16 research outputs found
Functional characterization of "Bartonella" effector protein - BepE during "in vivo" and "in vitro" infection
Get PDFThe 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
A novel mechanism-based pharmacokinetic-pharmacodynamic (PKPD) model describing ceftazidime/avibactam efficacy against β-lactamase-producing Gram-negative bacteria
No full textBACKGROUND: Diazabicyclooctanes (DBOs) are an increasingly important group of non β-lactam β-lactamase inhibitors, employed clinically in combinations such as ceftazidime/avibactam. The dose finding of such combinations is complicated using the traditional pharmacokinetic/pharmacodynamic (PK/PD) index approach, especially if the β-lactamase inhibitor has an antibiotic effect of its own.OBJECTIVES: To develop a novel mechanism-based pharmacokinetic-pharmacodynamic (PKPD) model for ceftazidime/avibactam against Gram-negative pathogens, with the potential for combination dosage simulation.METHODS: Four β-lactamase-producing Enterobacteriaceae, covering Ambler classes A, B and D, were exposed to ceftazidime and avibactam, alone and in combination, in static time-kill experiments. A PKPD model was developed and evaluated using internal and external evaluation, and combined with a population PK model and applied in dosage simulations.RESULTS: The developed PKPD model included the effects of ceftazidime alone, avibactam alone and an 'enhancer' effect of avibactam on ceftazidime in addition to the β-lactamase inhibitory effect of avibactam. The model could describe an extensive external Pseudomonas aeruginosa data set with minor modifications to the enhancer effect, and the utility of the model for clinical dosage simulation was demonstrated by investigating the influence of the addition of avibactam.CONCLUSIONS: A novel mechanism-based PKPD model for the DBO/β-lactam combination ceftazidime/avibactam was developed that enables future comparison of the effect of avibactam with other DBO/β-lactam inhibitors in simulations, and may be an aid in translating PKPD results from in vitro to animals and humans.</p
A translocated effector required for bartonella dissemination from derma to blood safeguards migratory host cells from damage by co-translocated effectors
Get PDFNumerous bacterial pathogens secrete multiple effectors to modulate host cellular functions. These effectors may interfere with each other to efficiently control the infection process. Bartonellae are Gram-negative, facultative intracellular bacteria using a VirB type IV secretion system to translocate a cocktail of Bartonella effector proteins (Beps) into host cells. Based on in vitro infection models we demonstrate here that BepE protects infected migratory cells from injurious effects triggered by BepC and is required for in vivo dissemination of bacteria from the dermal site of inoculation to blood. Human endothelial cells (HUVECs) infected with a ΔbepE mutant of B. henselae (Bhe) displayed a cell fragmentation phenotype resulting from Bep-dependent disturbance of rear edge detachment during migration. A ΔbepCE mutant did not show cell fragmentation, indicating that BepC is critical for triggering this deleterious phenotype. Complementation of ΔbepE with BepEBhe or its homologues from other Bartonella species abolished cell fragmentation. This cyto-protective activity is confined to the C-terminal Bartonella intracellular delivery (BID) domain of BepEBhe (BID2.EBhe). Ectopic expression of BID2.EBhe impeded the disruption of actin stress fibers by Rho Inhibitor 1, indicating that BepE restores normal cell migration via the RhoA signaling pathway, a major regulator of rear edge retraction. An intradermal (i.d.) model for B. tribocorum (Btr) infection in the rat reservoir host mimicking the natural route of infection by blood sucking arthropods allowed demonstrating a vital role for BepE in bacterial dissemination from derma to blood. While the Btr mutant ΔbepDE was abacteremic following i.d. inoculation, complementation with BepEBtr, BepEBhe or BIDs.EBhe restored bacteremia. Given that we observed a similar protective effect of BepEBhe on infected bone marrow-derived dendritic cells migrating through a monolayer of lymphatic endothelial cells we propose that infected dermal dendritic cells may be involved in disseminating Bartonella towards the blood stream in a BepE-dependent manner
From FIC to BID : target identification and functional characterization of "Bartonella" effector proteins
Get PDFPathogens belonging to the genus Bartonella employ a unique stealth infection strategy that
involves evasion from the host immune system, replication in the endothelium and persistence in
erythrocytes. A key factor in colonization of the replicative niche is the manipulation of nucleated
cells to the benefit of bacterial uptake, survival, proliferation or spreading. To this end,
Bartonella spp. translocate a set of bacterial effectors via a VirB/VirD4 type IV secretion system
(T4SS) into the host cell. Upon translocation, several Bartonella effector proteins (Beps) hijack
host cell signaling cascades, thus, subverting host cellular functions to promote pathogenicity, yet
their underlying mechanism remains largely elusive.
Although pathogenicity factors evolved independently in radiating lineages of Bartonellae, Beps
share a common domain architecture. The C-terminal part of all Beps consists of a Bartonella
intracellular delivery domain (BID) and a positively charged tail region that primarily serve as a
bi-partite secretion signal. Apart from translocation, some BID-domains acquired additional
functions and interfere with host cell signaling resulting in cytoskeletal rearrangements during
pathogen entry. The N-terminal part is less conserved and can harbor phospho-tyrosine motifs,
additional BID-domains or share the ancestral domain architecture with a filamentation induced
by cAMP (FIC) domain. This domain was recently shown to catalyze the transfer of an AMPmoiety
onto target proteins, a process called AMPylation or adenylylation. Although the FICdomain
is widely distributed and can be found in all kingdoms of life, the only identified targets
are small GTPases of the Ras superfamily. In this study, we aimed to identify target proteins of
different Beps and to gain insights into their molecular function.
In Research Article I, we describe that BepA of B. henselae elevates intracellular cAMP-levels
by activating eukaryotic adenylyl cyclase (AC) synergistically with the a-subunit of stimulating
heterotrimeric G-protein (Gas). Further we could show that BepA is a conditional activator of AC
and directly interacts with at least one of the catalytically active cytosolic AC domains.
Furthermore, we established a mass spectrometry based strategy to identify targets of post
translational modifications on the example of AMPylation that is presented in Research
Article II. To this end, we used stable isotope-labeled ATP in in vitro AMPylation assays on
crude cell lysates which results in the formation of reporter ion clusters in subsequent LC-MS
analysis. Applying this strategy on an exemplary Fic protein, Bep2 of B. rochalimae, we
identified vimentin as a target protein. As vimentin is not structurally related to small GTPases,
we exhibit cytoskeletal components as a new target class of Fic protein-mediated AMPylation.
Taken together, Bartonella effector proteins target a plethora of host cell proteins and are thereby
manipulating key elements of host cell signaling. Therefore, they developed a high level of
versatility in their target proteins and molecular mechanisms ranging from complex formation to
posttranslational modifications. We hypothesize that both of these attributes play fundamental
roles in the establishment of chronic infections. Furthermore, the understanding of these basic
functionalities will be useful in the development of cell biology tools or of innovative
therapeutics
Ectopic expression of BepE<i><sub>Bhe</sub></i> in HUVECs prevents cell fragmentation.
No full text<p>(<b>A, B</b>) HUVECs of an early passage were transduced with lentiviruses for the expression of the depicted GFP-fusion proteins. The mixed culture of transduced and non-transduced cells were infected with the indicated <i>Bhe</i> strains (MOI = 200). Infected cells were either fixed and stained for microscopy or analyzed for the survival by FACS at 48 hpi. (<b>A</b>) Representative microscopy images (scale bar = 100 µm). F-actin is represented in red (Phalloidin), DNA in blue (DAPI), GFP in green. (<b>B</b>) Protection by GFP-fused BepE and its derivatives against fragmentation induced by <i>Bhe</i> Δ<i>bepDEF</i> mutant strains. GFP-positive cell were quantified by FACS and normalized to the uninfected cell population. One representative experiment (n = 3) with the mean of triplicate samples +/− SD are presented. Statistical significance was determined using Student's <i>t</i>-test. <i>P</i><0.05 was considered statistically significant.</p
Expression of BepE<i><sub>Bhe</sub></i> homologues in the <i>Bhe</i> Δ<i>bepDEF</i> inhibit the cell fragmentation phenotype.
No full text<p>(<b>A</b>) Subconfluent monolayers of HUVECs were infected for 48 h with MOI = 200 of the <i>Bhe</i> Δ<i>bepDEF</i> mutant complemented with the indicated Bep-expression plasmids followed by fixation, immunocytochemical staining and confocal laser scanning microscopy. F-actin is represented in red and DNA in blue (scale bar = 50 µm). (<b>B</b>) Quantification of cell fragmentation at 48 h post infection was performed as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004187#ppat-1004187-g001" target="_blank">Fig. 1C and D</a> and presented as mean of triplicate samples +/− SD. Statistical significance was determined using Student's <i>t</i>-test. <i>P</i><0.05 was considered statistically significant. Data from one representative experiment (n = 3) are presented.</p
BepE is essential for <i>Bartonella tribocorum</i> (<i>Btr</i>) to establish bacteremia after <i>intradermal</i> (<i>i.d.</i>) infection of the rat reservoir host.
No full text<p>(<b>A</b>) Domain organization of BepE orthologues in <i>Btr</i> and <i>Bhe</i>. The BepE homologues from <i>Bartonella</i> species depicted in the figure (BepE<i><sub>Bhe</sub></i>, BepD<i><sub>Btr</sub></i>, BepE<i><sub>Btr</sub></i>) were aligned using Geneious Pro 5.3.4. The amino acid sequence alignment with pairwise % identity is indicated. The tyrosine-containing N-termini and BID domains were aligned independently. (<b>B</b>) <i>Btr</i> Δ<i>bepDE</i> is not able to reach the blood of rats infected by the <i>i.d.</i> route. Rats (n = 5) were inoculated in the ear dermis with either <i>Btr</i> wild-type or <i>Btr</i> Δ<i>bepDE</i>. Blood was drawn at the indicated days post infection (dpi), diluted and plated on sheep blood supplemented Columbia agar plates (CBA) for counting of colony forming units (CFUs). (<b>C</b>) Complementation of the <i>Btr</i> Δ<i>bepDE</i> mutant with BepE is sufficient to restore bacteremia in rats infected by the <i>i.d.</i> route. Groups of rats (n≥3) were infected with the indicated strains by the <i>i.v.</i> or <i>i.d.</i> route. Blood was drawn at 16 dpi and CFUs were recovered as described for B. The graph represents CFUs/ml of blood for individual animals (circles) and their cohort mean (line). Statistical significance was determined using Student's <i>t</i>-test. <i>P</i><0.05 was considered statistically significant. (<b>D</b>) Heterologous complementation of <i>Btr</i> Δ<i>bepDE</i> with p<i>BIDs.E<sub>Bhe</sub></i> is sufficient to rescue the abacteremia phenotype following infection by the <i>i.d.</i> route. The infections were performed as described for (C). Data represented for BIDs.E<i><sub>Bhe</sub></i> complementation were acquired in separate experiment from the other data shown. <i>P</i><0.05 was considered statistically significant.</p
Dendritic cells are infected by <i>Bartonella</i>.
No full text<p>(<b>A</b>) Effector translocation by <i>Bhe</i> into mouse bone marrow-derived dendritic cells (BMDCs). Balb/c mouse BMDCs were infected with corresponding MOIs and strains. “Effector”, Bla-BID, translocation efficiency was assessed as the % of infected cells that converted CCF2-AM blue emission into green detected by Leica DM-IRBE inverted fluorescence microscope. The bars represent the mean of triplicate samples +/− SD. Data from one representative experiment (n = 2) are presented. (<b>B</b>) Migration of BMDCs is inhibited in a trans-well assay by <i>Bhe</i> Δ<i>bepDEF</i> infection. BMDCs were pre-infected with MOI = 50 of the indicated bacterial strains. Infected cells were embedded in collagen and mounted in a trans-well migration system that was prior seeded with a confluent monolayer of iLECs (immortalized lymphatic endothelial cells). BMDCs that migrated though the iLECs were quantified after 24 h. The data normalized to uninfected condition. The bars represent the mean of triplicate samples +/− SD. Statistical significance was determined using Student's <i>t</i>-test. <i>P</i><0.05 was considered statistically significant. Data from one representative experiment (n = 3) are presented.</p
BepE<i><sub>Bhe</sub></i> localizes to cell-to-cell contacts and is recruited to the plasma membrane of HUVECs following translocation via the T4SS or by ectopic expression.
No full text<p>(<b>A</b>) Subconfluent monolayers of HUVECs were infected with MOI = 100 of the indicated bacterial strains for 24 h or left uninfected. After fixation and subsequent immunocytochemical staining the specimen was analyzed by confocal laser scanning microscopy. F-actin is represented in blue (Phalloidin) and VE-cadherin staining in red (anti-VE-cadherin). Translocation of the effector protein into the infected cells was detected by anti Myc-staining depicted in green (scale bar = 20 µm). (<b>B</b>) HUVECs of an early passage were transduced with lentiviruses directing expression of either GFP or GFP-BepE<i><sub>Bhe</sub></i>. Cells were stained with wheat germ agglutinin (WGA, red) and fixed. Confocal images were acquired in <i>xy</i>- and <i>xz</i>-planes (scale bar = 50 µm). (<b>C</b>) <i>gfp-bepE<sub>Bhe</sub></i>-transduced HUVECs were subjected to live cell imaging using an MD ImageXpress Micro automated microscope. Snapshots of gray scale images at different time points as depicted by the time stamps (format: dd:hh:mm) are presented (scale bar = 50 µm). The arrows are pointing to the regions of transient enrichments of BepE<i><sub>Bhe</sub></i> in migrating HUVECs.</p
