Bright individuals: Applications of fluorescent protein-based reporter systems in single-cell cellular microbiology.

Activation and function of virulence functions of bacterial pathogens are highly dynamic in time and space, and can show considerable heterogeneity between individual cells in pathogen populations. To investigate the complex events in host-pathogen interactions, single cell analyses are required. Fluorescent proteins (FPs) are excellent tools to follow the fate of individual bacterial cells during infection, and can also be deployed to use the pathogen as a sensor for its specific environment in host cells or host organisms. This Resources describes design and applications of dual fluorescence reporters (DFR) in cellular microbiology. DFR feature constitutively expressed FPs for detection of bacterial cells, and FPs expressed by an environmentally regulated promoter for interrogation of niche-specific cues or nutritional parameters. Variations of the basic design allow the generation of DFR that can be used to analyze, on single cell level, bacterial proliferation during infection, subcellular localization of intracellular bacteria, stress response, or persister state. We describe basic considerations for DFR design and review recent applications of DFR in cellular microbiology.

Fluorescent protein-based reporters reveal stress response of intracellular Salmonella enterica at level of single bacterial cells.

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.

Proteomics of intracellular Salmonella enterica reveals roles of Salmonella pathogenicity island 2 in metabolism and antioxidant defense.

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.

A versatile remote control system for functional expression of bacterial virulence genes based on the tetA promoter.

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.

Cancer evolution, mutations, and clonal selection in relapse neuroblastoma.

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.

Single molecule analyses reveal dynamics of Salmonella translocated effector proteins in host cell endomembranes.

The facultative intracellular pathogen Salmonella enterica remodels the host endosomal system for survival and proliferation inside host cells. Salmonella resides within the Salmonella-containing vacuole (SCV) and 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 proteins translocated into host cells. A subset of effectors is associated with, or integral in SCV and SIF membranes. How effectors reach their subcellular destination, and how they interact with endomembranes remodeled by Salmonella remains to be determined. We deployed self-labeling enzyme tags to label translocated effectors in living host cells, and analyzed their single molecule dynamics. Translocated effectors diffuse in membranes of SIF with mobility comparable to membrane-integral host proteins in endomembranes. Dynamics differ between various effectors investigated and is dependent on membrane architecture of SIF. In the early infection, host endosomal vesicles are associated with Salmonella effectors. Effector-positive vesicles continuously fuse with SCV and SIF membranes, providing a route of effector delivery by translocation, interaction with endosomal vesicles, and ultimately fusion with the continuum of SCV/SIF membranes. This mechanism controls membrane deformation and vesicular fusion to generate the specific intracellular niche for bacterial survival and proliferation.

Flow Cytometry-Based Single Cell Analyses of Bacterial Adaptation to Intracellular Environments.

Since decades, flow cytometry (FC) is a powerful technique to perform single cell analyses with high accuracy and throughput. Moreover, FC is the method of choice to study bacterial cell heterogeneity and complements single-cell imaging techniques. The complex experimental approaches for FC sample preparation and the subsequent FC adjustment and gating strategy demand careful considerations to be successful when analyzing complex microbial populations, especially when liberated populations of intracellular bacterial pathogens, or bacterial pathogens inside intact host cells are analyzed. Here, we provide a set of experimental protocols for FC sample preparation of (1) in vitro cultured bacterial cells, (2) liberated intracellular bacteria from host cells, or (3) preparation of intact infected phagocytic or epithelial cells commonly used as host cells in infection biology. Since sample preparation, cytometer adjustment, and gating strategy are essential for experimental success, we aim to provide our expertise to support application of FC by other researchers.

Exposure to stressors and antimicrobials induces cell-autonomous ultrastructural heterogeneity of an intracellular bacterial pathogen.

Despite their clonality, intracellular bacterial pathogens commonly show remarkable physiological heterogeneity during infection of host cells. Physiological heterogeneity results in distinct ultrastructural morphotypes, but the correlation between bacterial physiological state and ultrastructural appearance remains to be established. In this study, we showed that individual cells of Salmonella enterica serovar Typhimurium are heterogeneous in their ultrastructure. Two morphotypes based on the criterion of cytoplasmic density were discriminated after growth under standard culture conditions, as well as during intracellular lifestyle in mammalian host cells. We identified environmental conditions which affect cytoplasmic densities. Using compounds generating oxygen radicals and defined mutant strains, we were able to link the occurrence of an electron-dense ultrastructural morphotype to exposure to oxidative stress and other stressors. Furthermore, by combining ultrastructural analyses of Salmonella during infection and fluorescence reporter analyses for cell viability, we provided evidence that two characterized ultrastructural morphotypes with electron-lucent or electron-dense cytoplasm represent viable cells. Moreover, the presence of electron-dense types is stress related and can be experimentally induced only when amino acids are available in the medium. Our study proposes ultrastructural morphotypes as marker for physiological states of individual intracellular pathogens providing a new marker for single cell analyses.

The protected physiological state of intracellular Salmonella enterica persisters reduces host cell-imposed stress.

During infectious diseases, small subpopulations of bacterial pathogens enter a non-replicating (NR) state tolerant to antibiotics. After phagocytosis, intracellular Salmonella enterica serovar Typhimurium (STM) forms persisters able to subvert immune defenses of the host. Physiological state and sensing properties of persisters are difficult to analyze, thus poorly understood. Here we deploy fluorescent protein reporters to detect intracellular NR persister cells, and to monitor their stress response on single cell level. We determined metabolic properties of NR STM during infection and demonstrate that NR STM persisters sense their environment and respond to stressors. Since persisters showed a lower stress response compared to replicating (R) STM, which was not consequence of lower metabolic capacity, the persistent state of STM serves as protective niche. Up to 95% of NR STM were metabolically active at beginning of infection, very similar to metabolic capacity of R STM. Sensing and reacting to stress with constant metabolic activity supports STM to create a more permissive environment for recurrent infections. Stress sensing and response of persister may be targeted by new antimicrobial approaches.

Substrate selectivity of epidermal growth factor-receptor ligand sheddases and their regulation by phorbol esters and calcium influx.

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 alpha 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.

L1 is sequentially processed by two differently activated metalloproteases and presenilin/gamma-secretase and regulates neural cell adhesion, cell migration, and neurite outgrowth.

The immunoglobulin superfamily recognition molecule L1 plays important functional roles in the developing and adult nervous system. Metalloprotease-mediated cleavage of this adhesion molecule has been shown to stimulate cellular migration and neurite outgrowth. We demonstrate here that L1 cleavage is mediated by two distinct members of the disintegrin and metalloprotease family, ADAM10 and ADAM17. This cleavage is differently regulated and leads to the generation of a membrane bound C-terminal fragment, which is further processed through gamma-secretase activity. Pharmacological approaches with two hydroxamate-based inhibitors with different preferences in blocking ADAM10 and ADAM17, as well as loss of function and gain of function studies in murine embryonic fibroblasts, showed that constitutive shedding of L1 is mediated by ADAM10 while phorbol ester stimulation or cholesterol depletion led to ADAM17-mediated L1 cleavage. In contrast, N-methyl-d-aspartate treatment of primary neurons stimulated ADAM10-mediated L1 shedding. Both proteases were able to affect L1-mediated adhesion and haptotactic migration of neuronal cells. In particular, both proteases were involved in L1-dependent neurite outgrowth of cerebellar neurons. Thus, our data identify ADAM10 and ADAM17 as differentially regulated L1 membrane sheddases, both critically affecting the physiological functions of this adhesion protein.

RITA displays anti-tumor activity in medulloblastomas independent of TP53 status.

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.

Models of intestinal infection by Salmonella enterica: introduction of a new neonate mouse model.

Salmonella enterica serovar Typhimurium is a foodborne pathogen causing inflammatory disease in the intestine following diarrhea and is responsible for thousands of deaths worldwide. Many in vitro investigations using cell culture models are available, but these do not represent the real natural environment present in the intestine of infected hosts. Several in vivo animal models have been used to study the host-pathogen interaction and to unravel the immune responses and cellular processes occurring during infection. An animal model for Salmonella-induced intestinal inflammation relies on the pretreatment of mice with streptomycin. This model is of great importance but still shows limitations to investigate the host-pathogen interaction in the small intestine in vivo. Here, we review the use of mouse models for Salmonella infections and focus on a new small animal model using 1-day-old neonate mice. The neonate model enables researchers to observe infection of both the small and large intestine, thereby offering perspectives for new experimental approaches, as well as to analyze the Salmonella-enterocyte interaction in the small intestine in vivo.

Characterization of pancreatic glucagon-producing tumors and pituitary gland tumors in transgenic mice overexpressing MYCN in hGFAP-positive cells.

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.

Rhodobacter capsulatus XdhC is involved in molybdenum cofactor binding and insertion into xanthine dehydrogenase.

Rhodobacter capsulatus xanthine dehydrogenase (XDH) is a cytoplasmic enzyme with an (alphabeta)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.

Regulated ADAM10-dependent ectodomain shedding of gamma-protocadherin C3 modulates cell-cell adhesion.

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.