The Mycobacterium tuberculosis protein O-phosphorylation landscape.

Bacterial phosphosignalling has been synonymous with two-component systems and their histidine kinases, but many bacteria, including Mycobacterium tuberculosis (Mtb), also code for Ser/Thr protein kinases (STPKs). STPKs are the main phosphosignalling enzymes in eukaryotes but the full extent of phosphorylation on protein Ser/Thr and Tyr (O-phosphorylation) in bacteria is untested. Here we explored the global signalling capacity of the STPKs in Mtb using a panel of STPK loss-of-function and overexpression strains combined with mass spectrometry-based phosphoproteomics. A deep phosphoproteome with >14,000 unique phosphosites shows that O-phosphorylation in Mtb is a vastly underexplored protein modification that affects >80% of the proteome and extensively interfaces with the transcriptional machinery. Mtb O-phosphorylation gives rise to an expansive, distributed and cooperative network of a complexity that has not previously been seen in bacteria and that is on par with eukaryotic phosphosignalling networks. A resource of >3,700 high-confidence direct substrate-STPK interactions and their transcriptional effects provides signalling context for >80% of Mtb proteins and allows the prediction and assembly of signalling pathways for mycobacterial physiology.

Primary and multipassage culture of human fetal kidney epithelial progenitor cells.

Homogeneous, well-characterized cultures of kidney cells representative of defined cellular phenotypes comprising the developing and adult kidney provide important tools to investigate kidney biology. Further, the development of defined media for these culture systems provides opportunities to investigate the role of nutrients, hormones, and matrix components, as well as exogenous insults, in renal development, function, and toxicity. The current explosion in stem cell research has fueled an expanded effort to develop techniques to isolate and culture kidney progenitor and stem cells, which have the potential to treat various forms of renal disease. In this chapter, we outline methods to initiate and propagate long-term cultures of highly homogeneous fetal kidney epithelial progenitor cells. By utilizing a low calcium-containing serum-free culture medium together with a set of defined hormones and extracellular matrix, kidney epithelial progenitor cells can be cultured for more than 60 population doublings without loss of growth potential or phenotypic signs of differentiation. The cultures appear to represent early kidney epithelial progenitors based on cellular marker expression. The cells express the mRNA encoding the embryonic kidney mesenchyme/epithelial marker PAX-2, the stem cell protein CD133, the kidney embryonic progenitor protein CD24, as well as CD29 and CD44. The cells are negative for E-cadherin when grown under low calcium conditions (<0.05 mM); however, E-cadherin expression is induced when cells are cultured under normal calcium conditions (1.2 mM), suggesting that differentiation of the kidney epithelial progenitor culture can be modulated in part by altering the calcium concentration of the medium.