three rely mainly, if not absolutely, on the canonical secretory pathway from the ER over the Golgi apparatus to the plasma membrane. Our results indicate that COPII vesicles constitute a central hub for these processes

Molecular regulation of epithelial tube size

In nature, epithelial tubes are vital structures in organ design and are required for transport of gases and liquids in organs, such as the vascular system, the vertebrate lung and the kidneys. The tubular epithelium is single layered, but is often reinforced by layers of muscular support. It constitutes an apical side facing the lumen and a basal side that contacts surrounding tissues. To ensure optimal flow, it is critical that the tubes are correctly sized and shaped. Epithelial tube growth depends on apical membrane enlargements, as well as sub-apical rearrangements, but the mechanisms involved in the regulation of size and shape of epithelial tubes are yet to be revealed. In this thesis the Drosophila respiratory (tracheal) system has been used as a model organ to identify essential genes and clarify the mechanisms involved in the making and shaping of tubes. Through genetic and molecular analyses, new biological concepts have been uncovered. The main tracheal tube, the dorsal trunk (DT), expands three-fold in diameter during a short interval followed by tube elongation. In this thesis we have dissected the roles of five genes in tube regulation, called kkv, knk rtv, dBest2 and DAAM. Analysis of kkv, knk and rtv led us to identify an unprecedented need for luminal matrix components in modeling tube shape. A chitinous luminal matrix is deposited in newly formed tubes and constitutes an expanding cord inside the tube that is required for uniform tube diameter growth. kkv is required for chitin synthesis while knk and rtv are needed for chitin filament assembly. If chitin is missing or fail to form an organized matrix, the expanding tubes develop severe local dilations and constrictions. The subsequent tube elongation requires dBest2 and DAAM. dBest2 encodes an apical chloride channel and is essential for lumen growth during elongation, suggesting that elongation is driven by an increased luminal osmotic pressure. DAAM has a function in actin organization. In the wild type trachea, actin filaments arrange as sub-apical rings perpendicular to tube length, thus allowing for lumen elongation, but not diametrical expansion, upon the increase in lumen pressure. In DAAM mutants, the actin rings are disorganized, thus lumen elongation is inhibited. The luminal chitin matrix has a second role at this stage by preventing excess tube elongation. A balance between combinatorial physical forces exerted by the lumen and sub-apical actin cytoskeleton determines final tube size

A luminal glycoprotein drives dose-dependent diameter expansion of the Drosophila melanogaster hindgut tube.

International audienceAn important step in epithelial organ development is size maturation of the organ lumen to attain correct dimensions. Here we show that the regulated expression of Tenectin (Tnc) is critical to shape the Drosophila melanogaster hindgut tube. Tnc is a secreted protein that fills the embryonic hindgut lumen during tube diameter expansion. Inside the lumen, Tnc contributes to detectable O-Glycans and forms a dense striated matrix. Loss of tnc causes a narrow hindgut tube, while Tnc over-expression drives tube dilation in a dose-dependent manner. Cellular analyses show that luminal accumulation of Tnc causes an increase in inner and outer tube diameter, and cell flattening within the tube wall, similar to the effects of a hydrostatic pressure in other systems. When Tnc expression is induced only in cells at one side of the tube wall, Tnc fills the lumen and equally affects all cells at the lumen perimeter, arguing that Tnc acts non-cell-autonomously. Moreover, when Tnc expression is directed to a segment of a tube, its luminal accumulation is restricted to this segment and affects the surrounding cells to promote a corresponding local diameter expansion. These findings suggest that deposition of Tnc into the lumen might contribute to expansion of the lumen volume, and thereby to stretching of the tube wall. Consistent with such an idea, ectopic expression of Tnc in different developing epithelial tubes is sufficient to cause dilation, while epidermal Tnc expression has no effect on morphology. Together, the results show that epithelial tube diameter can be modelled by regulating the levels and pattern of expression of a single luminal glycoprotein

Transmission dynamics study of tuberculosis isolates with whole genome sequencing in southern Sweden

Epidemiological contact tracing complemented with genotyping of clinical Mycobacterium tuberculosis isolates is important for understanding disease transmission. In Sweden, tuberculosis (TB) is mostly reported in migrant and homeless where epidemiologic contact tracing could pose a problem. This study compared epidemiologic linking with genotyping in a low burden country. Mycobacterium tuberculosis isolates (n = 93) collected at Scania University Hospital in Southern Sweden were analysed with the standard genotyping method mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) and the results were compared with whole genome sequencing (WGS). Using a maximum of twelve single nucleotide polymorphisms (SNPs) as the upper threshold of genomic relatedness noted among hosts, we identified 18 clusters with WGS comprising 52 patients with overall pairwise genetic maximum distances ranging from zero to nine SNPs. MIRU-VNTR and WGS clustered the same isolates, although the distribution differed depending on MIRU-VNTR limitations. Both genotyping techniques identified clusters where epidemiologic linking was insufficient, although WGS had higher correlation with epidemiologic data. To summarize, WGS provided better resolution of transmission than MIRU-VNTR in a setting with low TB incidence. WGS predicted epidemiologic links better which could consolidate and correct the epidemiologically linked cases, avoiding thus false clustering

Tnc is a large protein detected in the lumen of developing epithelial tubes.

<p>(A) Comparison of Tnc from <i>D.melanogaster</i> (top) and predicted Tnc from <i>D.erecta</i> (middle) and <i>D.virilis</i> (bottom). The secreted proteins are drawn to scale, with the vWC-like domains in blue, PTS-domains in grey and the fibronectin-like domain in red. The PTS-domains of <i>D.melanogaster</i> Tnc show relatively low amino acid identity to those of <i>D.erecta</i> and <i>D.virilis</i> (represented as % below the domains), but their length and high P/T/S-content (indicated for the respective domains) are conserved between the species. (B and C) Anti-Tnc stains wild type embryos (B, arrowhead point to the hindgut), but not <i>tnc^13c</i> mutant embryos (C). (D–G) Tnc (magenta) localizes to the lumen (arrow) of the hindgut (D, stage 15), foregut (E, stage 14), trachea (F, stage 15) and the dorsal vessel (G, stage 16). Epithelial cells are visualized by anti-α-Spec (green) in D, E and G, and by anti-Fas3 (green) in F. Scale bars: 5 µm. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002850#pgen.1002850.s001" target="_blank">Figure S1</a>.</p

Tnc is required for hindgut lumen diameter expansion.

<p>(A–C) Wild type embryos were labelled for Crb (magenta) and Dg (green) to visualize the apical and basal surfaces of the hindgut epithelium. Arrows point to anterior and posterior Li borders, and stippled lines mark outer tube diameter. Crb-staining only is shown in A′–C′. At stage 13 (A, lateral view) the hindgut is narrow with an anterior hook pointing ventral. By stage 14, (B, dorsal view) the anterior hook has turned right and Crb-expressing border cells demarcate hindgut subdomains. From stage 14 to stage 16 (C, dorsal view) the hindgut expands in diameter and length. (D–F) <i>tnc^13c</i> mutant embryos stained for Crb revealed a normal hindgut lumen at stage 13 (D), slight reduction in lumen diameter at stage 14 (E) and a clear reduction in lumen diameter at stage 16 (F), compared to the wild type. White and open arrowheads in (C′) and (F) illustrate lumen diameter of Li and Si, respectively. (G) Drawings of the hindgut lumen at stages 14 and 16 with Si in red and Li in blue. Border cells (black lines) mark anterior and posterior boundaries of Li and separates dorsal Li (dLi) and ventral Li (vLi). (H) The graphs show mean lumen diameter of Si and Li at stages 14 and 16 in the wild type and at stage 16 in <i>tnc^13c</i> mutants. Li expands in diameter from 6.3 (+/−0.09) µm to 8.8 (+/−0.16) µm, and Si from 8.9 (+/−0.52) µm to 17.3 (+/−0.56) µm. * = P-value<0.05. Bars represent standard error of mean (n>5). Scale bar: 10 µm. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002850#pgen.1002850.s002" target="_blank">Figures S2</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002850#pgen.1002850.s003" target="_blank">S3</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002850#pgen.1002850.s004" target="_blank">S4</a>.</p

Tnc is a glycosylated intralumial matrix-component.

<p>(A) Tnc from wild type and <i>tnc^13c</i> mutant embryos and larvae (l) were detected on western blot and resided as high-molecular weight species in the stacking gel (stippled line indicates end of stacking gel). Anti-α-Tubulin was used as loading control. (B) Protein extracts from stage 16 wild type embryos were subjected to deglycosylation (no enz = no enzyme, N-Gly = N-Glycanase (PNGase F), O-Gly = O-Glycanase, O-Gly+ = O-Glycanase+Sialidase+β(1-4) Galactosidase+β-N-Acetylglucosaminidase). Addition of O-glycanase caused slightly faster migration of Tnc. (C–H) Embryos were co-labelled for the Tn antigen (green) and Crb (magenta) (C, D, F and G) or with VVA (E and H). Anti-Tn stains the wild type lumen with highest intensity in Si at stages 15 and 16 (C and D). The staining is reduced in mutant embryos (F and G). Arrows point to the Si/Li border. VVA also labels the wild type lumen (E) stronger than the mutant lumen (H). The embryos were processed in parallel and the hindgut was imaged at similar views with identical confocal settings. (I) Wild type embryos were prepared with Clark's fixation and stained for Tnc (I, green) and the Tn antigen (I′, magenta). The merged image (I″) shows partial overlap of the staining. Arrow points to the Si/Li border. (J) High magnification of the hindgut in (I), showing a striated pattern of Tnc-staining. Scale bars: 10 µm in I, 5 µm in J.</p