Short-term mucosal disruption enables colibactin-producing <i>E. coli</i> to cause long-term perturbation of colonic homeostasis

Colibactin, a bacterial genotoxin produced by E. coli strains harboring the pks genomic island, induces cytopathic effects, such as DNA breaks, cell cycle arrest, and apoptosis. Patients with inflammatory bowel diseases, such as ulcerative colitis, display changes in their microbiota with the expansion of E. coli. Whether and how colibactin affects the integrity of the colonic mucosa and whether pks+ E. coli contributes to the pathogenesis of colitis is not clear. Using a gnotobiotic mouse model, we show that under homeostatic conditions, pks+ E. coli do not directly interact with the epithelium or affect colonic integrity. However, upon short-term chemical disruption of mucosal integrity, pks+ E. coli gain direct access to the epithelium, causing epithelial injury and chronic colitis, while mice colonized with an isogenic & UDelta;clbR mutant incapable of producing colibactin show a rapid recovery. pks+ E. coli colonized mice are unable to reestablish a functional barrier. In turn, pks+ E. coli remains in direct contact with the epithelium, perpetuating the process and triggering chronic mucosal inflammation that morphologically and transcriptionally resembles human ulcerative colitis. This state is characterized by impaired epithelial differentiation and high proliferative activity, which is associated with high levels of stromal R-spondin 3. Genetic overexpression of R-spondin 3 in colon myofibroblasts is sufficient to mimic barrier disruption and expansion of E. coli. Together, our data reveal that pks+ E. coli are pathobionts that promote severe injury and initiate a proinflammatory trajectory upon contact with the colonic epithelium, resulting in a chronic impairment of tissue integrity

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

Toxoplasma and Eimeria co-opt the host cFos expression for intracellular development in mammalian cells

Successful asexual reproduction of intracellular pathogens depends on their potential to exploit host resources and subvert antimicrobial defense. In this work, we deployed two prevalent apicomplexan parasites of mammalian cells, namely Toxoplasma gondii and Eimeria falciformis, to identify potential host determinants of infection. Expression analyses of the young adult mouse colonic (YAMC) epithelial cells upon infection by either parasite showed regulation of several distinct transcripts, indicating that these two pathogens program their intracellular niches in a tailored manner. Conversely, parasitized mouse embryonic fibroblasts (MEFs) displayed a divergent transcriptome compared to corresponding YAMC epithelial cells, suggesting that individual host cells mount a fairly discrete response when encountering a particular pathogen. Among several host transcripts similarly altered by T. gondii and E. falciformis, we identified cFos, a master transcription factor, that was consistently induced throughout the infection. Indeed, asexual growth of both parasites was strongly impaired in MEF host cells lacking cFos expression. Last but not the least, our differential transcriptomics of the infected MEFs (parental and cFos-/- mutant) and YAMC epithelial cells disclosed a cFos-centered network, underlying signal cascades, as well as a repertoire of nucleotides- and ion-binding proteins, which presumably act in consort to acclimatize the mammalian cell and thereby facilitate the parasite development.Peer Reviewe

Developmental transcriptome of resting cell formation in Mycobacterium smegmatis

Functional classification of gene numbers up- and down-regulated during shock starvation in PBS. (PDF 1796 kb

TRANSVAC workshop on standardisation and harmonisation of analytical platforms for HIV, TB and malaria vaccines: 'how can big data help?'.

High-throughput analyses of RNA and protein expression are increasingly used for better understanding of vaccine-induced immunity and protection against infectious disease. With an increasing number of vaccine candidates in clinical development, it is timely to consider standardisation and harmonisation of sample collection, storage and analysis to ensure results of highest quality from these precious samples. These challenges were discussed by a group of international experts during a workshop organised by TRANSVAC, a European Commission-funded Research Infrastructure project. The main conclusions were: Platforms are rarely standardised for use in preclinical and clinical studies. Coordinated efforts should continue to harmonise the experimental set up of these studies, as well as the establishment of internal standards and controls. This will ensure comparability, efficiency and feasibility of the global analyses performed on preclinical and clinical data sets

Mycobacterium tuberculosis Invasion of the Human Lung: First Contact

Early immune responses to Mycobacterium tuberculosis (Mtb) invasion of the human lung play a decisive role in the outcome of infection, leading to either rapid clearance of the pathogen or stable infection. Despite their critical impact on health and disease, these early host–pathogen interactions at the primary site of infection are still poorly understood. In vitro studies cannot fully reflect the complexity of the lung architecture and its impact on host–pathogen interactions, while animal models have their own limitations. In this study, we have investigated the initial responses in human lung tissue explants to Mtb infection, focusing primarily on gene expression patterns in different tissue-resident cell types. As first cell types confronted with pathogens invading the lung, alveolar macrophages, and epithelial cells displayed rapid proinflammatory chemokine and cytokine responses to Mtb infection. Other tissue-resident innate cells like gamma/delta T cells, mucosal associated invariant T cells, and natural killer cells showed partially similar but weaker responses, with a high degree of variability across different donors. Finally, we investigated the responses of tissue-resident innate lymphoid cells to the inflammatory milieu induced by Mtb infection. Our infection model provides a unique approach toward host–pathogen interactions at the natural port of Mtb entry and site of its implantation, i.e., the human lung. Our data provide a first detailed insight into the early responses of different relevant pulmonary cells in the alveolar microenvironment to contact with Mtb. These results can form the basis for the identification of host markers that orchestrate early host defense and provide resistance or susceptibility to stable Mtb infection

IFNs Modify the Proteome of <i>Legionella</i>-Containing Vacuoles and Restrict Infection Via IRG1-Derived Itaconic Acid

Macrophages can be niches for bacterial pathogens or antibacterial effector cells depending on the pathogen and signals from the immune system. Here we show that type I and II IFNs are master regulators of gene expression during Legionella pneumophila infection, and activators of an alveolar macrophage-intrinsic immune response that restricts bacterial growth during pneumonia. Quantitative mass spectrometry revealed that both IFNs substantially modify Legionella-containing vacuoles, and comparative analyses reveal distinct subsets of transcriptionally and spatially IFN-regulated proteins. Immune-responsive gene (IRG)1 is induced by IFNs in mitochondria that closely associate with Legionella-containing vacuoles, and mediates production of itaconic acid. This metabolite is bactericidal against intravacuolar L. pneumophila as well as extracellular multidrug-resistant Gram-positive and -negative bacteria. Our study explores the overall role IFNs play in inducing substantial remodeling of bacterial vacuoles and in stimulating production of IRG1-derived itaconic acid which targets intravacuolar pathogens. IRG1 or its product itaconic acid might be therapeutically targetable to fight intracellular and drug-resistant bacteria

Functional Correlations of Pathogenesis-Driven Gene Expression Signatures in Tuberculosis

Tuberculosis remains a major health threat and its control depends on improved measures of prevention, diagnosis and treatment. Biosignatures can play a significant role in the development of novel intervention measures against TB and blood transcriptional profiling is increasingly exploited for their rational design. Such profiles also reveal fundamental biological mechanisms associated with the pathology of the disease. We have compared whole blood gene expression in TB patients, as well as in healthy infected and uninfected individuals in a cohort in The Gambia, West Africa and validated previously identified signatures showing high similarities of expression profiles among different cohorts. In this study, we applied a unique combination of classical gene expression analysis with pathway and functional association analysis integrated with intra-individual expression correlations. These analyses were employed for identification of new disease-associated gene signatures, identifying a network of Fc gamma receptor 1 signaling with correlating transcriptional activity as hallmark of gene expression in TB. Remarkable similarities to characteristic signatures in the autoimmune disease systemic lupus erythematosus (SLE) were observed. Functional gene clusters of immunoregulatory interactions involving the JAK-STAT pathway; sensing of microbial patterns by Toll-like receptors and IFN-signaling provide detailed insights into the dysregulation of critical immune processes in TB, involving active expression of both pro-inflammatory and immunoregulatory systems. We conclude that transcriptomics (i) provides a robust system for identification and validation of biosignatures for TB and (ii) application of integrated analysis tools yields novel insights into functional networks underlying TB pathogenesis

Epigenetics and proteomics join transcriptomics in the quest for tuberculosis biomarkers

CITATION: Esterhuyse, M. M. et al. 2015. Epigenetics and proteomics join transcriptomics in the quest for tuberculosis biomarkers. mBio, 6(5):e01187-15, doi:10.1128/mBio.01187-15.The original publication is available at http://mbio.asm.orgAn estimated one-third of the world’s population is currently latently infected with Mycobacterium tuberculosis. Latent M. tuberculosis infection (LTBI) progresses into active tuberculosis (TB) disease in ~5 to 10% of infected individuals. Diagnostic and prognostic biomarkers to monitor disease progression are urgently needed to ensure better care for TB patients and to decrease the spread of TB. Biomarker development is primarily based on transcriptomics. Our understanding of biology combined with evolving technical advances in high-throughput techniques led us to investigate the possibility of additional platforms (epigenetics and proteomics) in the quest to (i) understand the biology of the TB host response and (ii) search for multiplatform biosignatures in TB. We engaged in a pilot study to interrogate the DNA methylome, transcriptome, and proteome in selected monocytes and granulocytes from TB patients and healthy LTBI participants. Our study provides first insights into the levels and sources of diversity in the epigenome and proteome among TB patients and LTBI controls, despite limitations due to small sample size. Functionally the differences between the infection phenotypes (LTBI versus active TB) observed in the different platforms were congruent, thereby suggesting regulation of function not only at the transcriptional level but also by DNA methylation and microRNA. Thus, our data argue for the development of a large-scale study of the DNA methylome, with particular attention to study design in accounting for variation based on gender, age, and cell type.http://mbio.asm.org/content/6/5/e01187-15.abstract?sid=fe0ea1c7-6da2-4e53-b4a4-5cd8233777c7Publisher's versio

The adaptor molecule CARD9 is essential for tuberculosis control

The cross talk between host and pathogen starts with recognition of bacterial signatures through pattern recognition receptors (PRRs), which mobilize downstream signaling cascades. We investigated the role of the cytosolic adaptor caspase recruitment domain family, member 9 (CARD9) in tuberculosis. This adaptor was critical for full activation of innate immunity by converging signals downstream of multiple PRRs. Card9−/− mice succumbed early after aerosol infection, with higher mycobacterial burden, pyogranulomatous pneumonia, accelerated granulocyte recruitment, and higher abundance of proinflammatory cytokines and granulocyte colony-stimulating factor (G-CSF) in serum and lung. Neutralization of G-CSF and neutrophil depletion significantly prolonged survival, indicating that an exacerbated systemic inflammatory disease triggered lethality of Card9−/− mice. CARD9 deficiency had no apparent effect on T cell responses, but a marked impact on the hematopoietic compartment. Card9−/− granulocytes failed to produce IL-10 after Mycobaterium tuberculosis infection, suggesting that an absent antiinflammatory feedback loop accounted for granulocyte-dominated pathology, uncontrolled bacterial replication, and, ultimately, death of infected Card9−/− mice. Our data provide evidence that deregulated innate responses trigger excessive lung inflammation and demonstrate a pivotal role of CARD9 signaling in autonomous innate host defense against tuberculosis