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Bacterial pathogens deliver proteins in temporal and spatial coordinated manner to manipulate mammalian host cells. The facultative intracellular pathogen Salmonella enterica remodels the host endosomal system for survival and proliferation inside host cells. The pathogen resides in a membrane-bound compartment termed Salmonella-containing vacuole (SCV). By Salmonella- induced fusions of host endomembranes, the SCV is connected with extensive tubular structures termed Salmonella-induced filaments (SIF). The intracellular lifestyle of Salmonella critically depends on effector molecules translocated by the SPI2-encoded type III secretion system (SPI2-T3SS) into host cells. A subset of these effectors is associated with, or integral in SCV and SIF membranes. It remained to be determined how SPI2-T3SS effectors reach their subcellular destination, and how these effectors interact with endomembranes remodeled by Salmonella. We deployed self-labeling enzyme (SLE) tags as novel approach to label translocated effector proteins in living host cells, and analyzed their dynamics on single molecule level. We found that SPI2-T3SS effector proteins diffuse in membranes of SIF with mobility comparable to membrane-integral host proteins in endomembranes. Dynamics differed between various effector proteins investigated and was dependent on membrane architecture of SIF. In the early infection, we observed host endosomal vesicles associated with Salmonella effector proteins. Effector-positive vesicles continuously fused with SCV and SIF membranes, providing a route of effector delivery by SPI2-T3SS translocation, interaction with endosomal vesicles, and ultimately fusion with the continuum of SCV/SIF membranes. This novel mechanism controls membrane deformation and vesicular fusion to generate the specific intracellular niche for bacterial survival and proliferation.

Today, we are faced with increasingly occurring bacterial infections that are hard to treat and often tend to relapse. These recurrent infections can occur possibly due to antibiotic-tolerant persister cells. Antibiotic persistent bacteria represent a small part of a bacterial population that enters a non-replicating (NR) state arising from phenotypic switching. Intracellularly, after uptake by phagocytic cells, Salmonella enterica serovar Typhimurium (STM) forms persister cells that are able to subvert immune defenses of the host. However, the clear physiological state and perceptual properties are still poorly understood and many questions remain unanswered. Here we describe further development of fluorescent protein-based reporter plasmids that were used to detect intracellular NR persister cells and monitor the expression of stress response genes via extensive flow cytometric analyses. Moreover, we performed extensive measurements of the metabolic properties of NR STM at the early course of infection. Our studies demonstrate that NR STM persister cells perceive their environment and are capable respond to stress factors. Since persisters showed a lower stress response compared to replicating (R) STM, which was not a consequence of a lower metabolic capacity, the persistent status of STM serves as protective niche. Furthermore, up to 95% of NR STM were metabolically active at the beginning of infection additionally showing no difference in the metabolic capacity compared to R STM. The accessory capability of NR STM persisters to sense and to react to stress with constant metabolic activity may supports the pathogen to create a more permissive environment for recurrent infections.

Intracellular bacteria such as Salmonella enterica are confronted with a broad array of defense mechanisms of their mammalian host cells. The ability to sense host cell-imposed damages, and to mount efficient stress responses are crucial for survival and proliferation of intracellular pathogens. The various combinations of host defense mechanisms acting on intracellular bacteria and their individual response also explain the occurrence of distinct subpopulations of intracellular S. enterica such as dormant or persisting, slowly or rapidly replicating cells. Here we describe a set of fluorescence protein (FP)-based reporter strains that were used to monitor the expression of cytoplasmic or periplasmic stress response systems on a single cell level. This is mediated by a fast maturing FP as reporter for induction of stress response genes. We evaluated slower maturing FPs for a second function, i.e. the analyses of the status of intracellular proliferation of pathogens. The combination of two FPs allows, on a single cell level, the interrogation of stress response and intracellular proliferation. Application of these reporters to S. enterica allowed us to detect and quantify distinct intracellular subpopulations with different levels of stress response and proliferation.ImportanceSensing of, and responding to host-mediated damages are important defensive virulence traits of bacterial pathogens. Intracellular pathogens such as Salmonella enterica are exposed to various types of antimicrobial host cell defenses that impose, among other, periplasmic and cytosolic stresses. Intracellular S. enterica form distinct subpopulations that differ in proliferation rate, metabolic activity and persister formation. Here we deploy fluorescence protein-based reporter strains to monitor, on a single cell level, the response of intracellular S. enterica to periplasmic or cytoplasmic stress. A second fluorescent protein reports the biosynthetic capacity of individual intracellular S. enterica. The dual fluorescence reporters can be deployed to characterize by flow cytometry phenotypically diverse subpopulations and stress responses in intracellular bacteria.

Intracellular bacteria such as Salmonella enterica are confronted with a broad array of defence mechanisms of their mammalian host cells. The ability to sense host cell-imposed damages, and to mount efficient stress responses are crucial for survival and proliferation of intracellular pathogens. The various combinations of host defence mechanisms acting on intracellular bacteria and their individual response also explain the occurrence of distinct subpopulations of intracellular S. enterica such as dormant or persisting, slowly or rapidly replicating cells. Here we describe a set of fluorescence protein (FP)-based reporter strains that were used to monitor the expression of cytoplasmic or periplasmic stress response systems of single bacterial cells. This is mediated by a fast-maturing FP as reporter for induction of stress response genes. We evaluated slower maturing FPs for a second function, that is, the analysis of the status of intracellular proliferation of pathogens. The combination of two FPs allows, at level of single bacterial cells, the interrogation of stress response and intracellular proliferation. Application of these reporters to S. enterica allowed us to detect and quantify distinct intracellular subpopulations with different levels of stress response and proliferation.

Intracellular Salmonella enterica serovar Typhimurium (STM) deploy the Salmonella Pathogenicity Island 2-encoded type III secretion system (SPI2-T3SS) for the massive remodeling of the endosomal system for host cells. This activity results in formation of an extensive interconnected tubular network of Salmonella-induced filaments (SIFs) connected to the Salmonella-containing vacuole (SCV). Such network is absent in cells infected with SPI2-T3SS-deficient mutant strains such as ΔssaV. A tubular network with reduced dimensions is formed if SPI2-T3SS effector protein SseF is absent. Previous single cell live microscopy-based analyses revealed that intracellular proliferation of STM is directly correlated to the ability to transform the host cell endosomal system into a complex tubular network. This network may also abrogate host defense mechanisms such as delivery of antimicrobial effectors to the SCV. To test the role of SIFs in STM patho-metabolism, we performed quantitative comparative proteomics of STM recovered from infected murine macrophages. We infected RAW264.7 cells with STM wild type (WT), ΔsseF or ΔssaV strains, recovered bacteria 12 h after infection and determined proteome compositions. Increased numbers of proteins characteristic for nutritional starvation were detected in STM ΔsseF and ΔssaV compared to WT. In addition, STM ΔssaV, but not ΔsseF showed signatures of increased exposure to stress by antimicrobial defenses, in particular reactive oxygen species, of the host cells. The proteomics analyses presented here support and extend the role of SIFs for the intracellular lifestyle of STM. We conclude that efficient manipulation of the host cell endosomal system by effector proteins of the SPI2-T3SS contributes to nutrition, as well as to resistance against antimicrobial host defense mechanisms., Author summary The facultative intracellular bacterium Salmonella enterica has evolved sophisticated mechanisms to adapt to life inside a pathogen-containing vacuole in mammalian host cells. Intracellular Salmonella manipulate the host cell endosomal system resulting in formation of a complex network of tubular vesicles, termed Salmonella-induced filaments (SIFs). We applied quantitative proteomics to intracellular Salmonella in murine macrophages and compared the wild-type strain to mutant strains with aberrant SIF architecture, or no capacity for induction of SIF. We determined that those mutant strains contain higher amounts of transporters for nutrient uptake, and lower amounts of proteins for central carbon metabolism. These observations indicate response to nutrient restriction in absence of fully established SIF. In addition, the mutant strain unable to induce SIF formation showed increased amounts of proteins required for response to antimicrobial factors of the host cells. These data show that the massive remodeling of the endosomal system of host cells by intracellular Salmonella serves to essential needs, i.e. to enable access to nutrients for efficient proliferation of the pathogen, and to withstand hostile conditions within the pathogen-containing vacuole.

The expression of bacterial virulence factors is controlled in response to host or environmental factors and most virulence genes are not expressed under laboratory conditions. Investigations of molecular structures and cellular functions of bacterial virulence factors demand systems for experimentally controlled expression. We describe a simple and robust system that is based on the tetA promoter and the cognate repressor TetR. Expression under control of PtetA can be induced by non-antibiotic derivatives of tetracycline such as anhydrotetracycline (AHT). Tet-on expression cassettes can be used to replace native promoters of chromosomal genes or operons of interest. Tet-on plasmids allow episomal expression in homologous or heterologous host organisms. We demonstrate the application of Tet-on systems for the controlled induction of flagella assembly and motility, and for surface expression of adhesins of the chaperone/usher family of enteropathogenic Escherichia coli and autotransporter adhesins of Yersinia enterocolitica in Salmonella enterica and E. coli. Since inducer AHT can easily cross bacterial envelopes and mammalian cell membranes, the system can also be applied to control virulence genes in intracellular bacteria. We demonstrate the controlled synthesis, translocation and function of effector proteins of the type III secretion system of intracellular S. enterica.

The notion of cancer as a complex evolutionary system has been validated by in-depth molecular analyses of tumor progression over the last years. While a complex interplay of cell-autonomous programs and cell-cell interactions determines proliferation and differentiation during normal development, intrinsic and acquired plasticity of cancer cells allow for evasion of growth factor limitations, apoptotic signals, or attacks from the immune system. Treatment-induced molecular selection processes have been described by a number of studies already, but understanding of those events facilitating metastatic spread, organ-specific homing, and resistance to anoikis is still in its early days. In principle, somatic events giving rise to cancer progression should be easier to follow in childhood tumors bearing fewer mutations and genomic aberrations than their counterparts in adulthood. We have previously reported on the genetic events accompanying relapsing neuroblastoma, a solid tumor of early childhood. Our results indicated significantly higher single nucleotide variants in relapse tumors, gave hints for branched tumor evolution upon treatment and clonal selection as deduced from shifts in allelic frequencies between primary and relapsing neuroblastoma. Here, we will review these findings and give an outlook on dealing with intratumoral heterogeneity and sub-clonal diversity in neuroblastoma for future targeted treatments.

Current therapy of medulloblastoma, the most common malignant brain tumor of childhood, achieves 40-70% survival. Secondary chemotherapy resistance contributes to treatment failure, where TP53 pathway dysfunction plays a key role. MDM2 interaction with TP53 leads to its degradation. Reactivating TP53 functionality using small-molecule inhibitors, such as RITA, to disrupt TP53-MDM2 binding may have therapeutic potential. We show here that RITA decreased viability of all 4 analyzed medulloblastoma cell lines, regardless of TP53 functional status. The decrease in cell viability was accompanied in 3 of the 4 medulloblastoma cell lines by accumulation of TP53 protein in the cells and increased CDKN1A expression. RITA treatment in mouse models inhibited medulloblastoma xenograft tumor growth. These data demonstrate that RITA treatment reduces medulloblastoma cell viability in both in vitro and in vivo models, and acts independently of cellular TP53 status, identifying RITA as a potential therapeutic agent to treat medulloblastoma.

OA gold Amplification or overexpression of MYCN is involved in development and maintenance of multiple malignancies. A subset of these tumors originates from neural precursors, including the most aggressive forms of the childhood tumors, neuroblastoma and medulloblastoma. In order to model the spectrum of MYCN-driven neoplasms in mice, we transgenically overexpressed MYCN under the control of the human GFAP-promoter that, among other targets, drives expression in neural progenitor cells. However, LSL-MYCN;hGFAP-Cre double transgenic mice did neither develop neural crest tumors nor tumors of the central nervous system, but presented with neuroendocrine tumors of the pancreas and, less frequently, the pituitary gland. Pituitary tumors expressed chromogranin A and closely resembled human pituitary adenomas. Pancreatic tumors strongly produced and secreted glucagon, suggesting that they derived from glucagon- and GFAP-positive islet cells. Interestingly, 3 out of 9 human pancreatic neuroendocrine tumors expressed MYCN, supporting the similarity of the mouse tumors to the human system. Serial transplantations of mouse tumor cells into immunocompromised mice confirmed their fully transformed phenotype. MYCN-directed treatment by AuroraA- or Brd4-inhibitors resulted in significantly decreased cell proliferation in vitro and reduced tumor growth in vivo. In summary, we provide a novel mouse model for neuroendocrine tumors of the pancreas and pituitary gland that is dependent on MYCN expression and that may help to evaluate MYCN-directed therapies.

The apoptosis-inducing Fas ligand (FasL) is a type II transmembrane protein that is involved in the downregulation of immune reactions by activation-induced cell death (AICD) as well as in T cell-mediated cytotoxicity. Proteolytic cleavage leads to the generation of membrane-bound N-terminal fragments and a soluble FasL (sFasL) ectodomain. sFasL can be detected in the serum of patients with dysregulated inflammatory diseases and is discussed to affect Fas-FasL-mediated apoptosis. Using pharmacological approaches in 293T cells, in vitro cleavage assays as well as loss and gain of function studies in murine embryonic fibroblasts (MEFs), we demonstrate that the disintegrin and metalloprotease ADAM10 is critically involved in the shedding of FasL. In primary human T cells, FasL shedding is significantly reduced after inhibition of ADAM10. The resulting elevated FasL surface expression is associated with increased killing capacity and an increase of T cells undergoing AICD. Overall, our findings suggest that ADAM10 represents an important molecular modulator of FasL-mediated cell death.

Gamma-protocadherins (Pcdh gamma) are type I transmembrane proteins, which are most notably expressed in the nervous system. They are enriched at synapses and involved in synapse formation, specification, and maintenance. In this study, we show that Pcdh gamma C3 and Pcdh gamma B4 are specifically cleaved within their ectodomains by the disintegrin and metalloprotease ADAM10. Analysis of ADAM10-deficient fibroblasts and embryos, inhibitor studies, as well as RNA interference-mediated down-regulation demonstrated that ADAM10 is not only responsible for the constitutive but also for the regulated shedding of these proteins in fibroblasts and in neuronal cells. In contrast to N-cadherin shedding, which was activated by N-methyl-D-aspartic acid receptor activation in neuronal cells, Pcdh gamma shedding was induced by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid hydrate stimulation, suggesting differential regulation mechanisms of cadherin-mediated functions at synapses. Cell aggregation assays in the presence or absence of metalloprotease inhibitors strongly suggest that the ectodomain shedding events modulate the cell adhesion role of Pcdh gamma. The identification of ADAM10 as the protease responsible for constitutive and regulated Pcdh gamma shedding may therefore provide new insight into the regulation of Pcdh gamma functions.

Rhodobacter capsulatus xanthine dehydrogenase (XDH) is a cytoplasmic enzyme with an (alpha beta) 2 heterodimeric structure that is highly identical to homodimeric eukaryotic xanthine oxidoreductases. The crystal structure revealed that the molybdenum cofactor (Moco) is deeply buried within the protein. A protein involved in Moco insertion and XDH maturation has been identified, which was designated XdhC. XdhC was shown to be essential for the production of active XDH but is not a subunit of the purified enzyme. Here we describe the purification of XdhC and the detailed characterization of its role for XDH maturation. We could show that XdhC binds Moco in stoichiometric amounts, which subsequently can be inserted into Moco-free apo-XDH. A specific interaction between XdhC and XdhB was identified. We show that XdhC is required for the stabilization of the sulfurated form of Moco present in enzymes of the xanthine oxidase family. Our findings imply that enzyme-specific proteins exist for the biogenesis of molybdoenzymes, coordinating Moco binding and insertion into their respective target proteins. So far, the requirement of such proteins for molybdoenzyme maturation has been described only for prokaryotes

Signaling via the epidermal growth factor receptor (EGFR), which has critical roles in development and diseases such as cancer, is regulated by proteolytic shedding of its membrane-tethered ligands. Sheddases for EGFR-ligands are therefore key signaling switches in the EGFR pathway. Here, we determined which ADAMs (a disintegrin and metalloprotease) can shed various EGFR-ligands, and we analyzed the regulation of EGFR-ligand shedding by two commonly used stimuli, phorbol esters and calcium influx. Phorbol esters predominantly activate ADAM17, thereby triggering a burst of shedding of EGFR-ligands from a late secretory pathway compartment. Calcium influx stimulates ADAM10, requiring its cytoplasmic domain. However, calcium influx-stimulated shedding of transforming growth factor α and amphiregulin does not require ADAM17, even though ADAM17 is essential for phorbol ester-stimulated shedding of these EGFR-ligands. This study provides new insight into the machinery responsible for EGFR-ligand release and thus EGFR signaling and demonstrates that dysregulated EGFR-ligand shedding may be caused by increased expression of constitutively active sheddases or activation of different sheddases by distinct stimuli.