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

Evolutionary Conservation of Infection-Induced Cell Death Inhibition among Chlamydiales

Control of host cell death is of paramount importance for the survival and replication of obligate intracellular bacteria. Among these, human pathogenic Chlamydia induces the inhibition of apoptosis in a variety of different host cells by directly interfering with cell death signaling. However, the evolutionary conservation of cell death regulation has not been investigated in the order Chlamydiales, which also includes Chlamydia-like organisms with a broader host spectrum. Here, we investigated the apoptotic response of human cells infected with the Chlamydia-like organism Simkania negevensis (Sn). Simkania infected cells exhibited strong resistance to apoptosis induced by intrinsic stress or by the activation of cell death receptors. Apoptotic signaling was blocked upstream of mitochondria since Bax translocation, Bax and Bak oligomerisation and cytochrome c release were absent in these cells. Infected cells turned on pro-survival pathways like cellular Inhibitor of Apoptosis Protein 2 (cIAP-2) and the Akt/PI3K pathway. Blocking any of these inhibitory pathways sensitized infected host cell towards apoptosis induction, demonstrating their role in infection-induced apoptosis resistance. Our data support the hypothesis of evolutionary conserved signaling pathways to apoptosis resistance as common denominators in the order Chlamydiales

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Modulation of host signaling and cellular responses by Chlamydia

Modulation of host cell signaling and cellular functions is key to intracellular survival of pathogenic bacteria. Intracellular growth has several advantages e.g. escape from the humoral immune response and access to a stable nutrient rich environment. Growth in such a preferred niche comes at the price of an ongoing competition between the bacteria and the host as well as other microbes that compete for the very same host resources. This requires specialization and constant evolution of dedicated systems for adhesion, invasion and accommodation. Interestingly, obligate intracellular bacteria of the order Chlamydiales have evolved an impressive degree of control over several important host cell functions. In this review we summarize how Chlamydia controls its host cell with a special focus on signal transduction and cellular modulation

Pathogen – host cell interaction. - The role of the type III effector protein Tarp for the invasion of Chlamydia trachomatis

Chlamydia trachomatis, ein wichtiges humanpathogenes Bakterium, sekretiert während der Invasion in seine Wirtszelle das Typ III Effektorprotein Tarp. In silico Analysen offenbarten eine Homologie zwischen Tarp und Src-Kinasesubstraten. Diese Analyse ließ sich unter Verwendung verschiedener biochemischer Methoden bestätigen. So zeigte sich, dass sich die Tarp Tyrosinphosphorylierung durch einen Src-spezifischen Inhibitor blockieren lässt und dass Tarp durch rekombinantes Src in vitro phosphoryliert wird. In vivo konnte bestätigt werden, dass Tarp nicht in src-, fyn- und yes-defizienten Zellen phosphoryliert wird. Inhibiert man die Src-Kinasen während der Invasion, so führt dies zu einer um 60% abgeschwächten Chlamydieninfektion. Im zweiten Teil dieser Arbeit wurde die Interaktion von phosphoryliertem Tarp mit zellulären Komponenten untersucht. Experimente mit SH2-Domänenarrays haben eine Interaktion mit PLCγ, Shc, Lck und Shp-2 gezeigt. Die Interaktion zwischen Tarp und PLCγ, bzw. Tarp und Shc ließ sich mittels Immunpräzipitation bestätigen, eine morphologische Charakterisierung mittels Immunfluoreszenz im Anschluss zeigte, dass PLCγ mit den Bakterien während der Invasion kolokalisiert. Inhibiert man PLCγ während der Invasion verhindert dies die Inklusionsbildung der C. trachomatis-Infektion nahezu vollständig. Eine Inhibition nach der Invasion hingegen hatte nur einen sehr geringen Einfluss auf die Infektion. Im dritten Teil dieser Arbeit wurde untersucht, welche Bedeutung die Rekrutierung von PLCγ für den Invasionsprozess hat. Mit Hilfe konfokaler Echtzeitmikroskopie wurde der Phosphoinositolmetabolismus untersucht und es zeigte sich, dass Chlamydien eine PtdIns(3,4,5)-P3-angereicherte frühe Inklusion bilden. Biochemische Experimente ließen erkennen, dass die Bildung dieser frühen Inklusion PI3K- und PLCγ- abhängig ist, und dass die Inhibition dieser Enzyme zur lysosomalen Degradation der Bakterien führt. Die Bildung einer PtdIns(3,4,5)-P3-angereicherten frühen Inklusion stellt demnach einen bisher unbekannten bakteriellen Mechanismus zur Vermeidung der Degradation dar.Chlamydia trachomatis is an important pathogenic bacterium. Tarp, a type III effector protein, is secreted into the human host cell during invasion. In silico analysis revealed homology between Tarp and Src kinase substrates. This initial observation was confirmed using different biochemical methods. Inhibition with Src specific inhibitors blocks Tarp tyrosine phosphorylation and Tarp can be phosphorylated using recombinant Src. Using cells deficient in src, yes and fyn it was shown that Tarp phosphorylation is controlled by Src kinases in vivo. Inhibition of Src kinases during invasion leads to a 60 % reduced primary infection. The second part of this work deals with the interaction between phosphorylated Tarp and host cell components. Experiments using SH2-domain arrays show an interaction with PLCγ, Shc, Lck and Shp-2. The interaction between Tarp, PLCγ and Shc was confirmed using immunoprecipitation. A morphological characterization via immunofluorescence confirmed colocalisation between invading bacteria and PLCγ. Inhibition of PLCγ during invasion abrogates inclusion formation while this is not the case when PLCγ is inhibited at a later stage. The last part of this thesis deals with the question how PLCγ influences the invasion of Chlamydia trachomatis. Using live cell confocal microscopy the host phosphoinositide metabolism during infection was investigated. It was shown that Chlamydia induces strong PtdIns(3,4,5)-P3 formation and that the endosome surrounding the organism is enriched with this rare phospholipid. Biochemical experiments showed that the endosome formation is PI3K and PLCγ dependant and inhibition of these enzymes leads to lysosomal degradation of the bacteria. Thus, formation of the PtdIns(3,4,5)-P3 enriched early inclusion is a so far unknown bacterial mechanism to avoid lysosmal degradation within the host cell

Imbalanced oxidative stress causes chlamydial persistence during non-productive Human Herpes Virus co-infection

Both human herpes viruses and Chlamydia are highly prevalent in the human population and are detected together in different human disorders. Here, we demonstrate that co-infection with human herpes virus 6 (HHV6) interferes with the developmental cycle of C. trachomatis and induces persistence. Induction of chlamydial persistence by HHV6 is independent of productive virus infection, but requires the interaction and uptake of the virus by the host cell. On the other hand, viral uptake is strongly promoted under co-infection conditions. Host cell glutathione reductase activity was suppressed by HHV6 causing NADPH accumulation, decreased formation of reduced glutathione and increased oxidative stress. Prevention of oxidative stress restored infectivity of Chlamydia after HHV6-induced persistence. We show that co-infection with Herpes simplex virus 1 or human Cytomegalovirus also induces chlamydial persistence by a similar mechanism suggesting that Chlamydia -human herpes virus co-infections are evolutionary shaped interactions with a thus far unrecognized broad significance