Single object profiles regression analysis (SOPRA): a novel method for analyzing high-content cell-based screens.

BACKGROUND: High-content screening (HCS) experiments generate complex data from multiple object features for each cell within a treated population. Usually, these data are analyzed by using population-averaged values of the features of interest, increasing the amount of false positives and the need for intensive follow-up validation. Therefore, there is a strong need for novel approaches with reproducible hit prediction by identifying significantly altered cell populations. RESULTS: Here we describe SOPRA, a workflow for analyzing image-based HCS data based on regression analysis of non-averaged object features from cell populations, which can be run on hundreds of samples using different cell features. Following plate-wise normalization, the values are counted within predetermined binning intervals, generating unique frequency distribution profiles (histograms) for each population, which are then normalized to control populations (control-based normalization). These control-normalized frequency distribution profiles are analyzed using the Bioconductor R-package maSigPro, originally developed to analyze time profiles. However, statistically significant altered frequency distributions are also identified by maSigPro when integrating it into the SOPRA workflow. Finally, significantly changed profiles can be used to generate a heatmap from which altered cell populations with similar phenotypes can be identified, enabling the detection of siRNAs and compounds with the same 'on-target' profile and reducing the number of false positive hits. CONCLUSIONS: SOPRA is a novel analysis workflow for the detection of statistically significant normalized frequency distribution profiles of cellular features generated in high-throughput RNAi screens. For the validation of the SOPRA software workflow, a screen for cell cycle progression was used. We were able to identify such profiles for siRNA-mediated gene perturbations and chemical inhibitors of different cell cycle stages. The SOPRA software is freely available from Github.

ALPK1- and TIFA-Dependent Innate Immune Response Triggered by the Helicobacter pylori Type IV Secretion System.

Activation of transcription factor NF-κB is a hallmark of infection with the gastric pathogen Helicobacter pylori, associated with inflammation and carcinogenesis. Genome-wide RNAi screening revealed numerous host factors involved in H. pylori-, but not IL-1ß- and TNF-α-dependent NF-κB regulation. Pathway analysis including CRISPR/Cas9-knockout and recombinant protein technology, immunofluorescence microscopy, immunoblotting, mass spectrometry, and mutant H. pylori strains identified the H. pylori metabolite D-glycero-ß-D-manno-heptose 1,7-bisphosphate (ßHBP) as a cagPAI type IV secretion system (T4SS)-dependent effector of NF-κB activation in infected cells. Upon pathogen-host cell contact, TIFA forms large complexes (TIFAsomes) including interacting host factors, such as TRAF2. NF-κB activation, TIFA phosphorylation, and TIFAsome formation depend on a functional ALPK1 kinase, highlighting the ALPK1-TIFA axis as a core innate immune pathway. ALPK1-TIFA-mediated NF-κB activation was independent of CagA protein translocation, indicating that CagA translocation and HBP delivery to host cells are distinct features of the pathogen's T4SS.

HIF-1α is involved in mediating apoptosis resistance to Chlamydia trachomatis-infected cells.

Chlamydiae are obligate intracellular Gram-negative bacteria that cause widespread diseases in humans. Due to the intimate association between bacterium and host, Chlamydia evolved various strategies to protect their host cell against death-inducing stimuli, allowing the bacterium to complete its development cycle. An RNA interference (RNAi)-based screen was used to identify host cell factors required for apoptosis resistance of human epithelial cells infected with Chlamydia trachomatis serovar L2. Among the 32 validated hits, the anti-apoptotic Bcl-2 family member Mcl-1 was identified as a target. Protein network analyses implicated the transcription factor hypoxia-induced factor 1 alpha (HIF-1α) to be central to the regulation of many of the identified targets. Further mechanistic investigations showed that HIF-1α was stabilized within the host cell cytoplasm during early infection time points, followed by its translocation to the nucleus and eventual transcriptional activation of Mcl-1. siRNA-mediated depletion of HIF-1α led to a drastic decrease in Mcl-1, rendering the cell sensitive to apoptosis induction. Taken together, our findings identify HIF-1α as responsible for upregulation of Mcl-1 and the maintenance of apoptosis resistance during Chlamydia infection.

Tarp regulates early Chlamydia-induced host cell survival through interactions with the human adaptor protein SHC1.

Many bacterial pathogens translocate effector proteins into host cells to manipulate host cell functions. Here, we used a protein microarray comprising virtually all human SRC homology 2 (SH2) and phosphotyrosine binding domains to comprehensively and quantitatively assess interactions between host cell proteins and the early phase Chlamydia trachomatis effector protein translocated actin-recruiting phosphoprotein (Tarp), which is rapidly tyrosine phosphorylated upon host cell entry. We discovered numerous novel interactions between human SH2 domains and phosphopeptides derived from Tarp. The adaptor protein SHC1 was among Tarp's strongest interaction partners. Transcriptome analysis of SHC1-dependent gene regulation during infection indicated that SHC1 regulates apoptosis- and growth-related genes. SHC1 knockdown sensitized infected host cells to tumor necrosis factor-induced apoptosis. Collectively, our findings reveal a critical role for SHC1 in early C. trachomatis-induced cell survival and suggest that Tarp functions as a multivalent phosphorylation-dependent signaling hub that is important during the early phase of chlamydial infection.

A loss-of-function screen reveals Ras- and Raf-independent MEK-ERK signaling during Chlamydia trachomatis infection.

Chlamydiae are obligate intracellular bacterial pathogens that have a major effect on human health. Because of their intimate association with their host, chlamydiae depend on various host cell functions for their survival. Here, we present an RNA-interference-based screen in human epithelial cells that identified 59 host factors that either positively or negatively influenced the replication of Chlamydia trachomatis (Ctr). Two factors, K-Ras and Raf-1, which are members of the canonical Ras-Raf-MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase)-ERK (extracellular signal-regulated kinase) pathway, were identified as central components of signaling networks associated with hits from the screen. Depletion of Ras or Raf in HeLa cells increased pathogen growth. Mechanistic analyses revealed that ERK was activated independently of K-Ras and Raf-1. Infection with Ctr led to the Akt-dependent, increased phosphorylation (and inactivation) of Raf-1 at serine-259. Furthermore, phosphorylated Raf-1 relocalized from the cytoplasm to the intracellular bacterial inclusion in an Akt- and 14-3-3beta-dependent manner. Together, these findings not only show that Chlamydia regulates components of an important host cell signaling pathway, but also provide mechanistic insights into how this is achieved.

Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication.

Influenza A virus, being responsible for seasonal epidemics and reoccurring pandemics, represents a worldwide threat to public health. High mutation rates facilitate the generation of viral escape mutants, rendering vaccines and drugs directed against virus-encoded targets potentially ineffective. In contrast, targeting host cell determinants temporarily dispensable for the host but crucial for virus replication could prevent viral escape. Here we report the discovery of 287 human host cell genes influencing influenza A virus replication in a genome-wide RNA interference (RNAi) screen. Using an independent assay we confirmed 168 hits (59%) inhibiting either the endemic H1N1 (119 hits) or the current pandemic swine-origin (121 hits) influenza A virus strains, with an overlap of 60%. Notably, a subset of these common hits was also essential for replication of a highly pathogenic avian H5N1 strain. In-depth analyses of several factors provided insights into their infection stage relevance. Notably, SON DNA binding protein (SON) was found to be important for normal trafficking of influenza virions to late endosomes early in infection. We also show that a small molecule inhibitor of CDC-like kinase 1 (CLK1) reduces influenza virus replication by more than two orders of magnitude, an effect connected with impaired splicing of the viral M2 messenger RNA. Furthermore, influenza-virus-infected p27(-/-) (cyclin-dependent kinase inhibitor 1B; Cdkn1b) mice accumulated significantly lower viral titres in the lung, providing in vivo evidence for the importance of this gene. Thus, our results highlight the potency of genome-wide RNAi screening for the dissection of virus-host interactions and the identification of drug targets for a broad range of influenza viruses.

Reduced display of tumor necrosis factor receptor I at the host cell surface supports infection with Chlamydia trachomatis.

The obligate intracellular human pathogenic bacterium Chlamydia trachomatis has evolved multiple mechanisms to circumvent the host immune system. Infected cells exhibit a profound resistance to the induction of apoptosis and down-regulate the expression of major histocompatibility complex class I and class II molecules to evade the cytotoxic effect of effector immune cells. Here we demonstrate the down-regulation of tumor necrosis factor receptor 1 (TNFR1) on the surface of infected cells. Interestingly, other members of the TNFR family such as TNFR2 and CD95 (Fas/Apo-1) were not modulated during infection, suggesting a selective mechanism underlying surface reduction of TNFR1. The observed effect was not due to reduced expression since the overall amount of TNFR1 protein was increased in infected cells. TNFR1 accumulated at the chlamydial inclusion and was shed by the infected cell into the culture supernatant. Receptor shedding depended on the infection-induced activation of the MEK-ERK pathway and the metalloproteinase TACE (TNFalpha converting enzyme). Our results point to a new function of TNFR1 modulation by C. trachomatis in controlling inflammatory signals during infection.

Gene expression profiles of Chlamydophila pneumoniae during the developmental cycle and iron depletion-mediated persistence.

The obligate intracellular, gram-negative bacterium Chlamydophila pneumoniae (Cpn) has impact as a human pathogen. Little is known about changes in the Cpn transcriptome during its biphasic developmental cycle (the acute infection) and persistence. The latter stage has been linked to chronic diseases. To analyze Cpn CWL029 gene expression, we designed a pathogen-specific oligo microarray and optimized the extraction method for pathogen RNA. Throughout the acute infection, ratio expression profiles for each gene were generated using 48 h post infection as a reference. Based on these profiles, significantly expressed genes were separated into 12 expression clusters using self-organizing map clustering and manual sorting into the "early", "mid", "late", and "tardy" cluster classes. The latter two were differentiated because the "tardy" class showed steadily increasing expression at the end of the cycle. The transcriptome of the Cpn elementary body (EB) and published EB proteomics data were compared to the cluster profile of the acute infection. We found an intriguing association between "late" genes and genes coding for EB proteins, whereas "tardy" genes were mainly associated with genes coding for EB mRNA. It has been published that iron depletion leads to Cpn persistence. We compared the gene expression profiles during iron depletion-mediated persistence with the expression clusters of the acute infection. This led to the finding that establishment of iron depletion-mediated persistence is more likely a mid-cycle arrest in development rather than a completely distinct gene expression pattern. Here, we describe the Cpn transcriptome during the acute infection, differentiating "late" genes, which correlate to EB proteins, and "tardy" genes, which lead to EB mRNA. Expression profiles during iron mediated-persistence led us to propose the hypothesis that the transcriptomic "clock" is arrested during acute mid-cycle.

Tackling the intractable - approaching the genetics of Chlamydiales.

Chlamydia trachomatis and Chlamydia (Chlamydophila) pneumoniae are important human pathogens with significant socio-economic and medical impact. The development of an improved therapy or vaccine would represent a major break-through in the battle against these infections. Despite intense research on Chlamydiaceae, the molecular genetic analysis of these pathogens remains difficult as genetic manipulation still remains impossible. Even though several options for generating a universal genetic system are currently being pursued, the anticipated success of these approaches is uncertain. As an alternative approach, random chemical mutagenesis is currently pursued which could allow spotlighting critical chlamydial pathogenesis features in the near future. Another research track lies in the identification of immunogenic peptides which could serve two goals: Immunogenic peptides could provide a basis for generating an efficient antichlamydial vaccine. Further, they also might offer an efficient tool to diagnose acute and chronic chlamydial infections. Both are currently pursued by applying the autodisplay approach that facilitates the exposure of whole peptide libraries on the Escherichia coli cell surface, thus allowing immediate detection and gene tracking through antibody binding. Finally, global transcriptome analysis is an approach to circumvent the genetic intractability of Chlamydiaceae. Current analysis indicates that gene expression takes place in an ordered manner throughout the course of the developmental cycle and, as expected, gene expression appears to be directly linked to host cell responses. Moreover, recent microarray analysis in C. pneumoniae corroborated the notion that distinct mRNA species are being carried-over by the infectious elementary bodies (EBs). These and other recent observations on the chlamydial gene expression patterns offer unique opportunities to interfere with the onset, the course, and the persistency of chlamydial infections by paving the ways towards the development of novel diagnostic and therapeutic treatment regimens.