A Critical Region in the FlaA Flagellin Facilitates Filament Formation of the Vibrio cholerae Flagellum.

Vibrio cholerae is a Gram-negative bacterium with a monotrichous flagellum that causes the human disease cholera. Flagellum-mediated motility is an integral part of the bacterial life cycle inside the host and in the aquatic environment. The V. cholerae flagellar filament is composed of five flagellin subunits (FlaA, FlaB, FlaC, FlaD, and FlaE); however, only FlaA is necessary and sufficient for filament synthesis. flaA is transcribed from a class III flagellar promoter, whereas the other four flagellins are transcribed from class IV promoters. However, expressing flaA from a class IV promoter still facilitated motility in a strain that was otherwise lacking all five flagellins (ΔflaA-E). Furthermore, FlaA from V. parahaemolyticus (FlaAVP; 77% identity) supported motility of the V. cholerae ΔflaA-E strain, whereas FlaA from V. vulnificus (FlaAVV; 75% identity) did not, indicating that FlaA amino acid sequence is responsible for its critical role in flagellar synthesis. Chimeric proteins composed of different domains of FlaAVC and FlaD or FlaAVV revealed that the N-terminal D1 domain (D1N) contains an important region required for FlaA function. Further analyses of chimeric FlaAVC-FlaD proteins identified a lysine residue present at position 145 of the other flagellins but absent from FlaAVC that can prevent monofilament formation. Moreover, the D1N region of amino acids 87 to 153 of FlaAVV inserted into FlaAVC allows monofilament formation but not motility, apparently due to the lack of filament curvature. These results identify residues within the D1N domain that allow FlaAVC to fold into a functional filament structure and suggest that FlaAVC assists correct folding of the other flagellins.IMPORTANCEV. cholerae causes the severe diarrheal disease cholera. Its ability to swim is mediated by rotation of a polar flagellum, and this motility is integral to its ability to cause disease and persist in the environment. The current studies illuminate how one specific flagellin (FlaA) within a multiflagellin structure mediates formation of the flagellar filament, thus allowing V. cholerae to swim. This knowledge can lead to safer vaccines and potential therapeutics to inhibit cholera.

Efficient generation of marmoset primordial germ cell-like cells using induced pluripotent stem cells.

Reconstitution of germ cell fate from pluripotent stem cells provides an opportunity to understand the molecular underpinnings of germ cell development. Here, we established robust methods for induced pluripotent stem cell (iPSC) culture in the common marmoset (Callithrix jacchus [cj]), allowing stable propagation in an undifferentiated state. Notably, iPSCs cultured on a feeder layer in the presence of a WNT signaling inhibitor upregulated genes related to ubiquitin-dependent protein catabolic processes and enter a permissive state that enables differentiation into primordial germ cell-like cells (PGCLCs) bearing immunophenotypic and transcriptomic similarities to pre-migratory cjPGCs in vivo. Induction of cjPGCLCs is accompanied by transient upregulation of mesodermal genes, culminating in the establishment of a primate-specific germline transcriptional network. Moreover, cjPGCLCs can be expanded in monolayer while retaining the germline state. Upon co-culture with mouse testicular somatic cells, these cells acquire an early prospermatogonia-like phenotype. Our findings provide a framework for understanding and reconstituting marmoset germ cell development in vitro, thus providing a comparative tool and foundation for a preclinical modeling of human in vitro gametogenesis.