Spatially resolved whole transcriptome profiling in human and mouse tissue using Digital Spatial Profiling.

Emerging spatial profiling technology has enabled high-plex molecular profiling in biological tissues, preserving the spatial and morphological context of gene expression. Here, we describe expanding the chemistry for the Digital Spatial Profiling platform to quantify whole transcriptomes in human and mouse tissues using a wide range of spatial profiling strategies and sample types. We designed multiplexed in situ hybridization probes targeting the protein-coding genes of the human and mouse transcriptomes, referred to as the human or mouse Whole Transcriptome Atlas (WTA). Human and mouse WTAs were validated in cell lines for concordance with orthogonal gene expression profiling methods in regions ranging from ∼10-500 cells. By benchmarking against bulk RNA-seq and fluorescence in situ hybridization, we show robust transcript detection down to ∼100 transcripts per region. To assess the performance of WTA across tissue and sample types, we applied WTA to biological questions in cancer, molecular pathology, and developmental biology. Spatial profiling with WTA detected expected gene expression differences between tumor and tumor microenvironment, identified disease-specific gene expression heterogeneity in histological structures of the human kidney, and comprehensively mapped transcriptional programs in anatomical substructures of nine organs in the developing mouse embryo. Digital Spatial Profiling technology with the WTA assays provides a flexible method for spatial whole transcriptome profiling applicable to diverse tissue types and biological contexts.

Identification of Small-Molecule Modulators of Diguanylate Cyclase by FRET-Based High-Throughput Screening.

The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) is a key regulator of cellular motility, the cell cycle, and biofilm formation with its resultant antibiotic tolerance, which can make chronic infections difficult to treat. Therefore, diguanylate cyclases, which regulate the spatiotemporal production of c-di-GMP, might be attractive drug targets for control of biofilm formation that is part of chronic infections. We present a FRET-based biochemical high-throughput screening approach coupled with detailed structure-activity studies to identify synthetic small-molecule modulators of the diguanylate cyclase DgcA from Caulobacter crescentus. We identified a set of seven small molecules that regulate DgcA enzymatic activity in the low-micromolar range. Subsequent structure-activity studies on selected scaffolds revealed a remarkable diversity of modulatory behavior, including slight chemical substitutions that reverse the effects from allosteric enzyme inhibition to activation. The compounds identified represent new chemotypes and are potentially developable into chemical genetic tools for the dissection of c-di-GMP signaling networks and alteration of c-di-GMP-associated phenotypes. In sum, our studies underline the importance of detailed mechanism-of-action studies for inhibitors of c-di-GMP signaling and demonstrate the complex interplay between synthetic small molecules and the regulatory mechanisms that control the activity of diguanylate cyclases.

Human Diversity in a Cell Surface Receptor that Inhibits Autophagy.

Mutations in genes encoding autophagy proteins have been associated with human autoimmune diseases, suggesting that diversity in autophagy responses could be associated with disease susceptibility or severity. A cellular genome-wide association study (GWAS) screen was performed to explore normal human diversity in responses to rapamycin, a microbial product that induces autophagy. Cells from several human populations demonstrated variability in expression of a cell surface receptor, CD244 (SlamF4, 2B4), that correlated with changes in rapamycin-induced autophagy. High expression of CD244 and receptor activation with its endogenous ligand CD48 inhibited starvation- and rapamycin-induced autophagy by promoting association of CD244 with the autophagy complex proteins Vps34 and Beclin-1. The association of CD244 with this complex reduced Vps34 lipid kinase activity. Lack of CD244 is associated with auto-antibody production in mice, and lower expression of human CD244 has previously been implicated in severity of human rheumatoid arthritis and systemic lupus erythematosus, indicating that increased autophagy as a result of low levels of CD244 may alter disease outcomes.

A direct screen for c-di-GMP modulators reveals a Salmonella Typhimurium periplasmic ʟ-arginine-sensing pathway.

Cyclic-di-GMP (c-di-GMP) is a bacterial second messenger that transduces internal and external signals and regulates bacterial motility and biofilm formation. Some organisms encode more than 100 c-di-GMP-modulating enzymes, but only for a few has a signal been defined that modulates their activity. We developed and applied a high-throughput, real-time flow cytometry method that uses a fluorescence resonance energy transfer (FRET)-based biosensor of free c-di-GMP to screen for signals that modulate its concentration within Salmonella Typhimurium. We identified multiple compounds, including glucose, N-acetyl-d-glucosamine, salicylic acid, and ʟ-arginine, that modulated the FRET signal and therefore the free c-di-GMP concentration. By screening a library of mutants, we identified proteins required for the c-di-GMP response to each compound. Furthermore, low micromolar concentrations of ʟ-arginine induced a rapid translation-independent increase in c-di-GMP concentrations and c-di-GMP-dependent cellulose synthesis, responses that required the regulatory periplasmic domain of the diguanylate cyclase STM1987. ʟ-Arginine signaling also required the periplasmic putative ʟ-arginine-binding protein ArtI, implying that ʟ-arginine sensing occurred in the periplasm. Among the 20 commonly used amino acids, S. Typhimurium specifically responded to ʟ-arginine with an increase in c-di-GMP, suggesting that ʟ-arginine may serve as a signal during S. Typhimurium infection. Our results demonstrate that a second-messenger biosensor can be used to identify environmental signals and define pathways that alter microbial behavior.

Delivery of cardiolipins to the Salmonella outer membrane is necessary for survival within host tissues and virulence.

The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer that serves as a barrier to the environment. During infection, Gram-negative bacteria remodel their OM to promote survival and replication within host tissues. Salmonella rely on the PhoPQ two-component regulators to coordinate OM remodeling in response to environmental cues. In a screen for mediators of PhoPQ-regulated OM remodeling in Salmonella Typhimurium, we identified PbgA, a periplasmic domain-containing transmembrane protein, which binds cardiolipin glycerophospholipids near the inner membrane and promotes their PhoPQ-regulated trafficking to the OM. Purified-PbgA oligomers are tetrameric, and the periplasmic domain contains a globular region that binds to the OM in a PhoPQ-dependent manner. Thus, PbgA forms a complex that may bridge the envelope for regulated cardiolipin delivery. PbgA globular region-deleted mutant bacteria are severely attenuated for pathogenesis, suggesting that increased cardiolipin trafficking to the OM is necessary for Salmonella to survive within host tissues that activate PhoPQ.

Pseudomonas aeruginosa in vitro phenotypes distinguish cystic fibrosis infection stages and outcomes.

RATIONALE: Pseudomonas aeruginosa undergoes phenotypic changes during cystic fibrosis (CF) lung infection. Although mucoidy is traditionally associated with transition to chronic infection, we hypothesized that additional in vitro phenotypes correlate with this transition and contribute to disease. OBJECTIVES: To characterize the relationships between in vitro P. aeruginosa phenotypes, infection stage, and clinical outcomes. METHODS: A total of 649 children with CF and newly identified P. aeruginosa were followed for a median 5.4 years during which a total of 2,594 P. aeruginosa isolates were collected. Twenty-six in vitro bacterial phenotypes were assessed among the isolates, including measures of motility, exoproduct production, colony morphology, growth, and metabolism. MEASUREMENTS AND MAIN RESULTS: P. aeruginosa phenotypes present at the time of culture were associated with both stage of infection (new onset, intermittent, or chronic) and the primary clinical outcome, occurrence of a pulmonary exacerbation (PE) in the subsequent 2 years. Two in vitro P. aeruginosa phenotypes best distinguished infection stages: pyoverdine production (31% of new-onset cultures, 48% of intermittent, 69% of chronic) and reduced protease production (31%, 39%, and 65%, respectively). The best P. aeruginosa phenotypic predictors of subsequent occurrence of a PE were mucoidy (odds ratio, 1.75; 95% confidence interval, 1.19-2.57) and reduced twitching motility (odds ratio, 1.43; 95% confidence interval, 1.11-1.84). CONCLUSIONS: In this large epidemiologic study of CF P. aeruginosa adaptation, P. aeruginosa isolates exhibited two in vitro phenotypes that best distinguished early and later infection stages. Among the many phenotypes tested, mucoidy and reduced twitching best predicted subsequent PE. These phenotypes indicate potentially useful prognostic markers of transition to chronic infection and advancing lung disease.

Pseudomonas aeruginosa phenotypes associated with eradication failure in children with cystic fibrosis.

BACKGROUND: Pseudomonas aeruginosa is a key respiratory pathogen in people with cystic fibrosis (CF). Due to its association with lung disease progression, initial detection of P. aeruginosa in CF respiratory cultures usually results in antibiotic treatment with the goal of eradication. Pseudomonas aeruginosa exhibits many different phenotypes in vitro that could serve as useful prognostic markers, but the relative relationships between these phenotypes and failure to eradicate P. aeruginosa have not been well characterized. METHODS: We measured 22 easily assayed in vitro phenotypes among the baseline P. aeruginosa isolates collected from 194 participants in the 18-month EPIC clinical trial, which assessed outcomes after antibiotic eradication therapy for newly identified P. aeruginosa. We then evaluated the associations between these baseline isolate phenotypes and subsequent outcomes during the trial, including failure to eradicate after antipseudomonal therapy, emergence of mucoidy, and occurrence of an exacerbation. RESULTS: Baseline P. aeruginosa isolates frequently exhibited phenotypes thought to represent chronic adaptation, including mucoidy. Wrinkly colony surface and irregular colony edges were both associated with increased risk of eradication failure (hazard ratios [95% confidence intervals], 1.99 [1.03-3.83] and 2.14 [1.32-3.47], respectively). Phenotypes reflecting defective quorum sensing were significantly associated with subsequent mucoidy, but no phenotype was significantly associated with subsequent exacerbations during the trial. CONCLUSIONS: Pseudomonas aeruginosa phenotypes commonly considered to reflect chronic adaptation were observed frequently among isolates at early detection. We found that 2 easily assayed colony phenotypes were associated with failure to eradicate after antipseudomonal therapy, both of which have been previously associated with altered biofilm formation and defective quorum sensing.

c-di-GMP heterogeneity is generated by the chemotaxis machinery to regulate flagellar motility.

Individual cell heterogeneity is commonly observed within populations, although its molecular basis is largely unknown. Previously, using FRET-based microscopy, we observed heterogeneity in cellular c-di-GMP levels. In this study, we show that c-di-GMP heterogeneity in Pseudomonas aeruginosa is promoted by a specific phosphodiesterase partitioned after cell division. We found that subcellular localization and reduction of c-di-GMP levels by this phosphodiesterase is dependent on the histidine kinase component of the chemotaxis machinery, CheA, and its phosphorylation state. Therefore, individual cell heterogeneity in c-di-GMP concentrations is regulated by the activity and the asymmetrical inheritance of the chemotaxis organelle after cell division. c-di-GMP heterogeneity results in a diversity of motility behaviors. The generation of diverse intracellular concentrations of c-di-GMP by asymmetric partitioning is likely important to the success and survival of bacterial populations within the environment by allowing a variety of motility behaviors. DOI: http://dx.doi.org/10.7554/eLife.01402.001.

The response threshold of Salmonella PilZ domain proteins is determined by their binding affinities for c-di-GMP.

c-di-GMP is a bacterial second messenger that is enzymatically synthesized and degraded in response to environmental signals. Cellular processes are affected when c-di-GMP binds to receptors which include proteins that contain the PilZ domain. Although each c-di-GMP synthesis or degradation enzyme metabolizes the same molecule, many of these enzymes can be linked to specific downstream processes. Here we present evidence that c-di-GMP signalling specificity is achieved through differences in affinities of receptor macromolecules. We show that the PilZ domain proteins of Salmonella Typhimurium, YcgR and BcsA, demonstrate a 43-fold difference in their affinity for c-di-GMP. Modulation of the affinities of these proteins altered their activities in a predictable manner in vivo. Inactivation of yhjH, which encodes a predicted c-di-GMP degrading enzyme, increased the fraction of the cellular population that demonstrated c-di-GMP levels high enough to bind to the higher-affinity YcgR protein and inhibit motility, but not high enough to bind to the lower-affinity BcsA protein and stimulate cellulose production. Finally, PilZ domain proteins of Pseudomonas aeruginosa demonstrated a 145-fold difference in binding affinities, suggesting that regulation by binding affinity may be a conserved mechanism that allows organisms with many c-di-GMP binding macromolecules to rapidly integrate multiple environmental signals into one output.

Asymmetrical distribution of the second messenger c-di-GMP upon bacterial cell division.

The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP) regulates cellular motility and the synthesis of organelles and molecules that promote adhesion to a variety of biological and nonbiological surfaces. These properties likely require tight spatial and temporal regulation of c-di-GMP concentration. We have developed genetically encoded fluorescence resonance energy transfer (FRET)-based biosensors to monitor c-di-GMP concentrations within single bacterial cells by microscopy. Fluctuations of c-di-GMP were visualized in diverse Gram-negative bacterial species and observed to be cell cycle dependent. Asymmetrical distribution of c-di-GMP in the progeny correlated with the time of cell division and polarization for Caulobacter crescentus and Pseudomonas aeruginosa. Thus, asymmetrical distribution of c-di-GMP was observed as part of cell division, which may indicate an important regulatory step in extracellular organelle biosynthesis or function.

Analysis of the genome of the Escherichia coli O157:H7 2006 spinach-associated outbreak isolate indicates candidate genes that may enhance virulence.

In addition to causing diarrhea, Escherichia coli O157:H7 infection can lead to hemolytic-uremic syndrome (HUS), a severe disease characterized by hemolysis and renal failure. Differences in HUS frequency among E. coli O157:H7 outbreaks have been noted, but our understanding of bacterial factors that promote HUS is incomplete. In 2006, in an outbreak of E. coli O157:H7 caused by consumption of contaminated spinach, there was a notably high frequency of HUS. We sequenced the genome of the strain responsible (TW14359) with the goal of identifying candidate genetic factors that contribute to an enhanced ability to cause HUS. The TW14359 genome contains 70 kb of DNA segments not present in either of the two reference O157:H7 genomes. We identified seven putative virulence determinants, including two putative type III secretion system effector proteins, candidate genes that could result in increased pathogenicity or, alternatively, adaptation to plants, and an intact anaerobic nitric oxide reductase gene, norV. We surveyed 100 O157:H7 isolates for the presence of these putative virulence determinants. A norV deletion was found in over one-half of the strains surveyed and correlated strikingly with the absence of stx(1). The other putative virulence factors were found in 8 to 35% of the O157:H7 isolates surveyed, and their presence also correlated with the presence of norV and the absence of stx(1), indicating that the presence of norV may serve as a marker of a greater propensity for HUS, similar to the correlation between the absence of stx(1) and a propensity for HUS.

Evidence for globally shared, cross-reacting polymorphic epitopes in the pregnancy-associated malaria vaccine candidate VAR2CSA.

Pregnancy-associated malaria (PAM) is characterized by the placental sequestration of Plasmodium falciparum-infected erythrocytes (IEs) with the ability to bind to chondroitin sulfate A (CSA). VAR2CSA is a leading candidate for a pregnancy malaria vaccine, but its large size ( approximately 350 kDa) and extensive polymorphism may pose a challenge to vaccine development. In this study, rabbits were immunized with individual VAR2CSA Duffy binding-like (DBL) domains expressed in Pichia pastoris or var2csa plasmid DNA and sera were screened on different CSA-binding parasite lines. Rabbit antibodies to three recombinant proteins (DBL1, DBL3, and DBL6) and four plasmid DNAs (DBL1, DBL3, DBL5, and DBL6) reacted with homologous FCR3-CSA IEs. By comparison, antibodies to the DBL4 domain were unable to react with native VAR2CSA protein unless it was first partially proteolyzed with trypsin or chymotrypsin. To investigate the antigenic relationship of geographically diverse CSA-binding isolates, rabbit immune sera were screened on four heterologous CSA-binding lines from different continental origins. Antibodies did not target conserved epitopes exposed in all VAR2CSA alleles; however, antisera to several DBL domains cross-reacted on parasite isolates that had polymorphic loops in common with the homologous immunogen. This study demonstrates that VAR2CSA contains common polymorphic epitopes that are shared between geographically diverse CSA-binding lines.

MobilomeFINDER: web-based tools for in silico and experimental discovery of bacterial genomic islands.

MobilomeFINDER (http://mml.sjtu.edu.cn/MobilomeFINDER) is an interactive online tool that facilitates bacterial genomic island or 'mobile genome' (mobilome) discovery; it integrates the ArrayOme and tRNAcc software packages. ArrayOme utilizes a microarray-derived comparative genomic hybridization input data set to generate 'inferred contigs' produced by merging adjacent genes classified as 'present'. Collectively these 'fragments' represent a hypothetical 'microarray-visualized genome (MVG)'. ArrayOme permits recognition of discordances between physical genome and MVG sizes, thereby enabling identification of strains rich in microarray-elusive novel genes. Individual tRNAcc tools facilitate automated identification of genomic islands by comparative analysis of the contents and contexts of tRNA sites and other integration hotspots in closely related sequenced genomes. Accessory tools facilitate design of hotspot-flanking primers for in silico and/or wet-science-based interrogation of cognate loci in unsequenced strains and analysis of islands for features suggestive of foreign origins; island-specific and genome-contextual features are tabulated and represented in schematic and graphical forms. To date we have used MobilomeFINDER to analyse several Enterobacteriaceae, Pseudomonas aeruginosa and Streptococcus suis genomes. MobilomeFINDER enables high-throughput island identification and characterization through increased exploitation of emerging sequence data and PCR-based profiling of unsequenced test strains; subsequent targeted yeast recombination-based capture permits full-length sequencing and detailed functional studies of novel genomic islands.

Acquisition and evolution of the exoU locus in Pseudomonas aeruginosa.

ExoU is a potent Pseudomonas aeruginosa cytotoxin translocated into host cells by the type III secretion system. A comparison of genomes of various P. aeruginosa strains showed that that the ExoU determinant is found in the same polymorphic region of the chromosome near a tRNA(Lys) gene, suggesting that exoU is a horizontally acquired virulence determinant. We used yeast recombinational cloning to characterize four distinct ExoU-encoding DNA segments. We then sequenced and annotated three of these four genomic regions. The sequence of the largest DNA segment, named ExoU island A, revealed many plasmid- and genomic island-associated genes, most of which have been conserved across a broad set of beta- and gamma-Proteobacteria. Comparison of the sequenced ExoU-encoding genomic islands to the corresponding PAO1 tRNA(Lys)-linked genomic island, the pathogenicity islands of strain PA14, and pKLC102 of clone C strains allowed us to propose a mechanism for the origin and transmission of the ExoU determinant. The evolutionary history very likely involved transposition of the ExoU determinant onto a transmissible plasmid, followed by transfer of the plasmid into different P. aeruginosa strains. The plasmid subsequently integrated into a tRNA(Lys) gene in the chromosome of each recipient, where it acquired insertion sequences and underwent deletions and rearrangements. We have also applied yeast recombinational cloning to facilitate a targeted mutagenesis of ExoU island A, further demonstrating the utility of the specific features of the yeast capture vector for functional analyses of genes on large horizontally acquired genetic elements.

A novel two-component system controls the expression of Pseudomonas aeruginosa fimbrial cup genes.

Biofilm formation by the opportunistic pathogen Pseudomonas aeruginosa requires the expression of a number of surface adhesive components. The expression of surface organelles facilitating biofilm formation is controlled by environmental signals acting through transcriptional regulatory networks. We analysed the expression of a family of P. aeruginosa adhesins encoded by three distinct fimbrial gene clusters (cupA, cupB and cupC). Using transposon mutagenesis, we have identified several regulatory loci that upregulated cupB and cupC transcription. One such locus contains three components, RocS1, RocR and RocA1, which represent a variant of a classical two-component signal transduction pathway. RocS1 is a sensor kinase, RocA1 is a DNA binding response regulator that activates cup genes, and RocR is an antagonist of RocA1 activity. Using a two-hybrid assay, we have shown that RocS1 interacts with receiver domains of both RocA1 and RocR. Expression of the Cup system in response to environmental stimuli is accomplished by a novel mechanism in which the sensor kinase activates its cognate response regulator through a phosphorelay pathway, while an additional repressor protein modulates this interaction.

A signaling network reciprocally regulates genes associated with acute infection and chronic persistence in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa causes a variety of acute and chronic infections. We identified a gene whose inactivation results in attenuation of virulence due to premature activation of genes involved in biofilm formation and coordinate repression of genes required for initial colonization. This gene, retS, encodes a hybrid sensor kinase/response regulator with an unconventional arrangement of functional domains. Genome-wide transcriptional profiling indicates that the retS gene is required for expression of the Type III secretion system and other virulence factors and for repression of genes responsible for exopolysaccharide components of the P. aeruginosa biofilm matrix. These disparate phenotypes are suppressed by transposon insertions in genes encoding the GacS/GacA/rsmZ signal transduction pathway, a highly conserved system involved in the control of diverse adaptive functions. This study defines RetS as a pleiotropic regulator of multiple virulence phenotypes that orchestrates genes required for acute infection and genes associated with chronic persistence.

Conservation of genome content and virulence determinants among clinical and environmental isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a ubiquitous environmental bacterium capable of causing a variety of life-threatening human infections. The genetic basis for preferential infection of certain immunocompromised patients or individuals with cystic fibrosis by P. aeruginosa is not understood. To establish whether variation in the genomic repertoire of P. aeruginosa strains can be associated with a particular type of infection, we used a whole-genome DNA microarray to determine the genome content of 18 strains isolated from the most common human infections and environmental sources. A remarkable conservation of genes including those encoding nearly all known virulence factors was observed. Phylogenetic analysis of strain-specific genes revealed no correlation between genome content and infection type. Clusters of strain-specific genes in the P. aeruginosa genome, termed variable segments, appear to be preferential sites for the integration of novel genetic material. A specialized cloning vector was developed for capture and analysis of these genomic segments. With this capture system a site associated with the strain-specific ExoU cytotoxin-encoding gene was interrogated and an 80-kb genomic island carrying exoU was identified. These studies demonstrate that P. aeruginosa strains possess a highly conserved genome that encodes genes important for survival in numerous environments and allows it to cause a variety of human infections. The acquisition of novel genetic material, such as the exoU genomic island, through horizontal gene transfer may enhance colonization and survival in different host environments.