Functional characterization of "Bartonella" effector protein - BepE during "in vivo" and "in vitro" infection

The 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 translocated effector required for bartonella dissemination from derma to blood safeguards migratory host cells from damage by co-translocated effectors

Numerous 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

A novel mechanism-based pharmacokinetic-pharmacodynamic (PKPD) model describing ceftazidime/avibactam efficacy against β-lactamase-producing Gram-negative bacteria

BACKGROUND: 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 novel mechanism-based pharmacokinetic–pharmacodynamic (PKPD) model describing ceftazidime/avibactam efficacy against β-lactamase-producing Gram-negative bacteria

Abstract Background 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. </jats:sec

Ectopic expression of BepE<i>_Bhe</i> in HUVECs prevents cell fragmentation.

<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

BepE<i>_Bhe</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.

<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>_Bhe</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_Bhe</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>_Bhe</i> in migrating HUVECs.</p

The double deletion mutant <i>Bhe</i> Δ<i>bepCE</i> abolishes cell fragmentation.

<p>(<b>A</b>) Subconfluent monolayers of HUVECs were infected for 48 h with MOI = 200 of the <i>Bhe</i> strains depicted in the figure or were left uninfected. Samples were then fixed, stained immunocytochemically and analyzed by confocal laser scanning microscopy. F-actin is represented in red (Phalloidin) and DNA in blue (DAPI) (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

Dendritic cells are infected by <i>Bartonella</i>.

<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 protects host cells from fragmentation upon translocation via T4SS.

<p>(<b>A</b>) Subconfluent monolayers of HUVECs were infected with MOI = 100 of the indicated bacterial strains for 24 h. After fixation and subsequent immunocytochemical staining the specimen was analyzed by confocal laser scanning microscopy. F-actin is represented in red (Phalloidin) and DNA in Blue (DAPI). 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>) Subconfluent monolayers of HUVECs were infected with MOI = 200 or MOI = 200+200 in case of mixed infection depicted in the figure. 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 = 2) are presented. (<b>C</b>) Protein levels of the BepE<i>_Bhe</i> by overexpression in <i>Bartonella</i> strains. The anti-Myc western blot was obtained from total lysate of corresponding <i>Bartonella</i> strains depicted in figure.</p

Expression of BepE<i>_Bhe</i> homologues in the <i>Bhe</i> Δ<i>bepDEF</i> inhibit the cell fragmentation phenotype.

<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