1,721,092 research outputs found
Innate immune recognition of Salmonella and Francisella : two model intracellular bacterial pathogens
Full text linkThe innate immune system is the first line of host defense against invading pathogens. In multicellular organisms, specialized innate immune cells recognize conserved pathogen-associated molecular patters (PAMPs) with germ-line encoded pattern recognition receptors (PRR). Thereby, the organism discriminates between self and non-self and engages mechanisms to eliminate the invader. Beside PAMPs, PRRs recognize mislocalized self-molecules, so called danger-associated molecular patterns (DAMPs), which are indicators of tissue or cellular damage.
Upon PAMP or DAMP recognition, PRRs induce innate immune signaling pathways leading to the activation of pro-inflammatory genes and interferon production, which are important mediators of inflammation. Therefore the recognition of invading pathogen and thereby activation of innate immune signaling pathways determines the success of the immune system to eliminate the potential threat.
Innate immune signaling pathways largely depend on phosphorylation cascades. Today, global phosphorylation changes are analyzed by mass spectrometry, however the number of detected phosphopeptides remains unchanged despite technical improvements. Therefore, we investigated the issue of phosphopeptide detection in mass spectrometry.
The analyses of phosphopeptide-enriched samples have revealed lower signal intensities in MS1 spectra compared to total cell lysate samples, which results in poor phosphopeptide detection with mass spectrometry. Based on these observations, we hypothesized that the phosphate groups of phosphopeptides account for this poor detection. Indeed, we significantly increase the signal intensities in MS1 spectra after enzymatic removal of phosphate groups from phosphopeptides, and consequently we detect three-times more peptides in phosphatase-treated samples. Validation experiments elucidate that most of the newly detected peptides have been initially phosphorylated. Moreover, the newly detected peptides enlarge the activated signaling network upon Salmonella infection. Importantly, we identify known innate immune signaling pathways, which were missing in the analyses of phospho-enriched samples.
Taken together, the phosphate groups of phosphopeptides globally suppress peptide ionization efficacy and therefore account for the low phosphopeptide detection rate by mass spectrometry. By removing the phosphate groups, we identify three times more peptides after phosphatase treatment. The newly detected peptides enlarge the network of activated innate immune signaling pathways upon Salmonella infection and include signaling pathways that are important but have not been detected in phospho-enriched samples. Therefore our findings improve the analyses of innate immune signaling pathways by mass spectrometry and consequently the understanding of innate immunity.
One of the main mechanisms to eliminate invading microbes is by phagocytosis and degradation within phago-lysosomes. However, professional pathogens have developed various defense mechanisms to resist intracellular killing and can even use innate immune cells as replicative niches. For example, the bacterial pathogen Francisella tularensis causes a severe and life-threatening disease called tularemia in humans, because Francisella can survive and replicate in macrophages and dendritic cells. Critical for Francisella pathogenicity is the ability of the phagocytosed bacteria to escape from the phagosome to the host cytosol. Even though we know that genes encoded on the Francisella pathogenicity island (FPI) are essential for escaping from the phagosome, the mechanism is unknown. Homology analyses have suggested that the FPI encodes a type 6 secretion system (T6SS). However experimental evidence is missing, which show that the FPI encode a functional T6SS. Therefore, we investigated whether the FPI encodes a functional T6SS and what impact a functional T6SS has on Francisella virulence in vitro and in vivo.
We show that the FPI of Francisella novicida (F. novicida) encodes a functional T6SS that assembles exclusively at bacterial poles. T6SS function depends on the unfoldase ClpB, which specifically recognizes contracted T6SS sheaths leading to their disassembly. Furthermore we have characterized FPI genes that show no homology with known T6SSs. We have identified IglF, IglG, IglI and IglJ as structural components of the T6SS and PdpC, PdpD, PdpE and AnmK as potential T6SS effector proteins. Whereas PdpE and AnmK are dispensable for phagosomal escape, AIM2 inflammasome activation and virulence in mice, pdpC- and pdpD-deficient bacteria are impaired in all aforementioned analyses. This suggests that PdpC and PdpD are bacterial effector proteins involved in phagosomal escape and thereby in the establishment of a F. novicida infection.
Taken together, F. novicida uses its T6SS to deliver the effector proteins PdpC and PdpD into host cells. PdpC and PdpD are involved in phagosomal rupture and consequently in bacterial escape to the cytosol. These findings are a major breakthrough in the understanding of Francisella pathogenicity and could lead to new vaccination strategies to eradicate the life-threatening human disease Tularemia
Heterogeneous Salmonella-host encounters determine disease progression in a typhoid fever model
Full text linkAn infectious disease is an illness that is caused by invasion and multiplication of pathogenic microorganisms in body tissues of a host. The complex and heterogeneous tissue microenvironments provide different growth opportunities for pathogens with implications for disease outcome. However, the heterogeneous host environment has been largely neglected, because suitable methods to analyze single-cell host-pathogen encounters in vivo were largely lacking.
In this work, we addressed the implications of heterogeneous host-Salmonella encounters on disease progression in the well characterized mouse typhoid fever model. Specifically, we combined Salmonella biosensors with high resolution microscopy, flow cytometry and proteomics to reveal the fate of Salmonella in the diverse host microenvironments of spleen.
We showed that neutrophils and inflammatory monocytes are recruited to Salmonella infectious foci in spleen where they produce large amounts of reactive oxygen species (ROS), reactive nitrogen species (RNS) and lipids. We revealed that neutrophils and monocytes kill Salmonella with ROS generated through NADPH oxidase and myeloperoxidase (MPO), whereas RNS play a negligible role for infection control. However, ROS can also cause substantial collateral damage in host tissue. We showed that MPO protects the host against self-damage by converting diffusible long-lived ROS into highly reactive ROS with short reach that remain confined to the pathogen microenvironment. Some Salmonella escape to resident red pulp macrophages, which impose only sublethal oxidative bursts on Salmonella. Although macrophages are a primary niche for Salmonella survival, a specific subset of IFN activated macrophages can kill Salmonella with guanylate binding protein associated mechanisms. In addition, we showed that Salmonella growth in spleen is heterogeneous, independently of regional factors or host cell types. Overall, our analysis revealed numerous different Salmonella-host cell encounters in spleen with divergent outcomes. Local failures to control bacterial replication appear next to regions where the host successfully eradicates the pathogen.
Together, these data show that disease progression does not necessarily reflect an overall weak host immune response, but rather result from disparate host-pathogen encounters
A moonlighting enzyme imposes second messenger bistability to drive lifestyle decisions in E. coli.
Full text linkBacteria preferentially colonize surfaces and air-liquid interfaces as matrix embedded communities called biofilms. Biofilms exhibit specific physiological properties, including general stress tolerance, increased antibiotic recalcitrance and tolerance against phagocytic clearance. Together this largely accounts for increased biofilm persistence, chronic infections and infection relapses. One of the principle regulators of biofilm formation is c-di-GMP, a bacterial second messenger controlling various cellular processes. Cellular levels of c-di-GMP are controlled by two antagonistic enzyme families, diguanylate cyclases and phosphodiesterases. But despite the identification and characterization of an increasing number of components of the c-di-GMP network in different bacterial model organisms, details of c-di- GMP mediated decision-making have remained unclear. In particular, how cells shuttle between specific c-di-GMP regimes at the population and single cell level is largely unknown and moreover how these transitions are deterministically made in time and space, given that bacterial networks of diguanylate cyclases and phosphodiesterases show a high degree of complexity.
Here we describe a novel mechanism regulating c-di-GMP mediated biofilm formation in E. coli. This mechanism relies on the bistable expression of a key phosphodiesterase that acts both as catalyst for c- di-GMP degradation and as a transcription factor promoting its own production. Bistability results from two interconnected positive feedback loops operating on the catalytic and gene expression level. Based on genetic, structural and biochemical analyses we postulate a simple substrate-induced switch mechanism through which this enzyme can sense changing concentration of c-di-GMP and convert this information into a bistable c-di-GMP response. This mechanism may explain how cellular heterogeneity of small signaling molecules is generated in bacteria and used as a bet hedging strategy for important lifestyle transitions
Hijacking Pseudomonas aeruginosa active transporters across the outer membrane: Challenges and opportunities for drug transport
Full text linkAntimicrobial resistance is a serious public health threat worldwide. The emergence of multi-drug resistance bacteria challenges the development of novel antibiotics. Pseudomonas aeruginosa (P. aeruginosa) is an Gram-negative opportunistic pathogen that infects burn, wound and cystic fibrosis (CF) patients. P. aeruginosa is intrinsically resistant to many antibiotics, and further acquired resistance limits treatment options. The high level of resistance to antibiotics arises mainly from the tight control of P. aeruginosa over influx and efflux of molecules across its outer membrane. P. aeruginosa outer membrane includes a large number of different types of porins and efflux pumps that enable nutrient acquisition and antibiotic resistance. Among them, P. aeruginosa UCBPP-PA14 encodes 35 TonB-dependent transporters (TBDTs), which are defined as high-affinity active transporters and permit the transport of siderophores, heme, heavy metals and carbohydrates. Several studies showed high levels of TBDTs expression under iron deprivation and their importance for P. aeruginosa growth in vivo, but, except for the high-affinity siderophores and heme transporters, few have determined the contribution of single TBDT in vivo. In a Trojan horse approach, mimetics of essential substrates complexed to drugs, including siderophore-antibiotics conjugates and non-iron metalloporphyrins, are employed to induce the expression of associated TBDT(s) and to increase the antibiotic transport through TBDTs, hijacking the bacterial transport machinery. In spite of promising antibacterial activity in vitro, P. aeruginosa was able to rapidly develop resistance against the Trojan horse conjugates by facile inactivation of the TBDT involved in their transport. To circumvent this resistance mechanism, basic research on the processes associated with P. aeruginosa transport capabilities and substrate specificities in vivo is seriously needed to guide rational development of novel and effective therapeutics. In order to prevent facile inactivation of TBDT, we aim at identifying essential P. aeruginosa TBDTs that contribute to the bacterial fitness in vivo. We addressed this aim (i) by quantifying the abundance of TBDTs in vitro, in preclinical and human patients samples and (ii) by evaluating the relevance of TBDTs in vivo. We thus developed an ultrasensitive targeted proteomic approach to determine absolutely the abundance of TBDTs in vitro and in various hosts. Proteomic analyses revealed a clear disctinction between TBDTs expression in vitro and in vivo, suggesting that there is a urgent need for suitable in vitro medium that more faithfully reflects the in vivo reality. Expression data also highlighted a subset of TBDTs, including endogenous siderophore, heme, non-iron metal and some xenosiderophore transporters, that was highly abundant among the different in vivo conditions. Based on these data, we generated different mutants of the abundant TBDTs and evaluated them in competitive fitness assays in vivo. Overall, these data suggested that P. aeruginosa primarily used its high-affinity siderophore pyoverdine for iron scavenging, whereas uptake capabilities for its other endogenous siderophores, xenosiderophores, heme-associated substrates and possibly copper and zinc uptake were all dispensable for in vivo fitness in an intranasal model. In conclusion, implications of these results in the development of future compounds implementing the Trojan horse approach were discussed
Modulation of host immune responses by Bartonella effector proteins
Full text linkStarted in May 2011, my first project was collaborated with the group of Prof. Kempf in the Institute of Medical Microbiology and Infection Control at the University Hospital of the Johann Wolfgang Goethe-University in Frankfurt. Two pathogenicity factors of Bartonella henselae have been greater characterized in individual activities: the trimeric autotransporter Bartonella adhesin A (BadA) and the type IV secretion system VirB/D4 (VirB/D4 T4SS). In this study, we deeply investigated how these major virulence factors affect each other in their specific activities.
In the second project, I focused on the natural host interface with Bartonella, particularly on the bacterial defense mechanisms against host immunity. To achieve this, my work was divided into two directions. One was to identify which Bartonella effector proteins as immunomodulatory molecules involved in intracellular communications to subvert the immunological signaling cascade. The other was to find the primary niche of Bartonella entry and replication in the natural reservoir host. By understanding the Bartonella infection cycle, I aimed to explore how Bartonella manipulate host immunity towards its pathogenicity in vivo. Taken in vitro and in vivo results together, I sought to complete this project with a comprehensive insight into which Bep displays immunosuppressive properties, how it works and what are the consequences of its function on host immunity
Envelope stress response during Salmonella infection:role of σE
Full text linkBacterial infections are a major public health problem worldwide. Over the years, multi-drug resistant (MDR) pathogens emergence combined with approval absence of new antibiotics in clinics has resulted in untreatable infections. Thus, routine medical procedure constitutes a risk of infection for patients. Therefore, it is crucial to identify novel strategies to efficiently and effectively combat bacterial pathogens.
In vitro, it is challenging to reproduce the microenvironment that pathogens encounter in vivo. Consequently, important stress conditions are omitted, decreasing the ability to identify relevant inhibitor targets. The host immune response largely focuses on the pathogen envelope that constitutes the first line of defense for pathogens and a strong physical barrier. Pathogens possess several envelope stress response systems that enable an adapted response to host attacks, ensuring survival and growth. Inhibiting these systems may enhance host immunity and provide efficient infection control. The extracytoplasmic stress response factor sigma E (E), encoded by rpoE, is crucial for the virulence of several pathogens, including Salmonella. E regulates expression of more than 100 genes, including those that encode proteases, chaperones, and sRNAs to maintain envelope homeostasis. However, it remains unclear which E-regulated gene(s) is (are) critical for in vivo fitness. Here, we used an unbiased approach to identify target mutations that restore ∆rpoE survival by using a transposon library screen. While the parental strain ∆rpoE was cleared from infected mice, several transposon mutants with inactivated ompC survived, indicating partial fitness rescue. Clean mutations (i.e., ∆rpoE ∆ompC) reproduced the transposon effect, confirming the involvement of OmpC in ∆rpoE survival. OmpC could be the entry pore for a toxic molecule whose damages require E-mediated repair. Or, OmpC itself could be or generate a cargo in the periplasm. Further studies are required to understand the exact underlying molecular mechanisms, but the effect of ompC deletion on ∆rpoE fitness is remarkable. Another in vivo screen of the transposon library identified pnp as a target mutation. pnp encodes a major regulator involved in mRNAs degradation and cold shock resistance. Truncated pnp, in combination with ∆rpoE ∆ompC, almost reached WT-like fitness. Surprisingly, in vivo proteomics data demonstrated few differences between WT and mutant bacteria. This suggests that Salmonella can bypass E by a few minor alterations mediated by ompC and pnp deletions.
Collectively, we demonstrate that a small number of mutations can rescue the in vivo fitness of an avirulent regulatory mutant. Synergizing with host studies allows the identification of inhibitor targets that would not be found with our standard in vitro mimicking conditions. We therefore invalidated rpoE as an inhibitor target. Our approach could be more widely used in the future to evaluate other major systems such as the master regulator phoP or the general stress response sigma factor rpoS before potential inhibitors reach the clinics
A quantitative analysis of Salmonella Typhimurium metabolism during infection
Full text linkIn this thesis, Salmonella metabolism during infection was investigated. The goal was to gain a quantitative and comprehensive understanding of Salmonella in vivo nutrient supply, utilization and growth.
To achieve this goal, we used a combined experimental / in silico approach. First, we generated a reconstruction of Salmonella metabolism ([1], see 2.1). This reconstruction was then combined with in vivo data from experimental mutant phenotypes to build a comprehensive quantitative in vivo model of Salmonella metabolism during infection (unpublished data, see 2.2). The data indicated that Salmonella resided in a quantitatively nutrient poor environment, which limited Salmonella in vivo growth. On the other hand, the in vivo niche of Salmonella was qualitatively rich with at least 45 different metabolites available to Salmonella. We then used the in vivo model of infection to analyze reasons for the preponderance of Salmonella genes with undetectable virulence phenotype (unpublished data, see 2.3). Our data indicated that host supply with diverse nutrients resulted in large-scale inactivity of numerous Salmonella metabolic pathways. This together with extensive metabolic redundancy was the main cause of the massive Salmonella gene dispensability during infection. To verify this hypothesis experimentally, an unbiased method for large scale mutagenesis was developed (unpublished data, see 2.4). Results from 20 Salmonella mutator lines indicate that Salmonella can tolerate at least some 2700 to 3900 mutations, emphasizing again that a vast majority of Salmonella genes are dispensable in a defined environment
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Interferon signalling in the liver : implications for the natural course and therapy of hepatitis C
Full text linkHepatitis C virus is a global health concern, estimated to infect 2-3% of the world's population. Inter-individual differences in the course of infection and response to therapy, highlighted by recent genomewide association studies, point to the crucial role of the host immune system in the efficient control of infection. Ongoing progress in the studies of the role of innate immunity during hepatitis C virus infection has improved our understanding of the intricacies of the host-virus interactions. In this work I present and discuss results of three studies aimed to dissect interferon signalling in the liver in the context of natural course or therapy of hepatitis C virus infection.
Interferon-based therapies are in clinical use for treatment of diseases such as hepatitis C virus infection or multiple sclerosis. Interferon-induced regulators of the Jak-STAT signalling are known to involve in negative feedback loops and affect the response to exogenously administered interferon alpha. In this context it is important to understand which interferon subtypes are potent inducers of the negative regulators and whether all interferons are equally sensitive to the inhibitory mechanisms. To tackle this question we attempted to characterize and compare response patterns to interferons alpha, beta and lambda in a setting of continuous and repeated stimulation (see Section 3.1).
The acute phase of hepatitis C virus infection in humans (first 6 months after transmission) is characterized by high rates of spontaneous clearance and excellent treatment response (>90% cure rate). As the infection at that stage is mostly asymptomatic, it is rarely diagnosed and, in comparison to the chronic phase of hepatitis C virus infection, little is known about the human liver response to acute hepatitis C virus infection and the host-virus interactions during this time. In the second part of this PhD project we made use of the acute hepatitis C liver biopsies collected over the course of several years at the University Hospital of Basel to describe human hepatic response to acute hepatitis C virus infection and gain an insight into the mechanism of improved cure rate compared to chronic hepatitis C (see Section 3.2).
Chronic hepatitis C is currently treated with combination therapies based on pegylated interferon- alpha. A significant proportion of patients fails to respond to the current treatment options, probably due to the refractory state of the preactivated endogenous interferon system in the liver. Several compounds are currently in clinical development with the aim to improve the treatment outcome of pegylated interferon�alpha nonresponders. In the last part of this work we investigated in vivo the mode of action
of a novel synthetic TLR9 agonist which is a clinical candidate for anti-hepatitis C virus therapy
and characterized the hepatic response to this compound (see Section 3.3)
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