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Mass spectrometry based proteomics has become the method of choice for pinpointing and monitoring thousands of post-translational modifications, predominately phosphorylation sites, in cellular signaling studies. Critical for achieving this analytical depth is the enrichment of phosphorylated peptides prior to liquid chromatography-mass spectrometry (MS) analysis. Despite the high prevalence of this modification, the numbers of identified phosphopeptides lag behind those achieved for unmodified peptides, and the cause for this still remains controversial. Here, we use an effective phosphatase protocol that considerably improves global ionization efficiency and, therefore, the overall sensitivity and coverage of standard phosphoproteomics studies. We demonstrate the power of our method on the model system of Salmonella-infected macrophages by extending the current quantitative picture of immune signaling pathways involved in infection. In combination with sensitive, label-free targeted MS for phosphorylation site validation, our approach is ideally suited to exploring cellular phosphorylation based signaling networks in high detail.

Recognition of pathogens by the innate immune system relies on germline-encoded pattern recognition receptors (PRRs) that recognize unique microbial molecules, so-called pathogen-associated molecular patterns (PAMPs). Nucleic acids and their derivatives are one of the most important groups of PAMPs, and are recognized by a number of surface-associated as well as cytosolic PRRs. Cyclic GMP-AMP synthase (cGAS) recognizes the presence of pathogen- or host-derived dsDNA in the cytosol and initiates type-I-IFN production. Here, we describe a methodology that allows for evaluating the association of cGAS with released bacterial dsDNA during Francisella novicida infection of macrophages, by fluorescence confocal microscopy. This method can be adapted to the study of cGAS-dependent responses elicited by other intracellular bacterial pathogens and in other cell types.

Transcriptional regulators play an important role for the survival of Neisseria meningitidis within its human host. We have recently shown that FarR acts as transcriptional repressor of the adhesin nadA in N. meningitidis. Here, we examined the FarR regulon by microarray analyses, qRT-PCR, and electrophoretic mobility shift assays, revealing that FarR is a highly specific repressor of nadA. We demonstrate by reporter gene fusion assays that alterations of the FarR binding site within the nadA promoter are sufficient to induce transcription of nadA. Furthermore, farR expression is growth phase-dependent. The highest transcription rate was observed in the late-exponential growth phase of meningococci. Upon contact with active components of the complement system in normal human serum, expression of farR is slightly downregulated. Concluding, we present FarR as an exquisitely specialized, growth phase-dependent, possibly complement-responsive transcriptional regulator in N. meningitidis., (Copyright © 2011 Elsevier GmbH. All rights reserved.)