Single-cell guided prenatal derivation of primary fetal epithelial organoids from human amniotic and tracheal fluids.

Isolation of tissue-specific fetal stem cells and derivation of primary organoids is limited to samples obtained from termination of pregnancies, hampering prenatal investigation of fetal development and congenital diseases. Therefore, new patient-specific in vitro models are needed. To this aim, isolation and expansion of fetal stem cells during pregnancy, without the need for tissue samples or reprogramming, would be advantageous. Amniotic fluid (AF) is a source of cells from multiple developing organs. Using single-cell analysis, we characterized the cellular identities present in human AF. We identified and isolated viable epithelial stem/progenitor cells of fetal gastrointestinal, renal and pulmonary origin. Upon culture, these cells formed clonal epithelial organoids, manifesting small intestine, kidney tubule and lung identity. AF organoids exhibit transcriptomic, protein expression and functional features of their tissue of origin. With relevance for prenatal disease modeling, we derived lung organoids from AF and tracheal fluid cells of congenital diaphragmatic hernia fetuses, recapitulating some features of the disease. AF organoids are derived in a timeline compatible with prenatal intervention, potentially allowing investigation of therapeutic tools and regenerative medicine strategies personalized to the fetus at clinically relevant developmental stages.

Generation of Distinct Differentially Culturable Forms of Burkholderia following Starvation at Low Temperature.

Bacteria have developed unique mechanisms to adapt to environmental stresses and challenges of the immune system. Here, we report that Burkholderia pseudomallei, the causative agent of melioidosis, and its laboratory surrogate, Burkholderia thailandensis, utilize distinct mechanisms for surviving starvation at different incubation temperatures. At 21°C, Burkholderia are present as short rods which can rapidly reactivate and form colonies on solid media. At 4°C, Burkholderia convert into coccoid forms that cannot be cultured on solid agar but can be resuscitated in liquid media supplemented with supernatant obtained from logarithmic phase cultures of B. thailandensis, or catalase and Tween 80, thus displaying characteristics of differentially culturable bacteria (DCB). These DCB have low intensity fluorescence when stained with SYTO 9, have an intact cell membrane (propidium iodide negative), and contain 16S rRNA at levels comparable with growing cells. We also present evidence that lytic transglycosylases, a family of peptidoglycan-remodeling enzymes, are involved in the generation of coccoid forms and their resuscitation to actively growing cells. A B. pseudomallei ΔltgGCFD mutant with four ltg genes deleted did not produce coccoid forms at 4°C and could not be resuscitated in the liquid media evaluated. Our findings provide insights into the adaptation of Burkholderia to nutrient limitation and the generation of differentially culturable bacteria. IMPORTANCE Bacterial pathogens exhibit physiologically distinct forms that enable their survival in an infected host, the environment and following exposure to antimicrobial agents. B. pseudomallei causes the disease melioidosis, which has a high mortality rate and is difficult to treat with antibiotics. The bacterium is endemic to several countries and detected in high abundance in the environment. Here, we report that during starvation at low temperature, B. pseudomallei produces coccoid forms that cannot grow in standard media and which, therefore, can be challenging to detect using common tools. We provide evidence that the formation of these cocci is mediated by cell wall-specialized enzymes and lytic transglycosylases, and that resuscitation of these forms occurs following the addition of catalase and Tween 80. Our findings have important implications for the disease control and detection of B. pseudomallei, an agent of both public health and defense interest.

Mycobacterial phosphatase PstP regulates global serine threonine phosphorylation and cell division.

Protein phosphatase PstP is conserved throughout the Actinobacteria in a genetic locus related to cell wall synthesis and cell division. In many Actinobacteria it is the sole annotated serine threonine protein phosphatase to counter the activity of multiple serine threonine protein kinases. We used transcriptional knockdown, electron microscopy and comparative phosphoproteomics to investigate the putative dual functions of PstP as a specific regulator of cell division and as a global regulator of protein phosphorylation. Comparative phosphoproteomics in the early stages of PstP depletion showed hyperphosphorylation of protein kinases and their substrates, confirming PstP as a negative regulator of kinase activity and global serine and threonine phosphorylation. Analysis of the 838 phosphorylation sites that changed significantly, suggested that PstP may regulate diverse phosphoproteins, preferentially at phosphothreonine near acidic residues, near the protein termini, and within membrane associated proteins. Increased phosphorylation of the activation loop of protein kinase B (PknB) and of the essential PknB substrate CwlM offer possible explanations for the requirement for pstP for growth and for cell wall defects when PstP was depleted.