225 research outputs found
Target identification for prevention and therapy of "Salmonella" infections
No full textIn this thesis we investigated maintenance requirements of Salmonella during chronic infections, and determined the relevant antigen properties that might facilitate the development of Salmonella vaccines. Overall, we wanted to achieve two goals:
a)To identify suitable targets to eradicate persisting Salmonella.
b)Identify antigen properties for developing enteric fever vaccine.
To achieve the first goal we established a practical Salmonella mouse infection model for identifying bacterial maintenance functions essential for persistency.Using this model, we evaluated twelve Salmonella defects. Our data revealed extremely relaxed environment of Salmonella during persistency compared to the acute infection. On the other hand, we identified that unsatuarated/ cyclopropane fatty acid synthesis pathway might contain suitable targets for antimicrobial chemotherapy of chronic infections.
To achieve the second goal, we tested thirty seven in vivo expressed antigens for immunogenicity and protectivity in a mouse typhoid fever model. We identified novel Salmonella antigens that conferred partial protection against virulent Salmonella in a typhoid fever model. The identified antigens had high sequence conservation among several Salmonella serovars suggesting that these antigens might be suitable as vaccine candidates against systemic Salmonella infection caused by diverse serovars. Using model antigens expressed in Salmonella and autologous antigens, our data also revealed that surface associated antigens might be promising for inducing both humoral and cellular immunity to Salmonella, as recognition of such antigens might enable uniquely detection and destruction of live Salmonella. This may provide a strategy to discover additional protective antigens for Salmonella and other intracellular pathogens
Identification of protective antigens for vaccination against systemic salmonellosis
No full textThere is an urgent medical need for improved vaccines with broad serovar coverage and high efficacy against systemic salmonellosis. Subunit vaccines offer excellent safety profiles but require identification of protective antigens, which remains a challenging task. Here, I review crucial properties of Salmonella antigens that might help to narrow down the number of potential candidates from more than 4000 proteins encoded in Salmonella genomes, to a more manageable number of 50-200 most promising antigens. I also discuss complementary approaches for antigen identification and potential limitations of current pre-clinical vaccine testing
Heterogeneity of inflammation and host metabolism in a typhoid fever model
No full textSystemic infections can lead to severe inflammation and altered host metabolism. These host responses are being extensively studied, but their spatial relationships in infected tissues remain largely unknown.
The goal of this thesis was to investigate the spatial organization of metabolic and inflammatory patterns in Salmonella-infected tissues and to elucidate the impact of host heterogeneity on host-Salmonella interactions in a murine typhoid fever model.
We showed that antimicrobial effector mechanisms such as generation of ROS and RNS occurred predominantly in granulomatous lesions. However, a substantial fraction of Salmonella resided outside of these lesions and was therefore not covered by these antimicrobial regions. Heterogeneous exposure to RNS induced distinct, locally adapting Salmonella subpopulations (see chapter 2.1).
We also investigated host metabolic enzyme activities in various tissue regions. Using a novel combination of immunostaining with enzyme histochemistry, we showed that granulomas had a distinct metabolic profile with a high capacity for generation of NADPH, an essential substrate for local generation of bacteriostatic/bactericidal ROS and RNS. Indeed, adaptation of GFP-based live/dead discrimination revealed extensive Salmonella killing that predominantly occurred in granulomas (see chapter 2.2).
The spatial segregation of live Salmonella from regions with massive killing also offered a potential explanation why surface-associated Salmonella antigens, but not internal antigens that are inaccessible in live Salmonella, were required for protective immunity (see chapter 2.3).
In conclusion, this thesis revealed markedly heterogeneous conditions in Salmonella-infected host tissues that had profound impact on disease mechanisms, infection control, and immunity
A quantitative analysis of Salmonella Typhimurium metabolism during infection
No full textIn 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
Functional characterization of "Bartonella" effector protein - BepE during "in vivo" and "in vitro" infection
No full textThe bartonellae is a family of gram-negative, fastidious, facultative intracellular, zoonotic bacteria. Most of the Bartonella species are highly adapted to establish asymptomatic bacteremia of their reservoir host within which the bacteria colonize erythrocytes as privileged host niche and develop long-lasting persistent infections. Bartonella uses a VirB type IV secretion system (T4SS) to translocate Bartonella effector proteins (Beps) into the infected cells. By using such a tool box it subverts host cellular functions in order to establish a safe niche for replication and survival.
This thesis aimed to elucidate the role of one of the effector proteins – BepE in the establishment of Bartonella infection by using in vivo and in vitro infection models. Started in December 2006, my primary aim was to establish a suitable model for pathogen - natural host interaction. In order to closely mimic the reservoir host infection by BartonelIa, I have adapted the rat intra-venous (i.v.) to intra-dermal (i.d.) infection model, inoculation of B. tribocorum (Btr) in the ear dermis of the animal. This route of infection reflects the natural way of Bartonella transmission by arthropods when the bacteria are inoculated in the skin of a mammal via the feces of a vector after animal scratches. The Btr wild-type i.d. infected animals developed blood stage infection, which started around 7-8 days post infection and lasted for 10 weeks. It was a long-term bacteremic infection without obvious clinical manifestations, a hallmark of the reservoir host infection by Batonellae. The time delay that Btr took to appear in blood could correspond to the way that bacteria need to pass from the derma to the lymphatic-blood system and to the possible interaction with the innate immune system. In summary, the rat i.d. model enabled us to distinguish Bartonella factors involved on two different phases of the infection: early phase, prior seeding into the blood and the blood stage. On those two stages bacteria have different environment to interact with, and assumably different strategies to cope with the host immune system. The rat i.d. infection model revealed BepE as a critical factor in the establishment of reservoir host bacteremia. The expression of BepEBtr could rescue the abacteremic phenotype of Btr ΔbepDE mutant and enabled the strain to reach the blood. Heterologous complementation of Btr ΔbepDE phenotype with BepEBhe suggests that this function of BepE is conserved between different species of Bartonellae. Even more, I could demonstrate that the C-terminal BID domains are having the specific function but putative phosphotyrosine-containing N-term of BepE does not play an essential role in the establishment of long-term bacteremic infection of the natural host by Bartonella.
Another phenotype of BepE but in vitro was observed during the infection of primary endothelial cells HUVECs with Bhe ΔbepE (and ΔbepDEF) mutant(s). Besides erythrocytes, endothelial cells represent another major target cell type for Bartonellae demonstrated as bacillary angiomatoses within the incidental host environment, mostly in immunocompromized human patients. HUVECs infected with Bhe stain that lacked BepEBhe revealed disturbed rear edge detachment during migration and followed with the fragmentation of cell body. This phenomenon was inhibited by pbepEBhe expression in Bhe ΔbepE (and ΔbepDEF) as well as, by T4SS independent expression of pbepEBhe in HUVECs by transfection prior the infection with Bhe ΔbepE (and ΔbepDEF). We found that the cell fragmentation of infected HUVECs is T4SS dependent and is a secondary effect of translocated Beps, potentially the Beps involved in the invasome formation. Further we conclude that the C-terminal BID domains of BepEBhe are sufficient to interfere with the cells fragmentation process. From this we could hypothesize that primary infected cells in i.d. infection model of rats may also undergo fragmentation or impaired migration when infected with Btr ΔbepDE and then Bartonella does not succeed to reach the blood system and colonize red blood cells.
Further, I introduced the i.d. in vivo infection of Rosa 26-loxP-egfp Balb/c mice and in vitro infection of mouse Bone Marrow-derived Dendritic Cells (BMDCs) with B. birtlesii (Bbi) strain that is expressing Cre-BID fusion protein. The in vitro model showed for the first time a Bartonella effector protein translocation in primary immune cells of the reservoir host. This finding builds a strong basis for the hypothesis that primary infected cells in vivo may be the DCs (Langerhance cells or dermal DCs) in the skin of infected animal. DCs are the sentinels of the immune system that constantly sample the environment for the “danger signal”. Thus, they represent one of the candidate cells in the derma to be targeted by Bartonella after inoculation of the bacteria from the feces of arthropod vector. Infected DCs could serve as Trojan horses to carry and disseminate Bartonella from derma to lymphatic–blood system
Innate immune recognition of Salmonella and Francisella : two model intracellular bacterial pathogens
No full textThe 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
No full textAn 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
Pathogen proteomes during infection : a basis for infection research and novel control strategies
No full textInfectious diseases cause tremendous mortality and morbidity worldwide. Rising antimicrobial resistance and the lack of new drugs cause an increasingly alarming crisis in infectious disease control. New system-level approaches are likely to help understand complex host/pathogen interactions as a basis for rational development of novel antibiotics and vaccines. Proteome analysis of pathogens in infected tissues comprehensively reveals functionally relevant pathogen activities during infection. It also highlights potential targets for antimicrobial chemotherapy as well as promising antigens for vaccination. Integration of these data with complementary large-scale data helps to further prioritize candidates for in-depth experimental analysis. Here, I discuss some of these approaches with a special emphasis on the model pathogen Salmonella
A moonlighting enzyme imposes second messenger bistability to drive lifestyle decisions in E. coli.
No full textBacteria 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
No full textAntimicrobial 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
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