SUMMARY Lung alveoli, which are unique to air-breathing organisms, have been challenging to generate from pluripotent stem cells (PSCs) in part because there are limited model systems available to provide the necessary developmental roadmaps for in vitro differentiation. Here we report the generation of alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, from human PSCs. Using multicolored fluorescent reporter lines, we track and purify human SFTPC+ alveolar progenitors as they emerge from endodermal precursors in response to stimulation of Wnt and FGF signaling. Purified PSC-derived SFTPC+ cells form monolayered epithelial “alveolospheres” in 3D cultures without the need for mesenchymal support, exhibit self-renewal capacity, and display additional AEC2 functional capacities. Footprint-free CRISPR-based gene correction of PSCs derived from patients carrying a homozygous surfactant mutation (SFTPB121ins2) restores surfactant processing in AEC2s. Thus, PSC-derived AEC2s provide a platform for disease modeling and future functional regeneration of the distal lung.

The accessory gene regulator (agr) of Staphylococcus aureus is a global regulator of the staphylococcal virulon, which includes secreted virulence factors and surface proteins. The agr locus is important for virulence in a variety of animal models of infection, and has been assumed by inference to have a major role in human infection. Although most human clinical S. aureus isolates are agr+, there have been several reports of agr-defective mutants isolated from infected patients. Since it is well known that the agr locus is genetically labile in vitro, we have addressed the question of whether the reported agr-defective mutants were involved in the infection or could have arisen during post-isolation handling. We obtained a series of new staphylococcal isolates from local clinical infections and handled these with special care to avoid post-isolation mutations. Among these isolates, we found a number of strains with non-haemolytic phenotypes owing to mutations in the agr locus, and others with mutations elsewhere. We have also obtained isolates in which the population was continuously heterogeneous with respect to agr functionality, with agr+ and agr− variants having otherwise indistinguishable chromosomal backgrounds. This finding suggested that the agr− variants arose by mutation during the course of the infection. Our results indicate that while most clinical isolates are haemolytic and agr+, non-haemolytic and agr− strains are found in S. aureus infections, and that agr+ and agr− variants may have a cooperative interaction in certain types of infections.

Summaryagr is a global regulator of staphylococcal virulence and other accessory gene functions, especially including the haemolysins. Lack of haemolysin production therefore generally represents a defect in agr function. An example of this is Staphylococcus aureus strain RN4220, a widely used laboratory strain that carries a nitrosoguanidine (MNNG)-induced mutation enabling it to accept DNA from Escherichia coli and other bacteria. We show here that the nonhaemolytic phenotype of RN4220 is caused by an extra A residue in a run of seven As at the 3 ′ ′ ′ ′ end of agrA ( agrA-8A). This causes a frameshift that results in the addition of three amino acyl residues to the Cterminal end of the protein. The 8A mutation does not inactivate the agr locus, but rather delays agr activation by 2-3 h, which results in failure to translate α α α α -and δ δ δ δ -haemolysins, and hence, in a non-haemolytic phenotype. This mutation turned out not to be an adventitious consequence of MNNG mutagenesis, but rather had arisen in RN450, the immediate parent of RN4220. RN450 had become haemolytically heterogeneous in storage, and its non-haemolytic variants had the 8A mutation. The same mutation was also identified in a clinical isolate in which a non-haemolytic variant had arisen during the course of infection. Haemolytic activity in the mutant laboratory strains could be restored by the addition of auto-inducing peptide (AIP) early in growth, indicating that delayed production of RNAIII is responsible for the failure to translate α α α α -and δ δ δ δ -haemolysins. Discovery of the 8A mutation has revealed the basis of the dissociation between agr activity and the non-haemolytic phenotype of RN4220, and has solved the long-standing mystery of the variable non-haemolytic phenotype of its immediate parent, RN450. The occurrence of this mutation in a clinical isolate indicates that it is not simply a laboratory phenomenon, and may represent a naturally occurring mechanism for the modulation of agr activity.

agr is a global regulatory system in the staphylococci, operating by a classical two-component signaling module and controlling the expression of most of the genes encoding extracellular virulence factors. As it is autoinduced by a peptide, encoded within the locus, that is the ligand for the signal receptor, it is a sensor of population density or a quorum sensor and is the only known quorum-sensing system in the genus. agr is conserved throughout the staphylococci but has diverged along lines that appear to parallel speciation and subspeciation within the genus. This divergence has given rise to a novel type of interstrain and interspecies cross-inhibition that represents a fundamental aspect of the organism's biology and may be a predominant feature of the evolutionary forces that have driven it. We present evidence, using a newly developed, luciferasebased agr typing scheme, that the evolutionary divergence of the agr system was an early event in the evolution of the staphylococci and long preceded the development of the nucleotide polymorphisms presently used for genotyping. These polymorphisms developed, for the most part, within different agr groups; mobile genetic elements appear also to have diffused recently and, with a few notable exceptions, have come to reside largely indiscriminately within the several agr groups.The agr operon encodes a global regulatory system in the staphylococci, central to the biology of the organism (reviewed by Novick) (36). It controls a large set of genes, including most of those encoding extracellular virulence factors and many others encoding cytoplasmic proteins with catabolic and other functions. agr is highly conserved throughout the staphylococci but has diverged in a way that closely parallels speciation and subspeciation within the genus. This divergence has given rise to a novel type of interstrain and interspecies cross-inhibition that may represent the selective forces that have driven its evolution.agr operates by a classical two-component signaling module (Fig. 1A). It is autoinduced by a peptide, encoded within the locus, that is the ligand for the histidine kinase component of the signaling module. agr is therefore a sensor of population density or a quorum sensor. The agr variants represent specificity groups that determine the response to cognate or heterologous autoinducing peptides (AIPs). Although an AIP always activates its cognate agr locus, it competitively crossinhibits agr activation in most heterologous combinations. This cross-inhibition results in a novel type of bacterial interference in which the expression of accessory genes, but not growth, is blocked. This interference has potential therapeutic implications that are presently under investigation. In Staphylococcus aureus there are four known agr specificity groups, characterized by major sequence variations in a central region of the locus that encodes the AIP, the enzyme that processes it, and the receptor domain of the histidine kinase (7,19,21). The sequences flanking this central variab...

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